Journal of Morphology 26 (1915)

From Embryology
Embryology - 15 Dec 2019    Facebook link Pinterest link Twitter link  Expand to Translate  
Google Translate - select your language from the list shown below (this will open a new external page)

العربية | català | 中文 | 中國傳統的 | français | Deutsche | עִברִית | हिंदी | bahasa Indonesia | italiano | 日本語 | 한국어 | မြန်မာ | Pilipino | Polskie | português | ਪੰਜਾਬੀ ਦੇ | Română | русский | Español | Swahili | Svensk | ไทย | Türkçe | اردو | ייִדיש | Tiếng Việt    These external translations are automated and may not be accurate. (More? About Translations)

Historic Disclaimer - information about historic embryology pages 
Mark Hill.jpg
Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)

Template:J Morphol. Volumes

JOURNAL OF MORPHOLOGY

Founded by C. O. Whitman


EDITED BY J. S. KINGSLEY

University of Illinois Urbana, Ill.

with the collaboration of Gary N. Calkins Edwin G. Conklin C. E. McClung

Columbia University Princeton University University of Pennsylvania

W. M. Wheeler William Patten

Bus.'iey Institution, Harvard University Dartmouth College


VOLUME 26 1915

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA

COMPOSED AND PRINTED AT THE WAVERLY PRESS

By the Williams & Wilkins Company

Baltimore, JId., U. S. A.

Contents

1915

No. 1. MARCH

Pauline H. Dederer. Oogenesis in Philosamia cynthia. Sixty-four figures (six plates) 1

William H. F. Addison and J. L. Appleton, Jr. The structure and growth of the incisor teeth of the albino rat. Twenty-nine figures 43

James G. Hughes, Jr. A peculiar structure in the electroplax of the stargazer, Astroscopus guttatus. Three figures 97

W. Rees Bremner Robertson. Chromosome studies. III. Inequalities and deficiencies in homologous chromosomes : their bearing upon synapsis and the loss of unit characters. Fourteen figures (three plates) 109

No. 2. JUNE

George W. Tannreuther. The embryology of Bdellodrilus philadelphicus. Twenty-six text figures and eight plates 143

E. E. Just. The morphology of normal fertilization in Platynereis megalops Three plates (thirty figures) 217

Wm. a. Kepner and J. R. Cash. Ciliated pits of Stpnostoma. Four figures 235

Huber GC. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day. (1915) J Morphol. 26: 247-358.

G. Carl Huber. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day. Thirty-two figures 247

Huber GC. The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova: end of the first to the end of the ninth day. (1915) J Morphol. 26: 359-391.

G. Carl Huber. The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova: end of the first to the end of the ninth day. Ten figures. 359

Marianna van Heraverden. Comment on Miss Beckwith's paper on "The genesis of the plasma-structure in Hydractinia cchinata" and reply by Miss Beckwith

No. 3. SEPTEMBER

E. A. Baumgartner. The development of the hypophysis in SqualusAcanthias. Forty-three figures ,391

J. Frank Daniel. The anatomy of Heterodontus francisci. II. The endoskeleton. Thirty-one figures (eight plates) 447

Robert W. Hegner. Studies on germ cells. IV. Protoplasmic differentiation in the oocytes of certain Hymenoptera. Ninety-seven figures (thirteen plates) 495

No. 4. DECEMBER

Edward Phelps Allis, Jr. The homologies of the hyomandibula of the gnathostome fishes. One figure 503

James L. Kellogg. Ciliary mechanisms of lamellibranchs with descriptions of anatomy. Seventy-two figures 625

A. T. Evans. The morphology of the frontal appendage of the male in the Phyllopod crustacean Thamnocephalus platyurus Packard. Nine figures. 703

Oogenesis In Philosamia Cynthia

Pauline H. Dederer,

From the Zoological Laboratory, Columbia University

Sixty-Four Figures (Six Plates)

Contents

Introduction 1

Material and technique 2

Maturation divisions 3

1. Observations 3

a. Chromosomes in the embryo 3

b. First maturation division 3

c. Second maturation division 6

d. Fertilization 7

2 . Conclusions and comparisons 8

Early oogenesis 10

1 . Observations 10

a. Growth of the ovary: General description 10

b. Early stages in the development of the ovary 11

c. Development of nurse cells 13

d. Development of egg cells 17

e. Degenerating cells in the ovary 20

f. Abnormal nuclei in the early ovary 20

2 . Conclusions and comparisons 20

3 . Literature on the early development of eggs and nurse cells 22

Summary 25

Literature cited 27

INTRODUCTION

From the standpoint of sex production the Lepidoptera are of especial interest as compared with other insects, because the experimental evidence of Doncaster ('06, '08) and Raynor ('06), Punnett and Bateson ('08) seems to demand the assumption that there are two kinds of eggs in the moth. The absence of visible dimorphism in the spermatozoa of the Lepidoptera also lends probability to this hypothesis. In other groups of insects studied, the spermatozoa are often dimorphic. The eggs have been assumed to be all ahke, and this condition has been demonstrated by Morrill ('09) for certain coreid Hemiptera, by Morgan ('09) for phylloxerans, by Stevens ('06 a, '09) and von Baehr ('08, '09) for aphids.

In studying the history of the male germ cells in the moth Philosamia cynthia (Dederer '07), the spermatocytes were found to contain exactly similar groups of chromosomes. The same facts had been determined by Stevens ('06 b) and Cook ('10) for various other Saturniidae. Doncaster ('12) believes that in Pieris brassicae there is no dimorphism, either in the male or female germ cells. Recently, however. Seller ('13) has decribed two kinds of eggs in a lepidopteran. These two papers will be discussed later. The present work was undertaken with special reference to the question of dimorphism in the eggs. I wish to express my indebtedness to Prof. E. B. Wilson for valuable advice and criticism during the course of the investigation.

MATERIAL AND TECHNIQUE

Carnoy's aceto-alcohol-chloroform mixture, saturated with sublimate, was used almost exclusively, since it readily penetrated the tough chorion of the eggs, which were left in the fluid from two to four hours. Flemming's and Bouin's fluids were used, but proved very unsatisfactory.- The eggs were then transferred to iodized 95 per cent alcohol, and the chorion was removed with needles. After dehydration in absolute alcohol, the eggs were placed in a mixture of alcohol and chloroform followed by pure chloroform for ten minutes. Immersion in melted paraffine for fifteen minutes was sufficient for perfect infiltration. The stains used were iron hematoxylin, and in a few cases safranin.

The maturation spindle remains in first metaphase until the entrance of the sperm. The following tabulation gives roughly the maturation stages at different intervals after the eggs are laid:

Eggs just laid to ^ hour laid first anaphase

Eggs laid 1 to 1| hours second metaphase

Eggs laid 1| hours second telophase

Eggs laid 2 to 2j hours fusion of pronuclei


In the study of the early stages of oogenesis, both caterpillars and pupae were used. The ovaries in the early pupal stage lie enveloped in the fat bodies just beneath the dorsal wall of the abdomen in the fifth segment. Upon removing the wall, the ovaries were located and transferred immediately to the fixing fluid. Flemming's, Bouin's and Carnoy's fluids were used, the latter being the only one good for all stages. The stains employed were safranin and iron hematoxylin.

MATURATION DIVISIONS 1. OBSERVATIONS

a. Chromosomes in the embryo

The number of chromosomes in the spermatogonia is 26 (Dederer '07), all rounded bodies, approximately equal in size. For determining the somatic number, sections were made of eggs several hours after fertilization. Ten counts from three different lots of eggs showed clearly 26 chromosomes. In polar view of metaphase (fig. 1) they appear as slightly elongated, often bipartite, bodies of comparatively slight difference in size. Owing to their similarity it is impossible to attempt any arrangement of the chromosomes in pairs, as has been done in some animals.

h. First maturation division

The mature eggs are oval bodies about 1.5 mm. long, each invested in a tough white chorion, which is flatter and broader at the animal pole. The flattened area appears to be due to the contact of the nurse cells in this region, while in the egg tubes.

Figure 32 is from a longitudinal section through the animal pole of an egg, showing a spindle with chromosomes in anaphase, within a dense granular mass, whose long protoplasmic processes reach out into the more reticular portion of the cytoplasm. This latter region is free from yolk, and is cone-shaped in form, the apex pointing inward, and extending as a sort of narrow vacuolated core, into the center of the egg. The remainder of the egg is filled with large yolk spheres. At the periphery of the egg appears a thin layer of dark granular protoplasm.

In earlier eggs, before or at the time of laying, a clear pale vitelline membrane may be seen beyond this. The polar bodies are formed in the dense granular layer, very near the middle of the anterior end, just within the cone-shaped area.

The earliest nuclear stage obtained after the growth of the egg, is the late prophase, found in eggs which had not been laid (fig. 3). The chromosomes, 13 in number, lie enclosed within the nuclear membrane, near the surface of the egg, in the same position as the first maturation spindle. The chromosomes are smooth, elliptical or dumb-bell shaped bodies, almost equal in size. Later the nuclear wall breaks down, the spindle fibers appear and the chromosomes become arranged upon them preparatory to division (figs. 4-5). When first placed upon the spindle, the chromosomes do not all show a dyad form, but later a median constriction appears in each one. The spindle lies obliquely to the surface of the egg. The spindle fibers can rarely be traced to a point of convergence, and no centrosomes nor asters appear. Various cytoplasmic bodies lie near or attached to the spindle (fig. 4), but they are not constant in size or number, and often cannot be detected. They are present only during the metaphase.

In figures 6 and 7 are shown two first division groups. There is but slight difference in the size of the chromosomes, and each one appears to be separated into two equal parts. On account of their small size they were at first interpreted as chroraosomes of the second division, but a further study showed that this was not the case. There was no trace of a first polar body, nor of sperm within the egg. Moreover, the eggs had not been laid, but were taken from a moth which had just begun laying. Figures 4 and 5 and numerous other undoubted first metaphase stages, with larger chromosomes, were obtained from the same lot of eggs. Restaining and extraction had practically no effect in altering the size difference which remains unexplained. In twelve cases I found chromosome groups similar in size to those shown in figures 6 and 7. These figures seem to indicate that the chromosomes divide equally in the first division but a definite statement is unwarranted, for the chromosomes are so small that a shght size difference might easily escape detection; moreover the variability in size in different groups, as seen in figures 5 and 6 would render any deductions in this respect extremely hazardous.

As the chromosomes approach the ends of the spindle, the fibers thicken enormously in the middle, forming a deeply staining cell plate, which in side view, gives the appearance of a band encircling the spindle (fig. 8). Henking ('92) described similar bodies in Pieris, which he considered as waste achromatic substance. With long extraction the cell plate appears very faint, while the chromosomes remain dark. Figure 9 shows another anaphase, in which 13 chromosomes may be counted at each pole. No lagging chromosomes were observed. In a late anaphase (fig. 10) another size peculiarity is observed, each chromosome being after division approximately as large as those of the metaphase stage. The irregularity in the form of the chromosomes in late anaphase was characteristic of this stage, and was equally apparent with either dark or light staining, although in the latter case the chromosomes appeared slightly smaller. Figure 11 is an oblique polar view of a similar stage; on account of the plane of the section the spindle cannot be seen. In figures 12 to 14 are shown polar views of spindles, the groups lettered a in each case being those entering the first polar body. Four chromosomes are in the center of each group, surrounded by a ring of nine. The polar body groups sometimes appear slightly smoother in outline than the egg groups (fig. 12), smaller, and bipartite in preparation for a second division. In attempting to compare chromosomes in the polar body with those in a similar position in the egg group, it is impossible to obtain any evidence either for or against an equal division of chromosomes. The variability is extreme, within both egg and polar groups, and in many cases it is very difficult to be sure of the actual chromosome outlines.

During the formation of the first polar body the spindle fibers elongate considerably, and the granular cytoplasm forms a conspicuous projection on the surface of the egg (fig. 33). There is apparently no first telophase, for no loss of contour or massing of the chromosomes was observed between the late anaphase and the second metaphase.

c. The second maturation division

In the second division two spindles appear, as shown in figure 34. Upon one are arranged the chromosomes of the first polar body; on the other, those of the second polar division. The old spindle fibers have disappeared, and the cell plate has assumed the form of irregular deep-staining masses. At a corresponding stage in the oogenesis of Bombyx mori, Henking ('92) figured a cell plate in a similar position, but it differed from this in being a single disc-shaped body or 'thelyid', which stained very faintly. The later constriction of the first polar body in P. cynthia, as in Bombyx, does not involve the cell plate but passes between it and the outer group of chromosomes.

In figure 15 A, B, drawn from adjacent sections, are shown 13 approximately equal chromosomes, arranged upon the two spindles, preparatory to a second division. It will be observed that the groups in the egg B and the first polar body A are at this time similar in the form and size of the chromosomes. The remnants of the cell plate are composed of deep-staining bodies, so large and definite as to give the appearance of chromosomes, but they are very irregular in form, and vary in size from large masses to very small granules. Figure 16 is another view of a similar stage. In figure 17, a polar view from three succeeding sections, the cell plate is composed of 15 large bodies, and numerous granules. One chromosome is missing from the egg group, B,

For various reasons second anaphase stages were extremely diflficult to find. A few cases, however, were obtained, which seem fairly clear. In figure 19 is shown a spindle in which 13 chromosomes are seen at one pole, 11 at the other. Thi^ latter group, which is incomplete, enters the second polar body. The 13 chromosomes of the egg nucleus are very small rounded bodies nearly equal in size. There is nothing to indicate a peculiarity in behavior of any of the chromosomes. In figure 20 polar views of two groups in anaphase are shown; here 13 approximately similar chromosomes appear in each. In figure 21, a polar view of an egg group, 13 chromosomes may be counted. Figures 23 and 24 are two oblique sections through spindles in anaphase. The groups in each case have been slightly displaced. There are 13 chromosomes in each group. These examples, while not numerous, are sufficient to show that the second polar body receives a group of chromosomes similar in number to those remaining in the egg. The small size of the chromosomes, and the lack of early anaphase stages, make it impossible, as in the case of the first division, to draw any conclusions as to the equal or unequal division of the chromosomes.

The first polar body was frequently observed in anaphase, during the stage figured above. After the second anaphase the egg chromosomes show a tendency to fusion (fig. 25) and it is impossible to distinguish separate chromosomes at either pole.

d. Fertilization

In sections through eggs in the first metaphase stage, several spermatozoa may be seen within the vitelline membrane, but only occasionally within the egg. In late anaphase, sections show that the spermatozoon has penetrated into the egg and is enveloped in a dark granular island of cytoplasm. Numerous eggs were found containing two or three spermatozoa. Thus in P. cynthia, as in many other insects, polyspermy appears to be normal. Shortly after entering the egg, the sperm appears as a long tapering rod. Later, it has the form of an oval, deepstaining vesicle surrounded by a clear area, and in contact with the female pronucleus, which lies nearer the surface of the egg. Subsequently the male pronucleus becomes spherical, the clear area disappearing. The chromatin in both nuclei is in the form of irregular flocculent masses, at first darker in the male pronucleus. Later it has the same staining capacity in both, so that it is impossible to distinguish male and female except by position.

At a later period, it is possible to count the chromosomes in each nucleus. In figure 35 the pronuclei lie near the surface of the egg where the second polar body appears. The surface cytoplasm merges with the remnants of the vitelline membrane, in which the polar body lies. Figure 27 is the same section enlarged. The pronuclear walls appear broken at the region of contact, or are so thin as to be invisible. Nine chromosomes are seen in the first section of the outer nucleus, 11 in the first section of the inner; they differ s ightly in size, and some are noticeably dyad in form. To the right of these are drawn portions of the nuclei from a succeeding section, showing 4 more chromosomes in the outer nucleus, 2 more n the inner, making 13 in each. No nucleoli are present. Within the polar body figured here remains of spindle fibers and a nuclear membrane are seen. Here too, 13 chromosomes appear. This is probably the second polar body, for the first becomes very vague after the second anaphase.

In figure 28, from an egg similar to the one described, nine or more chromosomes may be counted in the second polar body. The first polar body has apparently divided, the chromosomes in each appearing as vague granular areas. It seems probable that all three degenerate shortly after the fusion of the germ nuclei. There is no evidence that they remain included within the egg.

2. CONCLUSIONS AND COMPARISONS

The evidence obtained from the foregoing study indicates that in Philosamia cynthia the 13 chromosomes seen in the late prophase of the egg all divide in both maturation divisions. The male and female pronuclei at the time of their union each contain 13 chromosomes, giving the somatic number 26, which is found in the nuclei of the blastoderm. It appears to be certain that all of the eggs contain the same number of chromosomes, but the evidence for either the presence or absence of an XY-pair is not conclusive, on account of the variability in the size of the chromosomes in the metaphase and anaphase plates. In the early oogenesis, to be described later, there is no indication of the presence of a heterochromosome, either of equal or unequal parts, and from this we might suspect its absence in later stages. Although from the totality of the evidence, it appears probable that there is no difference in the chromosome groups, the matter will have to be left an open question.

Doncaster ('12) found that in Pieris brassicae both the male and female germ cells contain an equally paired heterochromosome which constitutes a chromatin nucleus during the growth period. He believed that in Abraxas a similar condition probably prevailed, and concluded that the chromosomes here do not provide any visible basis for the sex-limited transmission of characters." More recently, however ('13) he has found some females of Abraxas with 56 chromosomes, some w'th 55, and he beheves that there s a possibility of two kinds of eggs in this form.

Until the past year, the only recorded case of nuclear dimorphism in eggs (exclusive of parthenogenetic and sexual eggs) was that of the sea-urchin described by Baltzer ('09), in which the female appeared to be the heterogametic sex. Tennant, ('12) however, discovered that in other forms the male is heterogametic. Baltzer has recently ('13) announced that the results described in his former paper are erroneous, and he is convinced that the male is the heterogametic sex. This solves the apparent contradiction within the echinoderm group, the females being homozygous for sex in all cases described.

The latest case of heterogamy in the female is that recently described by Seller ('13) for the lepidopteran Phragmatobia fuliginosa. In the spermatocyte divisions, 27 small chromosomes are present, and a large one, which, though lagging somewhat, divides equally in both maturation divisions. In the first metaphase plate of the egg, 27 small chromosomes and a large chromosome, slightly segmented or lobed appear. After the first division, at one pole of the spindle are seen 27 small chromosomes and a large one; at the other pole, 28 small chromosomes and a large one. It is a matter of chance whether the polar body or the egg nucleus receives the extra chromosome. Seller interprets the extra small chromosome as a lobe of the large autosome which has separated from it during division, since in anaphase a small chromatin mass lies near one end of the large chromosome as if detached from it. Second divisions were not observed but Seller believes they are probably equational. He suggests the tentative interpretation that the extra small chromosome is the X chromosome. Unfortunately, only polar views of the first division are given, and these only of late anaphase, so it is impossible to determine how the extra chromosome arises. It is possible that the separation of this chromosome (described by Seller) inay be merely a temporary condition, followed by a union with the large one at the second metaphase, thus giving similar groups of chromosomes in all the oocytes. In view of the fact that in echinoderm eggs an apparently clear case of dimorphism has been found to be incorrect, it seems particularly necessary "to scrutinize carefully any evidence along this line.

EARLY OOGENESIS

A study of the early oogenesis of P. cynthia was undertaken in order to determine the origin of the haploid groups of chromosomes which enter the first polar metaphase. By analogy with spermatogenesis, pairing of the chromosomes in the egg should occur before the growth period. Although the material is unfavorable for the study of oogenesis as a whole, a seriation of stages was obtained, and several points of interest were observed in regard to the tlifferentiation of primitive ovarian -cells into eggs and nurse cells, and their later relation to each other.

1. OBSERVATIONS

a. Growth of the ovary: General description

The earliest ovaries obtained were from larvae fixed the latter part of August, a few days before the spinning of the cocoon. They are pear-shaped bodies, about 1 mm. in length, slightly smaller than a mature egg. Figure 29 is a lengthwise section through a larval ovary. The oval mass of connective tissue surrounds four egg strings which take a complicated course within the capsule. The strings open into a single slightlyexpanded chamber at the surface of the ovary, from which the oviduct arises. The earliest eggs are found near the opposite end of the ovary. Oogonial stages to very early eggs were found in this and similar ovaries. Figure 30 is of a January ovary, showing an increase in size and the growth of the egg strings. Two strings are broken away from the oviduct, but their points of attachment may be seen. The stages in this ovary ranged from a few spiremes to well-developed eggs, each with its five nurse cells contained in a separate chamber in the string. The ovaries of early June were practically identical in size with those of January. AH the cells by this time have differentiated into eggs and nurse cells. In early July, the ovaries are markedly different. Figure 31 shows portions of three egg strings from a pupa about three days before the time of emergence. The ovary now consists entirely of egg strings with a decidedly beaded appearance due to the growth of the eggs.

b. Early stages in the development of the ovary

A description is given below of the stages in the development of the eggs and nurse cells from the oogonia to the first metaphase of the egg.

, Stage a {fig. 37 a): The oogonial region containing cells in various stages of final oogonial divisions and in rest before these divisions. A polar view (fig. 2) of a metaphase plate shows 26 chromosomes. In anaphase stages no lagging chromosomes were observed, nor differential divisions, such as have been described in several insects (Buchner '09, Gtinthert '10).

Stage b {fig. 36): Post-oogonial nuclei. Here the chromatin assumes the form of deep-staining bodies with ragged and irregular outhnes. No constant number can be counted. The cells are connected by dense protoplasmic strands or tubes, which appear to originate from the spindle remains of the oogonial divisions. Gtinthert ('10) figured similar connections between eggs and nurse cells in the oogonia of Dytiscus. In figures 41 and 42 are shown two tubes with their branches appearing to terminate in rounded knobs, which are merely the upturned ends of the branches. The largest number of branches observed was 6 and this is probably the correct number as sections of later ovaries show that each egg cell is connected with five nurse cells.

Stage c (figs. 38 and 39). The chromosomes are transformed into smaller irregular fragments which later assume the form of pale delicate threads. There is no trace of the uncoiling, of convoluted threads from the chromatin masses to form the leptotene stage, as described by Davis ('08) and Wilson ('12) in the spermatogenesis of insects.

Stage d (fig. 40) •' The presynaptic leptotene. The threads now appear more definite and convoluted. A few irregular clumps of chromatin may be seen, but fewer detached fragments than before. Several free ends of the spireme are visible, but it is impossible to approximate the number of threads.

Stage e (figs. 37 b and 43): The synaptic stage or synizesis. A study of this period gives most unsatisfactory results. The contraction figure seems to follow immediately upon the leptotene stage. The spireme forms a deep-staining mass closely and intricately coiled. In some animals the synaptic knot shows two kinds of threads, thick and thin, indicating a possible parasynapsis. In P. cynthia all parts show the same diameter throughout.

Stage f (figs. 37 c and 44) •' Postsynaptic spireme. The threads now begin to spread out through the nuclear cavity. A few free ends are visible. In other cases the spireme might be interpreted as continuous. It stains deeply as before, and is of the same thickness throughout. Several writers have stated that the nuclei are not enclosed at this time, the cells forming a syncytium. In many sections of this material, cell boundaries were not to be seen, but in other cases, particularly when Flemming's fluid was used, they could be traced without any difficulty. The tubes connecting the cells stain very lightly at this stage, being only occasionally visible.

From this point on*, a gradual differentiation occurs between eggs and nurse cells, so that it is convenient to treat the two separately. The further nuclear changes in the nurse cells will be considered first.


c. Development of nurse cells

A condition shown in figure 45 succeeds that of the preceding figure. The spireme is spread out through the nuclear cavity, and appears to consist of about 13 segments, most of them looped, but without any indication of polarization. A small plasmosome is present. Somewhat later (fig. 46), 13 definite segments may be counted, and the loops show a tendency to straighten out into long rods. The threads are very definite, with fairly smooth outhne, and appear very slightly thicker than when first opening out. The hajDloid number was counted in at least twenty nuclei of this period. The plasmosome is slightly larger than before, and has no chromatin associated with it, nor is there any orientation of the segments with respect to it. Toward the latter part of this stage, the nucleus increases in size, and the chromatin segments gradually become thicker and more deeply-staining, . giving rise to the condition shown in figure 47. Some of the segments are in the form of curved rods, others are sharply bent. The plasmosome is larger at this time, and frequently vacuolated.

The condition of the thick thi-eads in figure 47 is similar in general appearance to the pachytene stage of other animals, but it is probably not equivalent in its origin, since the threads are formed, not by doubling, but by a gradual widening of the thinner threads. This stage represents the pachytene period only in the sense that it is subsequent to synapsis, and gives rise to the diplotene stage.

Stage g (figure 48): The diplotene stage. A longitudinal split now appears for the first time in all the chroniosomes, and shows very clearly in cells which lie directly in contact with those of the preceding period which show no split. They are differentiated from them also in length of the chromosomes, for the split threads are considerably shorter. Doncaster ('12) describes in Abraxas the double thi'ead as arising probably by a bending over of the chromosome, with a separation later at the bend, but this is certainly not the case in P. cynthia. There is no clue whatever to the relation of these double threads to the chromosomes of the oogonia, since the spht appears de novo, and also since there is no direct evidence that the chromatin threads have conjugated in synapsis. Unfortunately, therefore, P. cynthia cannot be added to the list of forms in which either parasynapsis or telosynapsis has been observed. The impression gained from a study of the material is that reduction is accomplished by a simple segmentation of a continuous spireme into the haploid number of threads.

Following upon figure 48, a progressive shortening of the segments occurs, the chromosomes often appearing extremely ragged. In a few cases, the halves- show a divergence at one point as if beginning to separate (fig. 49) . Very rarely the threads open in the middle while remaining united at the ends, thus forming a ring, but these forms are probably accidental and due to the fact that the chromosomes are soon to disintegrate. The later condition of this stage is shown in figure 50. The threads have by this time assumed a rod-like appearance. The lengthwise split is still apparent, but the chromosomes are extremely ragged and irregular in outline. Thirteen rods are present. At about this stage in Abraxas, Doncaster ('12) found the egg cells differentiated. In P. cynthia, as will be shown, the period is somewhat earlier.

Stage h (figs. 37 d and 51). In figure 51 almost all of the rods have shortened to bipartite chromosomes, the halves being rather widely separated from each other. Frequently a constriction in each half gives a tetrad form to the chromosome. In this figure, two are still rod-like, as in the preceding stage. The large plasmosome is frequently vacuolated. In figure 52, a later condition, the chromosomes are more irregular and stain less deeply. A few of the tetrads are broken into four separate flocculent pieces; others into irregular fragments. Some chromosomes remain bipartite as before. Within the cytoplasm a dark rounded mass indicates the end of the strand or tube which connects this cell with others.

With further growth of the cell, the nucleus increases in size, and the chromatin fragments multiply considerably. Giardina ('01), Debaisieux ('09) and Gunthert ('10) have figured in the Dytiscidae a markedly regular division of tetrads, each part giving rise to a whole tetrad, this process being repeated several times. In P. cynthia there is no evidence of any order in the fragmentation, for there is the greatest irregularity in the size and shape of the pieces.

The period of fragmentation marks the first broad phase in the history of the nurse cells. It is interesting to note that the cells have passed through a cycle of changes as if for maturation divisions, since they show the reduced number of chromosomes. These are destined, however, only for disintegration.

Stages j to I. At the beginning of this phase, the eggs and nurse cells are practically similar in size (fig. 37 e). The egg cell increases steadily in size during the growth period, the nurse cells, although increasing for a time, do not keep pace with the growth of the egg, and become relatively smaller as development proceeds.

Stage j (figs. 37 e and 53): At the close of fragmentation, numerous small dark granules lie within the nucleus near the periphery, together with a variable number of larger round bodies, which, although stained very deeply in some sections, are very pale in others, and appear to be of the nature of plasmosomes. The cell contents appear shghtly granular, or reticular, with a very darkly granular, flask-shaped area extending from the nuclei toward one end of the cell, appearing to perforate the cell wall in the form of a curved tube which enters the egg cell. Marshall ('07), Giinthert ('10) and others describe similar areas, but the tubular portion is not apparent. A later stage is shown in figure 54. The nuclear wall is less easily seen on the side nearest the tubes, for the granules are thickest at this point, and lie close to the dark granular cytoplasm. In addition to this mass of granules, the nucleus contains much smaller masses scattered near the periphery, and several small plasmosomes. A thin dark band of cytoplasm often encircles the nucleus, merging with the flask-shaped portion. Beyond this the cytoplasm appears reticular. Figure 55 A shows another cell in which the nuclear cavity is indented in two regions, giving a somewhat dumb-bell shape. During these changes in the shape of the nucleus, the plasmosomes are extremely variable. Figure 55 B is a plasmosome from a similar nucleus, very irregular in form and encrusted with chromatin granules. In some cases the granules adhere in such numbers as practically to obscure the plasmosome.

Figure 56 is typical of older nurse cells. The nuclear wall appears to be thrown into a number of folds, beset with chromatin granules, which frequently obscure the wall. The nuclear cavity contains as before, clumps of granules and plasmosomes. The cytoplasm immediately surrounding the nucleus has become much broader, forming a conspicuous dark ring which merges into the flask-shaped region. The reticular portion of the cytoplasm is smaller in extent. Frequently at this stage or later, there appear very pale delicate cytoplasmic lines in the flaskshaped region, converging down into the tubular portion, probably indicating a transfer of material into the egg cell. In figure 56 two nurse tubes are shown, the egg cell into which they open not being indicated. The tubes appear longer than in the early stages, and are irregularly constricted in places, often apparently forming a series of rings lying upon each other. There appears to be a thin homogeneous membrane forming a distinct wall to the tube; this is not a continuation of the cell wall, but is formed at the edge of the flask-shaped cytoplasm, and passes through the cell wall (fig. 54). In Eacles imperialis and Telea polyphemus a similar condition was observed, although the tubes here are not so piominent.

In figure 57 a later stage of the nurse cells is shown, drawn to the same scale as figure 56. Here the nurse cells are considerably larger than before, yet smaller than the egg. The flaskshaped region, circular granular region and nuclear cavity appear as previously indicated. The plasmosomes are covered with granules, and single strands of more prominent granules partly line and extend, down into the circular region. Only two nurse cells are figured here. The total number for each egg is five, which can be readily determined by following through a series of transverse sections. Gross ('03) also found five in other Lepidoptera. In figure 57 and other similar sections, the follicle cells are arranged in a layer around the groups of eggs and nurse cells, first definitely formed at the periphery of the egg string, then growing in at the base of the egg cell. Later they grow in between the egg and its nurse cells, separating them except in the region of the tubes.

Stage I. At the end of the growth period, when the nurse cells have become very small, no definite tubes are seen, the cytoplasm opening broadly into the egg. Later the follicle cells form a continuous layer over the egg, and the nurse cells may be seen as small degenerated masses of cytoplasm which eventually disappear.

d. Development of egg cells

The further history of the egg cells, beginning with their differentiation from the nurse cells in Stage f is given below.

Stage f: Postsynaptic spireme. Among the nurse cells of this stage (figs. 44-46) a few cells may be observed in every section, in which the spireme appears to be thicker than in the surrounding cells, more continuous and more closely convoluted^ as in figure 58. The nucleus is somewhat larger, and is very frequently distinguished by a pale yellowish tinge. Characteristic of this stage is the large plasmosome, frequently surrounded by a darker rim. The cytoplasm is also slightly greater in amount. A large number of sections were examined, and these differences appeared fairly constant. Careful study of cells in the earlier contracted spireme failed to reveal any criterion by which the egg cells might be identified at this time.

After the nurse cells are well differentiated, the spireme of the egg cell appears much less convoluted (fig. 59 A), spreading out through the nuclear cavity, which has increased considerably in size. In figure 59 B is shown a portion of the spireme which was not included in the first section. It is not possible to determine accurately if the spireme is continuous, but it is my belief that this is the case. During this period, one or two large plasmosomes may appear, and frequently two smaller bodies, probably of the same nature. The entire nucleus has the yellow tinge noted in the earliest stage of its differentiation.


The cells next to be described are taken from sections of ovaries fixed in January, later than the preceding sections. Figure 57, already referred to, shows a portion of an egg string just beyond its point of emergence from the ovary proper. Figure 60 is a nucleus from a similar egg. The spireme is typical for the eggs at this period; it is still convoluted as before, with no trace of a longitudinal split. The plasmosome varies considerably in form, consisting usually of a dark spherical portion, and a light portion, sometimes lobed and vacuolated. In this figure the plasmosome gives the appearance of breaking through the nuclear membrane, and in another egg near by a similar body was observed lying in the reticular cytoplasm at a little distance from the nucleus. A few other cases were observed on the same slide. The material appeared to be unusually well fixed, but as other ovaries failed to show a similar condition, this is probably not a normal occurrence.

It is con\'enient at this point to note more definitely the relation between the eggs and nurse cells during this period of growth. In figure 57 two nurse cells are seen connected with the egg, the flask-shaped region with its faint converging lines of protoplasm is confluent through the nurse tubes with a dark granular layer which surrounds the egg nucleus, broadening out into a conspicuous mass on the farther side of the nucleus. Here it sends long processes radiating out into the reticular portion of the cytoplasm. In most sections this finely granular region has a yellowish tinge, like the nucleus, markedly diff'erent from the reticular region of the cells. The mass frequently contains small vacuoles and deep-staining granules and is similar in appearance to the so-called yolk nuclei in various eggs; it seems probable that in P. cynthia the mass is of the same nature.

Paulcke ('00), Gross ('03), and others, have described whole nurse cells entering the egg during the growth period. This would be impossible in the moth, on account of the small diameter of the nurse tubes.

Stage g: Disappearance of the spireme in the later growth period. The next stages figured are sections from the ovary shown in figure 31, from a moth fixed a few days before the time of emergence. In a few of the youngest eggs in this material, the spireme is still vaguely discernible (fig. 61) in the form of a pale network of irregular threads joined together, not the coiled spireme of earlier eggs. Several dark bodies of irregular size and shape are characteristic of this period. In figure 62 — a slightly older nucleus in the same string — all traces of the spireme have disappeared. The nuclear cavity contains a pale body with a large vacuole, and numerous smaller rounded masses, which frequently stain veiy deeply. These are probably all plasmosomes. There is extreme variability in respect to their number, size and appearance, some being apparently homogeneous, others filled with vacuoles. Figure 63 is a nucleus of about the same age as figure 62.

As the eggs increase in size, the nuclei appear to have at one side a darker region, frequently crescentic (fig. 64), which seems to be connected with a dark granular protoplasmic strand running down into the cytoplasmic region of the egg, now coneshaped, as in the mature egg. The nucleus is party surrounded by yolk spheres, lying in faintly granular cytoplasm. The crescentic region merges gradually into the lighter granular portion of the nucleus, and suggests merely a condensation of the nucleoplasm here. Over twenty-five nuclei of this stage were examined, after varying degrees of extraction. In many cases the contents of the darker region were visible, and all showed the same condition of darker granules merging into lighter ones. No plasmosomes were to be seen, nor any trace of chromatin. The nuclei lie near the periphery of the eggs in the cytoplasmic region near the nurse cells, which at this time are reduced to shrunken remnants. Later the nuclear wall seems to fade out at the side nearest the periphery, and several bipartite rod-like chromosomes may be seen within the nucleus. At a slightly later period, the chromosomes, now shorter and more dumb-bell-shaped, appear to lie in a rounded area in which no distinct nuclear boundary is discernible. In the latest prophase (fig. 3) the nuclear boundary reappears, very faint, and very much smaller than the former nuclear area.

e. Degenerating cells in the ovary

Groups of degenerating cells are to be found in almost all of the ovaries examined, occurring chiefly in the region of the tetrads. Similar cells were also noted in the spireme region, but very rarely in the egg strings. These cells have the form of clear vesicles which contain one or more deep-staining spheres of chromatin material. A number of writers have noted this condition both in oogenesis and in spermatogenesis.

No cases of amitosis were observed in any of the germ cells. The nurse cells do not divide in any manner after the last oogonial divisions, nor do the egg cells, until the maturation divisions.

/. Abnormal nuclei in the early ovary

Certain abnormal conditions were obser\'ed in nurse cells of Stage g. In two ovaries, several cells showed, instead of 13 bipartite rods, from 15 to 19 rods. These differed further in the fact that no longitudinal split was evident. As it happened that these two ovaries were the first ones examined, the problem was very puzzling, for it appeared to indicate that the chromosomes were not of the reduced number. Normal nuclei, however, were found in the same material, and sections of about 40 other ovaries failed to show any abnormal cells. Doncaster ('12) mentions a somewhat similar abnormality, in which one cell showed the diploid, instead of the haploid number of chromatic threads.

2. CONCLUSIONS AND COMPARISONS

Differential divisions in the oogonia, which have been described for the Dytiscidae, are not found in P. c^ynthia. The germ cells all appear similar in size until the post-synaptic spireme stage, agreeing in this respect with Pieris and Abraxas (Griinberg '03, Doncaster '12), the bee (Paulcke '00), and the di^.gon-fly (McGill '06, Marshall '07). Doncaster finds a differentiation appearing a little later than in P. cynthia, when the chromatin threads shorten to form bipartite chromosomes in the nurse cells. The egg spireme is not continuous, but is composed of the haploid number of interlaced threads, which have not yet contracted. Marshall ('07) found a still later dilTerentiation in Platyphylax, a neuropteran, in which the tetrad stage is common to both kinds of cells, but the egg cell is larger, and the tetrads persist longer before disintegration.

The haploid number of chromatin segments is present in the nurse cells of P. cynthia, as in Pieris (Doncaster '12), indicating a preparation for division in these cells whose function is only nutritive. A number of writers, including Griinberg ('03), Gross ('03), Marshall ('07), and Woltereck ('98), figure tetrads in the nurse cells of various animals, but do not state whether the haploid number is present. They agree, however, that differentiation of eggs and nurse cells occurs after synapsis, which would imply that nurse cells as well as eggs must have undergone pseudo-reduction.

A transfer of material takes place from the nurse cells and the egg through the connecting tubes which in P. cynthia have very prominent walls. A markedly similar condition was observed by Giinthert ('10) in Dytiscus, where converging bundles of fibrils appear, beset with chromidia or chromatin granules which enter the egg. In this case there is no definite wall to the tubes, Griinberg ('03j states that in Pieris the egg sends a large blunt process up between the nearest nurse cells. Evidently there is considerable variation in the relation of eggs and nurse cells within the Lepidoptera, for in P. cynthia it is the nurse cells which send processes into the egg.

The history of the egg nucleus seems to show that the chromosomes lose their visible identity during the growth period. I am convinced of the accuracy of the results in this particular, on account of the very careful study given to this stage. More than half the nuclei from one individual were examined, and only in the very earliest eggs were traces of spiremes to be found. I examined also egg strings of other moths. In Clisiocampa the spireme persists relatively longer, being found in large eggs. As in P. cynthia, it becomes gradually fainter and more broken the older the eggs become, and finally disappears altogether.


Throughout the growth period the nucleus contains many small non-chromatic bodies, in a pale flocculent nucleoplasm, but no trace of a spireme. Sections of Rothschildia jorulla eggs and Actias luna showed an essentially similar condition.

The literature dealing with the condition of the chromosomes during the growth period contains a number of diverse results. In several groups of vertebrates and invertebrates the persistence of the chromosomes has been demonstrated by Grifhn ('99), Stevens ('04), Dublin ('05), Marshall f'07, '10), Ruckert ('02), Born ('94), Schockaert ('02), Winiwarter and Saintmont ('08), King ('08), and others. Deton ('09) found only a pale reticulum in the egg of Thysanozoon; nevertheless he believes, with Gregoire ('09) that, whatever the appearance, the chromosomes persist autonomously up to the maturation divisions. On the other hand, evidence that the chromosomes disappear as such during the growth period, is given by the work of Carnoy and Le Brun ('99), Hacker ('95), Woltereck ('98), Bonnevie ('06), Popoff ('07), Goldschmidt ('08), and Schleip ('09). The latter found an interesting condition in Cypris; in one form the chromosomes may be traced throughout the growth period, in another they disappear. With the second group Philosamia cynthia is to be included, as the facts observed inchcate a gradual disappearance of the spireme during the growth of the egg.

3. LITERATURE ON THE EARLY DEVELOPMENT OF EGGS AND

NURSE CELLS

In this list are included only a few of the papers dealing with the various early stages in the growth of the eggs and nurse cells in insects.

Lepidoptera. Doncaster ('12) describes the early oogenesis in Pieris brassicae and Abraxas grossulariata. In Pieris, after the oogonial divisions, when 30 chromosomes are seen in the equatorial plate, the nucleus enlarges and forms a reticulum, followed suddenly by the synizesis stage, in which a chromatin nucleolus appears. In the ensuing stage, a broken spireme of 14 separate threads is seen, the fifteenth element being represented by the double chromatin nucleolus, which is interpreted as an equally paired heterochromosome. When the threads shorten to chromosomes, this is indistinguishable from the others. In Abraxas these stages are similar, A distinction is noted here between eggs and nurse cells. In the former "the bivalent threads persist to the latest stage observed — possibly till the prophase of the polar divisions;" in the latter, the bivalent threads shorten into loops to form chromosomes. In Bombyx and Pieris, as described by Griinberg ('03), the germ cells are at first all alike, with nucleolus and granules in the nucleus. The next zone in the ovary shows spiremes, in which stage synapsis occurs. Cell boundaries are not figured here. This is followed by a differentiation zone, in which the egg nucleus is distinguished by a nucleolus and threads, the nurse cells by tetrads. Details of their origin are not given. Fragmentation of the tetrads is described, and the arrangement of nurse cells near the egg, followed by a transfer of granular material to the egg cell.

Neuroptera. The observations of Marshall ('07) on the ovary of Platyphylax are meagre as regards chromatin changes. From synapsis, beaded threads appear, showing a lengthwise split. These threads give rise to tetrads, which fragment. In cells destined to form eggs, the tetrads disappear later and the nuclei are slightly larger. The further history of the eggs and nurse cells is not given.

Hy7nenoptera. In the early ovary of the bee, Paulcke ('00) observed that the eggs and nurse cells are at first similar. Beyond the synapsis stage, the nurse cells differentiate, the chromatin fragmenting and the nucleus increasing in size. Later the cytoplasm of the egg may be seen projecting into the region of the nurse cells, which are very numerous. He believed that nurse cells entire might be taken into the egg, and that amitosis occurred.

Hemiptera. In a study of the early ovary of Protenor, Foot and Strobell ('11) distinguish three zones in the ovary: Zone A consists of nuclei with numerous granules; zone B of larger nuclei with granules and a nucleolus, arising by growth from A;

zone C contains very small nuclei similar to A, arising chiefly from the cells of zone B by amitosis, and giving rise to the ova. They believe amitosis plays an important role. No cell bomidaries appear in these zones. In young ova, leptotene threads -are seen, followed by a stage of broken spireme threads. These gradually disappear, and reappear later to form chromosomes. The figures given are chiefly photographs and do not adequately illustrate the points mentioned in their paper. The importance of amitosis has been questioned b}^ Gross ('01), who concluded from his studies on Hemiptera that nuclei which divide amitotically never divide again by mitosis.

Payne ('12) also discusses the origin of the ova in Gelastocoris, another hemipteran. He finds the same three kinds of cells, although their arrangement in zones is very indefinite. He is convinced that the eggs are not derived from the small nuclei of zone C, but from certain larger cells of zone B, which he finds in synapsis, and that there is no break in the continuity of the cells from oogonia to ova. This appears to be a more reasonable interpretation of the development of the eggs, and accords more closely with the conditions observed in other insects.

Coleoptera. Debaisieux ('09) has described the early oogenesis in Dytiscus marginalis, amplifying Giardina's work on the same form. Debaisieux discovered a synaptic and a diplotene stage between the zone of differentiation of eggs and nurse cells and the growth zone. In the latter, the chromatin of the nurse cells gives rise to tetrads which fragment, the chromatin of the egg cell remaining in the diplotene stage as before. His main conclusions are, (1) that the 'chromatic mass', which Giardina believed to be derived from certain chromosomes is not true chromatin, but a condensation of the reticulum left in the nucleus after the chromosomes of the last oogonial division are formed; (2) that the chromosomes persist autonomously up to the maturation divisions. In Giinthert's paper ('10) the chief point of interest is the description of difi'erential mitosis in the oogonia. When a cell divides, a 'chromatic mass' and the spherical remains of the spindle pass into one cell undivided. This becomes the egg cell, which again divides differentially as before. At the end of the fourth differential mitosis, there are 15 nurse cells and one egg cell, which enters the resting stage. The spindle remains of the nurse cells join that of the egg, forming a protoplasmic bridge between them. Gtinthert believes that differential divisions occur in many animals; and that the 'accessory body' described by Buchner ('09) in Gryllus is merely a 'chromatic mass' indicating a differential mitosis. The origin of nurse cells from the egg by a process of budding, as described by Will, is probably to be interpreted in the same way. In the later nurse cells of Dytiscus Gtinthert finds tetrads which subdivide regularly several times, freeing thousands of granules in the nucleus. When the nuclear wall breaks down, they migrate into the cytoplasm, where they increase by division, eventualh^ entering the egg.

Orthoptera. Buchner ('09) figures a leptotene stage in Gryllus after the oogonial divisions, followed by a diplotene stage during which the 'accessory' is much vacuolated. The probable significance of this body has been referred to above. After the diplotene stage the threads shorten into rods and tetrads. No further development of the egg is given.

SUMMARY

1. In Philosamia cynthia the 13 bivalent chromosomes of the late prophase all divide in both maturation divisions.

2. The male and female pronuclei at the time of their union each contain 13 chromosomes, making the somatic number 26, which is found in the nuclei of the blastoderm.

3. On account of the great variations in the size of the chromosomes in the metaphase and anaphase plates, there is no conclusive evidence for either the presence or the absence of an X Y-pair of chromosomes.

4. In the oogonia no differential divisions occur. The germ cells all appear similar through the presynaptic and synizesis stages.

5. In the post-synaptic spireme stage, the nuclei of the future nurse cells show the haploid number of threads, indicating a preparation for division, although the chromosomes are destined only for disintegration. In the egg cell the spireme is probably continuous. A plasmosome is present in both cases.

6. During the growth period, the chromosomes of the nurse cells fragment into numerous granules. The nuclear wall becomes much infolded, and is lined with the granules.

7. A transfer of material takes place from the nurse cells to the egg, through connecting tubes derived from the spindle remains of the final oogonial divisions. The egg cell increases in size at the expense of the nurse cells.

8. Amitosis does not occur among the germ cells. Degeneration of cells is common in the region of differentiation.

9. The egg nucleus remains in the spireme stage throughout the greater part of the growth period. There is no indication of a segmentation into the haploid number of threads.

10. Shortly before emergence, a few of the youngest cells in the ovary show a faint disintegrating sj^ireme. In most of the cells no trace of chromatin is present. This indicates that the chromosomes lose their visible identity during the growth period. It is impossible to demonstrate the form which the chromatin assumes during this period of its diffusion.

11. In the oldest cells of a late ovary the chromosomes reappear in the form of 13 short rods or dumb-bell-shaped bodies characteristic of the early metaphase groups of chromosomes.

March, 1914.

LITERATURE CITED

VON Baehr, W. B. 1908 Ueber die Bildung dcr Sexualzellen bei Aphididae. Zool. Anz., Bd. 33.

1909 Die Oogenese bei einigen viviparen Aphididen imd die Si)ermatogenese von Aphis saliceti, u.s.w. Arch. f. Zellf., Bd. 3. no. 2.

Baltzer, F. 1909 Die chromosomen von Strongylocentrotus lividus und Ecdiinus microtuberculatus. Arch. f. Zellf., Bd. 2.

BoNNEviE, K. 1906 Beobachtungen an den Keimzellen von Enteroxenos cstergreni. Jenaische Zeit., Bd. 41.

Born, G. 1894 Die Struktur des Keimblaschens im Ovarialci von Triton taeniatus. Arch. f. mikr. Anat., Bd. 43.

BucHNER, P. 1909 Das accessorische Chromosom in Spermatogenese und Ovogenese der Orthopteren, u.s.w., Arch. f. Zellf., Bd. 3, no. 3.

Carnoy, J. B., AND Lebrun, H. 1899 La vesicule germinative et les globules polaires chez les batraciens. III. La Cellule, torn. 16.

Cook, M. H. 1910 Spermatogenesis in Lepidoptera. Proc. Acad. Nat. Sci., Philadelphia, April.

Davis, H. S. 1908 Spermatogenesis in Acrididae and Locustidae. Bull. Mus. Comp. Zool. Hai"vard, vol. 52, no. 2.

Debaisieux, p. 1909 Les debuts de I'ovogenese dans le Dytiscus marginalis. La Cellule, tom. 25.

Dederer, p. H. 1907 Spermatogenesis in Philosamia cynthia. Biol. Hull, vol. 13, no. 2.

Deton, W. 1909 L'etape synaptique dans le Thysanozoon brocchii. La Cellule, tom. 25.

Donc'aster, L. 1908 Sex inheritance in the moth Abraxas grossulariata. Royal Soc. Evol. Comm., rept. 4.

1912 The chromosomes in the oogenesis and spermatogenesis of Pieris brassicae, and in the oogenesis of Abraxas grossulariata. Journ. Genetics, vol. 2, no. 3.

. 1913 On an inherited tendency to produce purely female families in Abraxas grossulariata, and its relation to an abnormal cliromosome nximber. Journ. Genetics, vol. 3, no. 1.

Doncaster, L., and Raynor, G. E. 1906 Breeding experiments with Lepidoptera. Proc. Zool. Soc, London, vol. 1.

Dublin, L. 1905 The history of the germ cells in Pedicellina americana. Ann. New York Acad. Sci., vol. 16.

Foot, K., and Strobell, E. C. 1911 Amitosis in the ovary of Protenor belfragei and a study of the chromatin nucleolus. Arch. f. Zellf., Bd. 7, no. 2.

GiARDiNA, A. 1901 Origine dell' oocite e delle cellule nutrici nel Dytiscus. Internat. Monatschr. f. Anat. u. Phys., Bd. 18.

GoLDSCHMiDT, R. 1908 Uebei" das Verhalten des Chromatins bei der Eireifimg und Befruchtung des Dicrocoelium lanceatum. Arch. f. Zellf., Bd. 1.

Gregoire, V. 1909 Les phenomenes de I'etape synaptique. La Cellule, torn. 25.

Griffin, B. B. 1899 Studies on the maturation, fertilization and cleavage of Thalassema and Zirphaea. Jour. Morph., vol. 15.

Gross, J. 1901 Untersuchungen liber das Ovarium der Hemipteren, zugleich ein Beitrag zur Amitosenfrage. Zeit. f. wiss. Zool., Bd. 69.

1903 Untersuchungen fiber die Histologic des Insectenovariums, Zool. Jahrb. Bd. 18.

Grunbero, K. 1903 Keim- und Nahrzellen in den Hoden und Ovarien der Lepidopteren. Zeit. f. wiss. Zool., Bd. 74.

GtixTHERT, T. 1910 Die Eibildung der Dytisciden. Zool. Jahrb., Bd. 30.

Hacker, V. 1895 Die Vorstadien der Eireifung. Arch. f. mikr. Anat., Bd. 45.

Henking, H. 1892 Untersuchungen iiber die ersten Enlwicklungsvorgange in den Eiern der Insekten, III. Zeit. f. wiss. Zool., Bd. 54.

King, H. D. 1908 The oogenesis of Bufo lentiginosus. Jour. Morph., vol. 19.

McGiLL, C. 1906 The behavior of the nucleoli during the oogenesis of the di-agon fly, with especial reference to synapsis. Zool. Jahrb., Bd. 23.

Marechal, J. 1907 L'ovogenese des selaciens et dc quelciues autres chordates. La Cellule, torn. 24.

Marechal, J., et de Saedeleer, A. 1910 Le premier developpement de I'ovocyte chcz les Raj ides. La Cellule, torn. 26.

Marshall, W. S. 1907 Cellular elements of the ovary of Platyphylax designatus Walk. Zeit. f. wiss. Zool., Bd. 86.

Morgan, T. H. 1909 A biological and cytological study of sex determination in Phylloxerans and Aphids. Jour. Lxp. Zool., vol. 7, no. 2.

Morrill, C. V. 1910 The chromosomes in the oogenesis, fertilization and cleavage of Coreid Hemiptera. Biol. Bull., vol. 19, no. 2.

Paulcke, W. 1900 Ueber die Differenzierung der Zellelemente im Ovarium der Bienen-konigin. Zool. Jahrb., Bd. 14.

Payne, F. 1912 L A further study of the chromosomes of the Reduviidae. IL The nucleolus in the young oocytes and origin of the ova in Gelastocoris. Jour. Morph., vol. 23, no. 2.

PopoFF, M. 1907 Eibildung bei Paludina vivipara und Chromidien bei Paludina und Helix. Arch. f. mikr. Anat., Bd. 60.

Punnett, R. C, and Bateson, W. 1908 The heredity of sex. Science, N. S., vol. 27, no. 698.


RucKERT, J. 1892 Zur Entwickelungsgeschichte des Ovarialeies bei Selachiorn. Anat. Anz., Bd. 7.

ScHLEip, W. 1909 Vergleichende Untersuchung der Eireifung, u.s.w. Arch, f. Zellforsch., Bd. 2.

ScHOCKAERT, R. 1902 L'ovogcuesc chez le Thysanozoon brocchi, II. La Cellule, torn. 20.

Seller, J. 1913 Das Verhalten der Geschlechtschromosomen bei Lepidopteren. Zool. Anz., Bd. 41, no. 6.

Stevens, N. M. 1904 Further studies on the ovogenesis of Sagitta. Zool. Jahrb., Bd. 21.

1906 a Studies on the germ-cells of Aphids. Carnegie Institution. Pub. Xo. 51.

1906 b Studies on spermatogenesis. II. A comparative study of the heterochromosomes in certain species of Coleoptera, Hemiptera, and Lepidoptera, with especial reference to sex determination. Carnegie Institution Pub. No. 36.

1909 An unpaired heterochromosome in the Aphids. Jour. I^xj). Zool., vol. 6.

Tennant, D. H. 1912 a The behavior of the chromosomes in cross-fertilized echinoid eggs. Jour. Morph., vol. 23.

1912 b Studies in cytology. I. A further study of the chromosomes of Toxopneustes variegatus. II. The behavior of the chromosomes in Arbacia-Toxopneustes crosses. Jour. Exp. Zool., vol. 12.

Wilson, E. B. 1912 Studies on chromosomes. VIII. Observations on the maturation-phenomena in certain Hemiptera and other forms, with considerations on synapsis and reduction. Jour. Exp. Zool., vol. 13, no. 3.

VON Winiwarter, H., et Saintmont, G. 1908 Nouvelles recherches sur I'ovogcnese de I'ovaire des mammiferes (chat). Arch, de Biol., tom. 24.

WoLTERECK, R. 1898 Zur Bilclung und Entwicklung des Ostracocleneies. Zeit. f. wiss. Zool., Bd. 64.


All the. figures (Philosamia cynthia) were drawn with the camera lucida. The enlargement is 2100 diameters, unless otherwise specified.

PLATE 1

EXPLANATION OF FIGURES

1 Metaphase from cell of embryo, showing 26 chromosomes.

2 Oogonial metapliase, showing 26 chromosomes.

3 Prophase of first oocyte division, showing 13 chromosomes.

4 to 7 Metaphasesof first oocyte division, side view, showing 13 chromosomes.

8 Anaphase of first oocyte division, showing cell plate and chromosomes.

9 Same; 13 chromosomes at each pole.

10 and 11 Late first anaphase, oblique polar view. The upper groups of chromosomes enter the first polar body.

12 to 14 Three sister anaphase groups of the first division; a, chromosomes of first polar body; 6 chromosomes remaining in the egg.


PLATE 2

EXPLANATION OF FIGT'RES .

15 Metaphase of second oocyte division, side view, from serial sections, showing cell plate between .4, 13 chromosomes of first polar boch', and B, 13 chromosomes in the egg.

16 Same, without cell plate.

17 Same, with cell plate; polar view.

18, 19, 22, 26 Anaphases of second oocA^te division, all incomplete except lower group in figure 19.

20 Sister anaphase groups of second division, showing a, 13 chromosomes of second polar body; h, 13 chromosomes in the egg.

21 Second anaphase group of 13 chromosomes in the egg.

23 and 24 Sister anaphase groups of second division; oblique polar view, the groups slightly displaced.

25 Telophase of second division; incomplete.


PLATE 3

EXPLANATION OF FIGURES

27 Copulation of the pronuclei, from serial sections, showing 13 chromosomes in each pronucleus B, C, and 13 in second polar body, A.

28 Polar bodies from egg of similar stage; the first one has divided, and shows shadowy chromosome outlines.

29 Ovary from a larva, longitudinal section. X 16.

30 Ovary from a pupa, fixed in January; total, X 16.

31 Same, fixed in July; total, X 16.


PLATE 4

EXPLANATION OF FIGURES

32 and 33 Anaphases of first oocyte division. X 700.

34 Metaphase of second oocyte division. X 700.

35 Copulation of pronuclei; same egg as figure 27. X 700.

36 Post-oogonial nuclei with chromatin masses. The cells are connected by protoplasmic strands or tubes.

37 Longitudinal section through portion of an egg string of a larval ovary. X 400. a. Stage a, oogonial region; b, Stage e, synizesis; c, Stage f, post-synaptic spireme; d, Stage h, dyad or tetrad chromosomes in nurse cells; e, eggs and nurse cells well differentiated.

38 and 39 Stage c; the chromatin masses are transformed into small irregular fragments which later assume a thread-like form.

40 Stage d; presynaptic leptotene.

41 and 42 Two groups of protoplasmic tubes with branches.


PLATE 5

EXPLANATION OF FIGURES

43 Stage e; synizesis.

44 to 47 Stage f ; post-sjoiaptic spireme of nurse cells. 48 and 49 Stage g; diplotene stage; 13 split rods.

50 to 52 Stage h; chromosomes begin to fragment.

53 and 54 Stage j ; young nurse cells, showing tubes entering egg cell. X 700.

55 A, nucleus of nurse cell with plasmosomes and chromatin granules; X 700. B, plasmosome enlarged.

56 Older nurse cells, with tubes. X 400.


PLATE 6

EXPLANATION OF FIGUKES

57 Portion of an egg string, showing an egg and two nurse cells. X 400.

58 and 59 Stage f; early and later post-synaptic stages of egg cell; spireme probably continuous.

60 Nucleus from an egg cell similar to the one shown in figure 57. X 700.

61 Stage g; spireme disappearing in the later-growth period; plasmosomes of varying size and form.

62 Slightly older nucleus; all traces of the spireme have disappeared.

63 Same. X 850.

64 Nucleus from a nearly mature egg, showing dark crescentic region. The nuclear cavity is filled with granules. X 400.



The Structure And Growth Of The Incisor Teeth Of The Albino Rat

William H. F. Addison And J. L. Appleton, Jr.

From the Anatomical Laboratory of the University of Pennsylvania and The Wistar Institute of Anatomy, Philadelphia

TWENTY-NINE FIGURES

CONTENTS

Introduction 43

Historical survey 44

Material and methods 46

Dentition of adult animal 46

Minute description of the incisors 52

Microscopic structure of enamel and dentine 55

Development of the incisors 59

Detailed description of development up to time of eruption 61

Eruption of the teeth 78

Changes in apex of tooth by use 81

Description of mature tooth and tooth-forming organs, in 5-month animal. . 83

Rate of growth of incisor teeth 88

Overgrowth of incisor teeth 89

Summary 91

Literature cited 95

INTRODUCTION

The incisor teeth of the Rodentia have long been regarded by the zoologist as having a high value for the understanding of many of the characteristics of this order. For instance, in 1888, Cope wrote "nearly all the peculiarities of the rodent dental system and manner of mastication are the mechanical consequences of an increase in length of the incisor teeth." Tullberg ('98-'99) gives the taxonomic position of the genus Mus, proceeding from the more general to the more specific grouping: Rodentia, Simplicidentati, Sciurognathi, Myomorphi, Myoidei, Muriformes, Myodontes, Muridae, Murini, Mus. A consideration of these terms merely from an etymological view suggests the importance of the teeth and jaws in the classification of the gnawing animals. The observations here recorded are based on the study of the celhilar processes involved in the formation, eruption and growth of the incisor teeth in a single rodent form — Mus norvegicus albinus. An additional interest was lent to the work by the fact of the increasing use of this animal for laboratory purposes, which makes it desirable to learn the time-relations of its life-processes, as a basis of comparison in various forms of experimental studies. Although the rodent incisors have been the object of much study, few observers have carried out their observations through the complete life-history, including developmental stages and adult structure, in one form of animal and this it has been our aim to do.

HISTORICAL SURVEY

Oudet ('23) proved the phenomenon of permanent growth in the incisor teeth of rodents by cutting off the teeth at the gingival margin and observing that they were regenerated. Retzius ('37) and others noted the overgrowth of these teeth in cases of malocclusion. MacGillavry ('76) observed the rate of growth of the incisors of a rabbit by making marks on the teeth and noting the gradual advance and disappearance of these marks, as the teeth grew out and were worn away.

Questions which have called forth much study and controversy are (1) does the rodent incisor belong to the milk or to the permanent dentition; and (2) which of the three incisors of the typical mammalian dental formula does it represent. Without exception, all who have studied the first question agree that the large rodent incisor belongs to the second or permanent dentition. These same studies show that abortive milk incisors occur in a varying degree in the several families of the Rodentia; and that they are slightly, if at all, represented in the Muridae. As to the second question. Cope on palaeontological evidence decided that the large rodent incisor was h. Adloff ('98) on embryological evidence confirmed this view. Freund ('92), Woodward ('94) and Stach ('10) beheved it to be I,. Weber ('04) has given a resume and extended bibliography of this work, up to the date of his writing.


The histology of the incisor was briefly described by Owen ('40-'45) and more completely studied by J. Tomes ('50). The latter found a considerable diversity of arrangement of the enamel prisms in the different families of the order, so that in many cases he was able correctly to refer a tooth to a particular family by a simple inspection of thin sections of its enamel. Von Brunn ('87) showed that at eruption the tip of the incisor of the albino rat is free from enamel, and Sachse ('94) confirmed this on Mus musculus. J. L. Williams ('96), in a comparative study of the formation of enamel, gives a number of good illustrations of the structure of the enamel and enamel-organ of the rat, prepared from microphotographs.

Ryder ('78) and Cope ('88), in harmony with their views on the "Origin of the Fittest," described the form and position of the rodent incisor as manifestations of a most efficient mechanical system; and studied the various effects on skull topography, necessitated by adaptation to this system.

The enamel organ of the albino rat was studied by von Brunn ('87) who described in some detail the differences in structure between its functional labial portion and its non-functional lingual side. He also described the early continuity of the lingual side of the enamel-organ and its later penetration by the surrounding connective tissue. Roetter ('89), studying Mus musculus, denied von Brunn's position in regard to the invasion of the lingual side of the enamel-organ by connective tissue, and Sachse ('94), also using Mus musculus, agreed with Roetter and described the continuity of the lingual portion as persisting through life.

The development of the rodent incisor has been studied especially by Roetter ('89), Sachse ('96) and Meyerheim ('98). Burckhardt ('06), in his description of the development of the persistently growing rodent incisor in O. Hertwig's Handbuch der Entwickelungslehre has followed chiefly Sachse's work upon Mus musculus. In both Weber ('04) and Hertwig ('06) are extensive bibliographies and in these may be found all references not fully given in our appended list of literature cited.

MATERIAL AND METHODS

The albino rat is a variety of Mus norvegicus, the common gray rat (Donaldson '12). This has been shown by similarity of skull measurements (Hatai '07) and of hemoglobin crystals (Reichert and Brown '10) and also by the fact that the two interbreed freely.

The material used was obtained from the rat colony of The Wistar Institute. Serial sections in paraffin or in paraffincelloidin were made of decalcified heads of fetuses taken at daily intervals from the 16th day onwards until birth, and of jaws of animals newly-born and at short intervals until one month, and of several older stages. Serial sections of fetuses younger than 16 days were examined in the collection of The Wistar Institute. Ground sections were made of the isolated teeth, and the petrifaction method of imbedding in Canada balsam was used to prepare the teeth and adjacent soft parts in situ. Also a series of prepared crania, some entire and some disarticulated, was made at selected ages, varying from birth to old age. The 'gold dust' method of Davison, as tested out for different ages at The Wistar Institute was used for the preparation of the former, and maceration in tap water for the latter. Schultze's clearing method was found useful in studying the early periods of calcification.

DENTITION OF ADULT ANIMAL

The dental formula of the albino rat is I ., C ^, P „, M ' .

There is only one set of teeth, and hence the dentition is monophyodont. The time of eruption of the various teeth extends over a period of 3^ weeks. The incisors are the first to appear, viz., at 8 to 10 days after bhth. The first and second molars erupt at about the 19th and 21st days respectively, and it is after this period that the young animals may be weaned and are able to maintain an independent existence, as far as food is concerned. The third molars are delayed until 2 weeks later and do not appear until about the 35th day.


Fig. 1 Cranium of a 5-month albino rat. X 2.

Fig. 2 Cranium of a 5-month albino rat, with the bony alveoli dissected away to show the entire length of the incisor teeth. X 2.

The incisors are permanently-growing (or rootless) teeth, while the molars have a definite limited period of development and acquire roots. A wide diastema separates the incisors from the molars as may be seen by reference to figm-e 1. The incisors are strongly curved and Owen ('40-'45) has described the lower incisor as being the smaller segment of a larger circle, and the upper incisor as the larger segment of a smaller circle. In the lower incisor of the albino rat this statement needs a slight modification. For while the curvature of the upper incisor is in one plane only, the lower incisor is a portion of a flattened spiral, possessing a curve in three planes. The upper incisor is a segment of a true circle (at 5 months about 210°) and in cases of overgrowth it has often been known to complete the cu'cle. In the case of the lower incisor, however, when we project it on the sagittal, frontal or coronal planes, it gives in each case a curve. It was the very evident curved projection seen on the sagittal plane to which Owen referred. Considering only this view, the lower incisor of a 5-month animal forms a segment of about four-fifths of a semicircle (140-145°).

TABLE 1 23 1 41 ! 10 15 , 5 ; 8 10

DAYS j DAYS I WEEKS WEEKS MONTHS MONTHS MONTHS

imii. ! m7n. : mm. \ mm. mm. mm. mm.

Xaso-occipital length 29 .7 32 .5 39 40 43 44 46 .5

Interzvgomatic 13.7 14 14.5 14.6 15. -> 15.1 15.5

Uppe/diastema 7.4 9.5 10 11.4 12 .3 12 .5 13

Upper incisor— total length 12.8 15 18.3 20.3 23.3 23.7 26.2

Upper incisor — extra-alveolar

length ! 5.1 i 5.5 j 7 8.4 8.7 9 9 3

Lower diastema ' 4.6 I 5 I 5.6 6 6.7 7 6.8

Lower incisor— totallength 18.1 21.7 25.5 26.4 29.4 29.9 31.3

Lower incisor — extra-alveolar

length 6.5: 7 I 10.5 11.4 11.6 12 12.4


Measurements of the incisors and skulls of animals of different ages, were made as shown in table 1.

The teeth were measm-ed along their convex surfaces by means of silk thread wet with water, and applied to the object to be measured. The thread was then cut with scissors at the end of the object, straightened on paper and measured to tenths of millimeters.

A consideration of table 1, shows in a definite way the peculiarities characteristic of the dentition, not only of the rat but of rodents in general. As is well known, these are the great development of the incisors, the wide diastema, and the consequent posterior position of the molar teeth as related to the rest of the skull. Cope ('88) wrote that he considered "the increase in the length of these teeth has been due to their continued use, as beUeved by Ryder." The effects of this increased elongation upon surrounding parts he described under several different headings, but reference will be made here only to one, viz., upon the shape of the glenoid cavity. "A peculiarity of the masticating apparatus is the lack of a postglenoid process, and the consequent freedom of the lower jaw to slide backward and forward in mastication. Appropriately to this motion, the condyle of the mandible is extended antero-posteriorly and the glenoid ca^dty is a longitudinal instead of a transverse groove."


Fig. 3 Thimble-shaped portion of the maxiUa bone, in which the basal end of the upper incisor is located. X 2.

The lower incisors are longer and more slender than the upper and extend far back in the mandible, beneath the lower molars, to near the sigmoid notch. The upper incisors are contained within the premaxilla and maxilla, the basal end occupying a thin-walled, thimble-shaped recess of bone (fig. 3) to be seen best in the disarticulated skull, and which is attached at only one limited region to the rest of the maxilla. In both upper and lower teeth, the intra-alveolar portions are longer than the extra-alveolar. When one compares the extra-alveolar lengths of the upper and lower teeth of the mature animal, the latter are always greater, and, as may be seen by reference to table 1, the difference in lengths becomes greater with increased age and size.

In both upper and lower incisors the bone is so contoured around their imbedded portions that their course may be easilj^ recognized. The basal end or foraminal apex of the lower incisor forms on the outer aspect of the mandible a marked rounded projection, directed upwards and backwards beneath the coronoid process, and sometimes extending sUghtly posteriorly beneath the sigmoid notch. Almost directly opposite this projection on the mesial aspect of the mandible is the inferior dental foramen. This projection marks the position of the growing end of the formative organs of the incisor in the adult. In the new-born anunal it is not present, nor at the end of the first month. By the age of 2| months it may be recognized, and thereafter it increases in prominence and constitutes a very evident feature of the bone. This region of the growing end of the tooth is protected b}'^ the zygomatic arch, and also by the overlying muscles.

The course of the upper incisor may also be readily followed in the prepared skull. Laterally it is covered with a thin rounded layer of bone. Mesially it forms an elevated, distinct ridge projecting markedly into the nasal fossa. In the adult the position of its basal or growing end is not so prominent as that of the lower incisor. As these incisor teeth are an indispensable part of the rodents' existence their importance demands protection from traumatism which might injure their growing pulp. Here in the upper incisors, this protection is afforded by a flange of the maxilla running parallel to the lateral wall of the cranium, as shown in figure 1 , as well as being encased in a separate thimble-shaped recess of bone (fig. 3), beneath, and separated by a narrow interval from, the outer layer of the maxilla. These details are in harmony with Cope's idea ('88) of the influence of the incisors in moulding the general topography of the rodent skull.

The diastema in the upper jaw is always longer than in the lower (fig. 1). By reference to table 1 it may be seen that in the mature animal the upper is nearly twice as long as the lower, but that in the younger stages the difference is not so great. The upper hair-covered lips are infolded into the diastema, dividing the oral cavity into an anterior and posterior compartment. This arrangement probably prevents the debris and splinters of gnawing from entering the main oral cavity.


The mandibular symphysis is formed of fibrous tissue and allows independent rotation of either ramus with its contained tooth. This lateral movement of the lower incisors appears to be under the control of the will of the animal. According to the observations of Jolyet and Chaker (75) this mobility has a definite purpose in mastication. They observed a rapid alter



Fig. 4 Cross-sections of the (a) upper and (b) lower incisor teeth of a 5-month albino rat, taken near the alveolar margins. These show the arrangement of the enamel and the dentine, and the difference in contour of the enamel in the upper and lower teeth. The mesial surface cf each tooth is towards the right side. X 15.

nate separation and approximation of the tips of the lower incisors in the act of attempting to bite into a match or other slender object offered to the animal. At the same time the upper incisors were held stationary.

Mention may be made here of a point of variation among the Rodentia in the relation of the angle of the lower jaw to the sheath of bone around the lower incisor. In the Myomorphi and Sciuromorphi the angle arises from the lower surface of the incisive sheath, while in Hystrix the angle arises entirely on the outer side.

Ryder (77) suggested a classification of rodents based on the shape of their incisors as seen in cross-section. In some genera the diameter of the teeth is less from side to side, than in the antero-posterior direction, while in others the reverse condition is found. The present form belongs to the former group, as is shown in figure 4. From the consideration of many rodents, Ryder deduced the general principle, that where the incisors are thicker in the antero-posterior direction, the gnawing habit is greatly developed.

MINUTE DESCRIPTION OF THE INCISORS

Enamel and dentine make up the hard tooth substance, enclosing the pulp. Owen, in his "Odontography" ('40-'45, p. 399) said that there existed a general investment of cementum over the whole tooth structure. J. Tomes ('50, p. 533) was not able to agree entirely but said that in most, if not in all, incisors of rodents cementum could be seen investing the posterior surface. In the rat, it is not apparent that there is any cementum at all. The enamel is usually colored with a pigment which is yellowish in the young but becomes orange-colored with age, and is usually more pronounced in the upper than in the lower incisors. At 13 days, there is as yet no color, but at 21 days a slight tinge of yellow is perceptible in the uppers, but none in the lowers. At 25 days the uppers are distinctly yellow, and the lowers have now acquired a slight color. At 38 days, these colors have intensified, the uppers having more pigment than the lowers; and in the mature animal the same relation continues, the uppers being orange-colored and the lowers yellow. The enamel is found principally on the labial side, and this accounts for the shape of the occlusal surface. For, the enamel being harder than the dentine, the latter is more easily worn away by the action of the opposing tooth, and the more resistant enamel remains as the cutting edge or point. The shape of the incisal end of the upper and lower teeth is different, being chisellike (scalpriform) in the upper, and more rounded and narrower in the lower. The incisal line is also usually different in the upper and lower teeth. In the former, it is often sHghtly concave from side to side, while in the latter it is convex (fig. 5). As is shown in figures 1 and 5 the occlusal surface is an elongated concave area on the lingual aspect of the teeth, and in the living animal extends practically to the gingival margin. Due to the difference in the curve of the upper and lower teeth, the occlusal surface of the lower teeth is always longer than that of the upper, and in the mature animal it is usually found to be nearly twice as long.



Fig. 5 Labial and lingual aspects of the extra-alveolar portions of the (a) upper and (b) lower incisors of a 5-month albino rat, showing the occlusal surfaces and incisal edges of the teeth, and the outline of the bony alveolar margins. X 2.


It follows that because these teeth are constantly growing, the occlusal surfaces are constantly being worn away. As we shall see, when discussing the growth of the teeth, the elongated temporo-mandibular articulation is important, in allowing the teeth to have either the position pictured in figure 1 or to have the opposite relation, with the lower teeth outside of the upper. Thus the very important factor in the animal's economy — the proper regulation of the length of the opposing incisors — is controlled by their own inter-action.


The pulp-chamber has the characteristic shape found in all permanently growing teeth, as is well seen, for instance, in the elephant's incisor. Its cross-area is greatest at the basal end of the tooth, and gradually diminishes anteriorly. The pulpchamber is found to extend in the tooth beyond the line of the gingivus, and very nearly to the occlusal surface. The shape


Fig. 6 Upper incisor of a o-month albino rat (X o) and cross-sections of it at different points (X 8), to show the relative cross-area of the dentine and of the pulp chamber at these regions. The dotted line indicates the position of the margin of the alveolus.


in cross-section of the pulp-chamber at different levels may be seen by reference to figure 6. The position of the filled-in pulpchamber is usually well marked on the occlusal surfaces as a line (fig. 5). In weathered specimens of rats' teeth from recent geological formations this last-formed part which fills in the pulpchamber at the end of the tooth, is usually found to be lacking, and is evidently not of the same hardness as the surrounding parts of the tooth.


MICROSCOPIC STRUCTURE OF ENAMEL AND DENTINE

Sections of enamel show two layers; an outer thin and an inner thicker layer, as noted by Owen ('40-'45, p. 399). The enamel rods run in different directions in the two layers as fully described by J. Tomes in 1850. In the inner layer the enamel rods appear to run in two sets, obliquely to one another, while in the outer layer the rods are all parallel. The outer layer has also been called the fibrous layer, and in its superficial part is situated the yellow or orange pigment which gives the color to the enamel.

Figures 7 and 8 show the arrangement of the enamel rods in the two la3^ers. In the inner or plexiform layer, when examined in cross section, the alternating series of enamel rods decussate, forming an angle varying between 70 and 90°. In longitudinal sections (fig. 26) these rods are slightly S-shaped, running outwards from the enamel-dentine surface at an angle of 50 to 54°, and inclining towards the anterior end of the tooth. Figure 8 is from a ground-section in which the enamel was broken during the process of preparation, and the broken edge shows distinctly the two sets of rods running at nearly right angles to each other. Under high magnification the rods are slightly notched.

In cross-sections of the outer fibrous layer, the rods are parallel and form in the mid-line of the tooth an angle of 90° with the outer surface. As one proceeds away from the mid-line of the tooth, whether mesially or laterally, the general tendency of the long axis of the rods as they pass from the dentine junction to the periphery, is to incline in the direction away from the mid-line of the tooth. The ameloblasts usually form an obtuse angle with the rods of the outer layer and seldom coincide in direction with them (fig. 7). In longitudinal sections the rods of the outer layer are not usually so distinctly seen as in cross-sections. In favorable longitudinal sections, however, they are seen to run quite obliquely, inclining towards the apex of the tooth, and forming an angle of 20 to 25° with the plane of the enamel-dentine junction. The pigment, as will be seen



Fig. 7 Portion of cross-section of lower incisor with enamel-organ, prepared by the petrifaction method, showing the decussation of the enamel-rods in the inner or plexiform layer and their jiarallel arrangement in the outer or fibrous layer. X 350.

Fig. 8 Small piece of enamel, showing the rods of the inner or plexiform layer running in two directions nearly at right angles to one another. X 350.


below, is confined to the outermost part of the fibrous layer. There appears to be no Nasmyth's membrane over the enamel, which means that there has been a complete transformation of the enamel matrix into enamel rods. The pigment extends about two-thirds of the total length of the upper tooth, and about one-half of the total length of the lower tooth, and hence it follows that the deposition of enamel is completed within the basal third of the upper and the basal half of the lower tooth. By examining cross-sections of the tooth at different regions (fig. 6) it would seem that the full thickness of the enamel is attained within even a smaller area at the basal end of the tooth.

The arrangement of the enamel over the labial aspect of the upper and lower teeth is shown in figure 4, drawn from crosssections of the teeth of a 5-month animal. In both teeth the sections were made just posterior to the alveolar border. In both upper and lower teeth the enamel is thickest over the labial aspect, and is continued over the adjacent mesial and lateral surfaces. In both, the enamel is continued farther on the lateral than on the mesial surfaces, and relatively farther on the lateral surface in the lower than in the upper tooth. In the upper tooth the enamel has a flattened external surface labially, while in the lower it has a rounded contour. In the upper there is a distinct labio-mesial and a labio-lateral angle, the enamel being somewhat thicker at the former. In the lower there is a labio-mesial angle, though less prominent than in the upper, and the labio-lateral angle is practically absent.

In a 5-month animal the thickness of the enamel and its constituent layers was measured in the mid-line of the teeth, as follows:

Upper Lower

Total thickness 100-110 lJO-150

Outer fibrous layer 30-40 20-30

Pigmented portion of outer fibrous layer 8-10-12 6-8

Inner plexiforni layer 70 120-125

It will be observed, however, in figure 4 that the enamel is not thickest in the mid-line of the upper tooth, but at the lateral and mesial angles. While the enamel of the upper tooth measures only 100 to llO^t in the mid-line, it measures 160 to 180/x at the region of these angles, and is, therefore, thicker here than the enamel of the lower tooth. The increased thickness at the angles is principally in the inner plexiform layer, the other layer being increased only slightly or not at all. The outer fibrous layer is distinctly thicker in the uppers and has a slightly wider band of pigment in it superficially. This, no doubt, is the basis of the more deeply pigmented appearance of the labial surface of the upper as compared with the lower teeth.

The dentine, unlike the enamel, grows continually thicker as one passes towards the outer end of the tooth. At the basal, growing, end it begins as an extremely thin layer. The thickness at different points is seen in figure 6. As the dentine increases in thickness, the pulp-chamber is in consequence proportionately reduced. At the distal end there is no longer any pulp-chamber and the site of its previous position has been filled in by the formation of a kind of secondary dentine. C. Tomes ('14) notes that "in some rodents the final closure of the axial tract takes place almost by a continuance of the formation of normal fine-tubed dentine, with very little secondary dentine of different structure, while in others there is a large area of dentine with vascular tracts in it." In the rat there is relatively little of this secondary dentine. It is laid down in irregular trabeculae, with the pulp tissue, including bloodvessels, at first within it. At the exposed surface, however, it forms a continuous granular mass with apparently no soft tissues in it (fig. 27). The ordinary dentine of the tooth is quite typical in structure, with numerous parallel dentinal tubules, each having many fine lateral branches. The tubules are slightly sinuous, and the lateral branches anastomose with those of neighboring tubules. Sometimes a tubule sends off at an acute angle a branch nearly equal in diameter to the continuation of the main tubule. This is usually in the dentine not covered by enamel. Where these large branches come off the diameter of the tubule is greater than elsewhere, measuring nearly 2^. Elsewhere the diameter varies from 1 to l.T/z. Slight differences may be seen between the tubules (a) in the dentine covered by enamel, and (b) in the dentine free from enamel. The tubules of the anterior region (a) of the dentine, covered by enamel, are more regularly parallel and have finer lateral branches than elsewhere. They also seem to taper slightly as one follows them towards the enamel. In the dentine not covered by enamel (b) the tubules are more sinuous and irregular, the irregularities marking the position of origin of the larger lateral branches. In all parts at the periphery of the dentine the tubules end in a great number of very fine anastomosing arching branches. As a consequence of the smaller diameter of the little tubules here, a narrow zone at the periphery of the dentine has usually a more homogeneous appearance than has the remainder. Towards the anterior end of the tooth, in the vicinity of the pulp-chamber, are vascular channels in the form of loops within the dentine. The tubules must necessarily take a curved course around these vascular channels, and thus the position of the vessels is more easily seen.

In the dentinal tubules Mummery ('12), Fritsch ('14) and others have demonstrated not only the processes of the odontoblasts, but also fine non-medullated nerve fibers. As to why the exposed dentine on the lingual aspect of the teeth is insensitive, there are no definite observations to decide. A contributing factor may be the compression which the pulp tissues undergo at the anterior end of the pulp-chamber, leading to the physiological cutting off of the nerve supply to the dentinal tubules.

DEVELOPMENT OF THE INCISORS

The times of the early stages of development of the incisors were seen as follows:

14-day fetus — slight thickening of oral epithelium

15-day fetus — distinct thickening and growth inwards of oral epithelium

16-day fetus — dental ledge and beginning of flask-shaped enamel organ

17-day fetus — dental papilla with crescentic enamel organ capping it

19-day fetus — both ameloblasts and odontoblasts differentiated

new-born animal — enamel and dentine formation begun

8 to 10 days — eruption of the tooth

Throughout life growth continues, and in the adult animal is on the average 2.2 mm. per week in the upper and 2.8 mm. per week in the lower incisor.


The structures to be described here, as in the development of the crowns of all teeth, are the enamel-organ with the ameloblasts, and the dental papilla (which becomes the pulp-substance) with the odontoblasts. There are two factors, however, which alter the usual history of the development of these structures, and especially of the enamel-organ. First, in permanently growing teeth of which these are examples, all these structures continue functional throughout life, so that the enamelorgan is also a persistent structure. The other factor and one correlated to some extent with the first, is that the enamel is formed on one side of the tooth only, and here only does the enamel-organ develop to its most highly differentiated functional condition.

The history of the development and growth of the tooth may be conveniently considered in two stages (1) pre-eruptive, and (2) post-eruptive. The pre-eruptive stage extends from the 14th or 15th day of fetal life until eruption of the tooth takes place between the 8th and 10th post-natal days. Until near the time of birth there is no formation of enamel and dentine, but from birth onwards these substances are laid down rapidly, so that at eruption, the teeth have their characteristic elongated narrow form. This pre-eruptive stage is characterized by the rapid elongation of the tooth-forming organs, and by the teeth attaining very similar relations to the other structures of the jaw which the imbedded portions of the erupted teeth possess. Thus, the anlage of the lower incisor appears under the oral epithelium in the anterior region of the mandible, and grows continually backwards, until its growing end reaches the region beneath the developing molars. At this time the growing end presumably reaches a region which, by reason of its increasing calcification, offers resistance to further progress. The result of the ever-continuing mitotic division and cell growth at the basal end, is the pushing of the whole tooth and its formative organs, in the opposite direction, and the consequent eruption of the tooth. During the latter half of this pre-eruptive stage, the anterior tip of the developing tooth structure is immediately beneath the oral epithelium, and remains at a fixed point, while the posterior end is continually growing backwards and changing its relations. At eruption this condition changes, and the posterior extremity becomes practically a fixed point from which the whole tooth moves forward. That there is, however, a gradual change in the position of the posterior end of the tooth may be seen in figure 9. As the jaw grows, the entire tooth not only grows to keep the same general relative position, to surrounding structures, but it may be seen that the growing end progresses gradually posteriorly. In the full-grown animal this end occupies a distinct outpushing of the bone (fig. 1).

During the post-eruptive period, which continues throughout life, this outward growth is continued at a regular rate, and at the same rate the outer end has to be worn away. This wearing-away process would soon result in the pulp becoming exposed were not the occlusal end of the pulp-chamber also being continually filled in. As may be seen from figure 6 the dentine continues to increase in thickness until near the end of the tooth. This means that the odontoblasts continue their regular functional activity until near the end of the tooth. However, the final fiUing-in of the pulp-chamber to form a continuous hard occlusal surface is accomplished by the deposition of a hard matrix between the pulp elements and by the probable calcification of the latter. The result is, that as the tooth is worn away, the soft pulp never becomes exposed. Although the pulp reaches very near to the end of the tooth, a hard substance always fills in the end of the pulp-chamber, and so protects the pulp beneath.

DETAILED DESCRIPTION OF DEVELOPMENT UP TO THE TIME OF ERUPTION

The anlage of the enamel-organ of each incisor arises as an epithelial ingrowth, distinct and separate from that for the molars.

In frontal sections of the 14-day fetus, there are slight diffuse thickenings of the oral epithelium in the four positions, which represent the sites of the future tooth-formations.


Fig. 9 Series of mandibles of the albino rat at ages \arying from birth to ten months, viewed from the lateral aspect. These show the changing relation of the basal end of the incisor to the rest of the mandible during this period.


At 15 days these thickenings have become more definite, and in the lower jaws especially have begun to push into the underlying mesenchyme, and may be described as the dental ledges or dental laminae.

At 16 days the ingrowths have continued to increase as broad masses of cells, pushing deeper into the underlying mesenchyme, and in the lower jaws the enamel organs may be distinguished as expanded structures, each connected by a slightly narrower mass of cells with the oral epithelium. In the upper jaws the differentiation of the enamel-organs from the remainder of the epithelial ingrowth is not so marked.

At 17 daj^s (fig. 10) the dental papillae are beginning, and the enamel-organs in both upper and lower jaws have a crescentic outline. In the enamel-organs there is already an indication of the differentiation into three layers. As seen in sagittal sections, the papillae develop on the posterior side of the enamelorgans, thus foreshadowing the axis of growth of the toothforming organs in the antero-posterior direction.

Eighteen-day fetus

Series of frontal sections of 18-day lower jaws, show that the enamel-organs are growing over the dental papillae more rapidly on the labial and lingual surfaces than elsewhere, and extend more posteriorly on these surfaces. There are thus two projections of the posterior margin of each enamel-organ as already noted by Meyerheim ('98). The labial process is broad and thin and extends more posteriorly than the lingual process, which is somewhat narrower and thicker. One may her-e remark, therefore, an early difference between the labial and lingual part of the enamel-organ. Other differences which will soon appear have not yet developed. Thus, the inner layer of the enamel-organ is made up of columnar elements which are still similar in all parts, both labially and lingually. In the dental papilla no columnar odontoblasts are yet seen.

The enamel-organ remains connected with the surface epithelium by a broad band of epithelial cells. In the lower jaw, immediately laterad to the line of j miction of this stalk of the enamel-organ to the surface epitheliimi, the lip furrow is a depression, the plane of which is continued into the underlying mesenchyme by an ingrowth of surface epithelium several layers of cells in thickness. It is by the subsequent splitting of this epithelial layer into two, that the separation of the lip will be effected.

Nineteen-day fetus

At 19 days, the enamel-organ in the upper jaw (fig. 11) is crescentic in outline in sagittal section, and in the lower jaw (fig. 12) is more elongated and conical in shape. At this age odontoblasts are first seen as columnar cells on the labial aspect of the mesodermal papilla. Three layers are recognizable in the enamel-organ, but the middle layer (enamel pulp), as has been also described by Sachse ('94) for the mouse, is extremely thin, and therefore is not present in the great quantity tj^pically seen in the development of rooted teeth. It appears as a more lightly stained zone between the inner and outer layers, and is thickest at the basal end of the enamel-organ as shown in figures 11 and 12. It averages about 20^i in thickness and is made up of stellate cells loosely arranged. Already there is an indication of a compact arrangement of the two or three rows of cells next the inner layer of the enamel-organ, which will result in the so-called stratum intermedium seen at later ages. This middle layer is also slightly more abundant at the anterior end in the region where the enamel-organ is continuous with the stalk which joins it to the oral epithelium.

At this age the enamel-organs in the lower jaws have a greater total length than those in the upper, and especially in the lower

Fig. 10 Longitudinal section of upper jaw of 17-day fetus, showing tooth anlage of incisor, with the enamel-organ longer labially than lingually when measured from the point of junction of the stalk of the enamel-organ. The dental papilla is on the posterior aspect of the enamel-organ. X 70.

Fig. 11 Longitudinal section of upper incisor anlage of 19-day fetus of albino rat, showing the crescentic outline of the enamel-organ, its greater length labially than lingually, and its thickened basal margin. X 70.

Fig. 12 Longitudinal section of lower incisor anlage of 19-day fetus of albino rat, showing the conical outline of the enamel-organ and its greater length than in the upper jaw^ at the same age. X 70.


jaws distinct differences may be made out between the oral and labial sides of the tooth-forming organs. These differences are:

(1) The enamel-organ is longer labially than on the oral side.

(2) The staining of the inner layer of the enamel-organ on the labial side is more intense, and here the cells are slightly longer than in other parts of the enamel-organ, measuring 24/x in length and assuming the typical appearance of ameloblasts. Measurements show the similar cells on the lingual side to be about 20m in length. It is also to be noted at this age that the site of the most advanced cells which are differentiating to become ameloblasts is not at the apex of the enamel-organ, as is the case in the development of rooted teeth. For as one follows the cells of the labial side of the enamel-organ from the apex towards the base, while at the apex the cells are columnar they become longer as one goes posteriorly, and then towards the base of the enamel-organ diminish again. So that the site of most advanced differentiation here is a short distance posterior to the apex on the labial side. This is true also of the developing odontoblasts which are longest opposite the tallest ameloblasts.

(3) The outer layer of the enamel-organ on the labial side is becoming slightly wavy in outline, and this denotes the beginning of the papillae, which form such a characteristic part of the mature functional enamel-organ (fig. 26).

(4) The odontoblasts are seen only on the labial side of the dental papilla.

Mitoses are abundant in all parts of the developing tissues.

Twenty-one-day fetus

Thus the anlage of the rodent incisor begins in the usual way, and for a short time continues along the typical mammalian course. From 19 days onward, however, the differences which have already begun, become more distinct and definite. At 21 days (end of gestation) the enamel-organ has become more definitely differentiated into a labial and a lingual region. Of the three constituent layers, the inner especially is strikingly different in these two parts. On the labial side at the anterior end, the organ has advanced to the condition where functional activity is beginning, while the oral side has remained stationary, or has actually retrogressed. Thus in the innermost layer on the labial side of the lower incisor, where the ameloblasts have begun to form enamel, these cells measure 30 to 34ju in length, while the non-functional cells on the oral side of the innermost layer are low columnar or cubical in shape and measure only 12 /j. in length (fig. 13). Comparison of these measurements with those at 19 days shows that the cells of the inner layer of the labial side of the enamel-organ have advanced in length from 24/i to 30 : or 34m, while the cells on the lingual side have decreased from 20 to 12ju. There is, therefore, a primary tendency for the cells of the inner layer to develop equally in all parts, but very soon the nonenamel-forming cells of the lingual side begin to retrogress, while the functional cells of the labial side continue to grow. This constitutes another point of contrast with the development of the crowns of rooted teeth. For here in the 21 -day fetus, when the enamel and dentine formation has just begun, these substances are thickest, not over the apex of the tooth-forming organs, as in the usual method, but at a short distance posterior to this point, on the labial surface. Thus, not only are the odontoblasts and the ameloblasts first differentiated on the labial side, posterior to the apex, but at this region enamel and dentine formation is also evidently first begun.

Over the apex of the dental papilla there is apparently a very thin outline of dentine deposited, but within this, in the tissues of the apex of the dental papilla, there is also beginning an irregular formation of a hard matrix. Between the cells of the pulp, trabeculae of a bone-like material are appearing. As development proceeds this substance increases until the final result is, as seen in figure 20, that the primary apex of the tooth has a bone-like structure, consisting of cells imbedded in lacunae within a dense matrix. This has been called by Tomes ('04) 'osteo-dentine.'

A similar difference between the labial and oral sides is noted in the cells on the margin of the dental papilla, which are becoming odontoblasts. In the basal half of the papilla (fig. 14), odontoblasts occur only on the labial side opposite the tall ameloblasts, the peripheral cells of the other sides being still irregular or cuboidal in shape. Farther forwards the odontoblasts are found also on the lateral and mesial surfaces of the dental papilla, but not on the lingual. In the apical one-fourth of the dental papilla odontoblasts occur all round, measuring 20 to 24/x in length, and are engaged in the formation of dentine (fig. 13). The dentine is thickest on the labial side.

In the region where enamel and dentine formation has begun no mitoses were seen in the formative ameloblastic and odontoblastic cells, but posteriorly, where the deposition of enamel and dentine has not yet commenced, many mitoses occur in the layers of developing ameloblasts and odontoblasts, as well as elsewhere. The nearer one approaches the basal margin of the enamel-organ the more numerous are the mitoses and it is apparent that it is principally in this region that growth by addition of new cells is taking place.

One day old

At the end of the first day of post-natal life, there has been great progress in the enamel and dentine formation, and the narrow, pointed outline of the tooth has been already laid down. In the upper jaw the teeth measure about 2.3 mm. in length and in the lower jaw about 3 mm. Definite changes in its relation to the oral epithelium have occurred also at the anterior end of the tooth. The original epithelial stalk connecting the enamelorgan with the oral epithelium has increased in size and the end

Fig. 13 Cross-section of developing lower incisor of 21-day fetus of albino rat, nearer the anterior extremity of the tooth than figure 14. Shows the greater thickness of the labial side of the enamel organ, as compared with that of the other sides, and shows odontoblasts around the entire periphery of the pulp. Enamel and dentine formation has begun. X 110.

Fig. 14 Cross-section of developing lower incisor of 21-day fetus of albino rat, posterior to the region shown in figure 13. No enamel or dentine yet formed at this point. Odontoblasts highest on the labial aspect, decreasing in height laterally but not yet differentiated as columnar elements on the lingual side. Enamel-organ thickest on the labial side. X 110.


of the tooth has apparently advanced somewhat into it. So this thick stratified layer of epithelium forms a close-fitting investment about the tooth apex, and is continuous posteriorly with the remainder of the enamel-organ. But in this epithelial cap there are no ameloblasts and consequently there can be no enamel over the osteodentine which forms the tip of the primitive tooth. This substance forming the tip of the unerupted tooth is a form of secondary dentine with its cells located in the lacunae of the matrix. Passing backwards, one comes to the ordinary dentine containing the vascular pulp with odontoblasts situated at the periphery of the pulp-chamber in a regular manner.

As the odontoblasts were first differentiated labially, and dentine formation began there before on the other side, the dentine of the labial side is thicker than on the lingual side. Thus at a point about the middle of the entire tooth structure, the dentine measured 54/^ labially and 20/x orally (fig. 15). Between the odontoblasts are numerous fine capillary loops. At this region may also be seen the characteristic structure of the enamel-organ (fig. 15). This extends all around the tooth, but is much thicker on the labial side than elsewhere. This difference in thickness is seen in all the constituent layers. In the inner layer, the tall ameloblasts of the labial surface measure iOfj., while the similarly situated cells on the other surfaces are cubical and measure only 10^. Comparing these with the previous stage described, it is seen that the cells on the labial surface have increased and those on the other surfaces have decreased. Of the middle layer on the labial side, the stratum intermedium is a distinct line of cuboidal cells, one to two rows in thickness, lying behind the ameloblasts. The other constituent — the original enamel pulp — is small in amount and is principally within the elevations of the outer layer, which form the beginning of the epithelial papillae. The cells of the outer layer, somewhat irregular in shape with round nuclei, are in a single row. Between the developing papillae (called by Sachse StLitzpapillen) are numerous capillary blood-vessels. On the other surfaces, practically nothing remains of the middle layer, although the outer layer still persists as a layer of flattened cells. Thus lingually the enamel-organ is represented by only two rows of cells — one representing the inner, the other the outer layer of the enamel-organ.



Fig. 15 Cross-section of developing lower incisor of 1-day albino rat, showing the great development of the ameloblasts on the labial side, and the thinness of the enamel-organ elsewhere. The space between the ameloblasts and the dentine is an artefect, and was formerly partly filled by the enamel, which has disappeared in the pi'ocess of decalcification. In the layer of odontoblasts are seen the nuclei of the endothelial cells of the walls of capillaries. X 110.


Two days old

Figure 16 shows a longitudinal section of a 2-day upper incisor. The epithelial enamel-organ is continuous over the whole tooth, but only shows its specialized functioning structure on the labial side. On the lingual side it is still intact and consists only of two rows of cuboidal or flattened epithelial cells. On the labial side, along the region where enamel has been formed (fig. 17) the


Fig. 16 Longitudinal section of upper incisor of 2-day albino rat showing the enamel-organ continuous over the labial surface and terminating posteriorly in the thickened margin. X 18.

Fig. 17 Small portion of preceding figure more highly magnified, to show the structure of the enamel-organ and the odontoblasts, a, outermost layer of enamel-organ and epithelial papillae; b, enamel pulp; c, stratum intermedium; d, layer of ameloblasts; e, layer of dentine; f, layer of odontoblasts. X 175.


ameloblasts measure about 40^. These are backed by two rows of darkly staining flattened cells composing the stratum intermedium. Next to these is the looser arrangement of stellate cells, comparable to the enamel pulp of ordinary tooth development, but with much smaller spaces between the cells. This tissue is covered by the layer of cells constituting the outer layer of the enamel-organ, and the two together constitute the epithelial papillae. At the summit of each of these papillae the cells of the outer layer are grouped in a more compact manner. With higher magnification processes can be seen running from the ameloblasts into the developing enamel — the so-called enamel processes of Tomes.

At the basal formative part of the enamel-organ the three original layers show distinctly. At the thickened basal margin of the enamel-organ, around its entire circumference, is a mass of rapidly dividing cells. As seen in figure 16 this thickened margin is more noticeable on the labial side. Its peripheral zone as seen in longitudinal sections is deeply staining and its cells, more or less columnar in shape, are compacted together. The interior, of more lightly stained appearance, is composed of oval or elongated cells, irregularly parallel, but more loosely arranged than the cells of the periphery. This region constitutes the site of origin of the cells of the ever-forming enamelorgan. From this pass forward the outer and inner layers, and between them, in larger quantity than is found more anteriorly, the tissue of the middle layer. This for a short distance is all enamel pulp and shows no differentiated layer of stratum intermedium.

In this formative region on the labial side, the inner layer consists of columnar cells, the future ameloblasts, in which many mitoses are seen. While the outer layer consists of cells which are columnar near the margin, a short distance anterior to this (150/x) they change shape, first to cubical, then to flattened cuboidal. Between the two layers are cells representing the enamel pulp. At this region there are no papillae, although numerous blood-vessels are seen alongside the outer layer of the enamel-organ. About 0.5 nmi. from the basal end this outer layer of the enamel-organ becomes sinuous, and low papillae are being formed.

On the lingual side, the structure of the basal end of the enamelorgan is similar, but somewhat simpler. Thus there are three layers at and near the basal margin, but soon, proceeding anteriorly, these become reduced to two by the disappearance of the middle layer. The lingual side then consists of two rows of cuboidal or flattened cells, one constituting the outer and the other the inner layer of the enamel-organ in this situation.

The dental papilla is made up of closely packed small stellate cells, with rounded nuclei. The mesenchymal cells which lie against the basal margin of the enamel-organ are rounded or irregular in shape, but within a short distance (0.5 mm.) anterior to this margin, the peripheral cells become first cubical and then columnar in shape. Where they are beginning to form dentine they measure 30m in length. From the odontoblasts processes enter the dentinal tubules of the dentine. The outer surfaces of the odontoblasts from which these processes arise show a distinct cuticular margin. Between the odontoblasts at short intervals capillaries form loops around the cells. These are evidently for the purpose of insuring an ample blood supply to these functionally active cells.

Four days old

By 4 days of age there has been continued growth, and deposition of enamel and dentine. The upper incisor measures 3.6 mm. in length and the lower 5 mm. The position of the apex of the tooth is in close relation to the oral epithelium (fig. 18). A thickened mass of epithelium, partly a derivative of the original stalk of the enamel-organ, and partly an ingrowth from the

Fig. 18 Longitudinal section of upper incisor of 4-day albino rat, showing the increased curvature of the outline of the tooth and the relation of the apex of the tooth to the ingrowth of the oral epithelium. X 16.

Fig. 19 Longitudinal section through basal end of labial side of enamelorgan of 4-day albino rat showing the region of the thickened margin, a, margin composed of mass of proliferating cells; b, region where three layers are seen; c, region where stratum intermedium becomes differentiated from rest of middle layer. Anterior to the region of this figure the epithelial papillae appear and the ameloblasts begin to form enamel. X 80.


surface epithelium surrounding the tip of the tooth, is a preparation for the eruption of the tooth, and will serve as a resistant ling of tissue through which the tooth will be pushed at eruption. It may be looked upon as a protective device, to prevent adjacent tissues from being carried out by the erupting tooth.

The typical enamel-organ seen on the labial side does not cover the apex, for the tall columnar cells give place here, first to cubical and then to flat squamous epithelial cells, which form but a part of the thick mass of stratified epithelium, constituting the epithelial sheath over the end of the dentine. The other layers of the functioning enamel-organ also lose their identity at the region where the ameloblasts cease to have their characteristic elongated form. As maintained by von Brunn ('87) and Sachse ('94), there is no enamel apparent over the dentine at the apex of the tooth.

The cells representing the enamel-organ on the lingual side can be traced forward for a short distance as a two-layered stratum. These cells are flattened, with oval nuclei. Beyond this point only a single regular row of cells is apparent, and about half way along the length of the tooth-structure, even this ceases to be definite, and apparently here the mesenchymal cells of the peridental tissues have grown between and scattered these cells. As a result of this activity of the mesenchymal cells in this region, the enamel-organ now ceases to exist as a complete conical investment of the tooth. Approaching the apex of the tooth on the lingual side, one finds the prolongation of the epithelial sheath as a thin layer of flattened cells which thickens as it passes forwards into the epithelial sheath.

The basal formative end of the enamel-organ consists of a thickened band of tissue, as shown in figure 18, and under higher magnification in figure 19. This end is thicker on the labial side than elsewhere and it curves inwards, as seen in longitudinal sections, thus considerably diminishing the diameter of the entrance to the pulp-chamber. The extremity of this mass of tissue (fig. 19, a), constitutes a common origin for the several layers of the enamel-organ and contains many dividing cells. A short distance (0.1 to 0.2 mm.) from the extremity (fig. 19, b)


the cells form three layers, inner, middle and outer. The inner and outer layers, made up of columnar elements, stain more darkly than the middle layer, and the inner is thicker than the outer. The middle layer consists of elongated cells with oval nuclei, arranged for the most part with theii* long axes parallel to the surface of the enamel-organ. Frequent mitoses are also to be seen here, especially in the inner layer.

In the region about 0.6 mm. anterior to this (fig. 19, c), where enamel formation has not yet begun, the innermost layer shows a single row of distinct tall columnar cells, the ameloblasts. The middle layer now shows two subdivisions (a) two or three layers of compacted flattened cells lying against the ameloblasts, and composing the stratum intermedium, and (b) a somewhat thicker stratum, lightly staining, of more loosely arranged cells, constituting the enamel pulp. The outermost layer is a single row of cubical cells, which form a straight continuous surface for the enamel-organ. Beyond this layer and in contact with it are numerous small blood-vessels. Passing still farther forwards, the outermost layer becomes more sinuous in outline, and blood-vessels occupy the depressions between the elevations. This arrangement shows the beginning formation of the typical epithelial papillae.

Seven days old

At 7 days the tip of the tooth is in the oral epithelium (fig. 20), and ready for eruption, being separated from the outside by only a thin layer of superficial cornified epithelium. The epithelial tissues immediately about the apex of the tooth show the appearance of pressure atrophy. The cell boundaries are more indistinct than elsewhere, the tissue takes the acid stain deeply, and there is increased granularity — evidently degenerative effects due to the pressure of the advancing tooth.

In the upper jaw, the basal end of the tooth in its backward growth has reached the region of the maxilla, into which it continues to grow, pushing before it a little pocket of thin bone. The average length of the upper teeth at this age is 5 mm., and of the lower teeth, 7 to 8 mm. Their pointed apices, and their comparatively slight curvature are shown in figure 24.



Fig. 20 Osteodentine of apex of tooth of 7-day albino rat imbedded in the surface epithelium, showing cells in the lacunae in the matrix. X 175.

ERUPTION OF TEETH

Eight to ten days

During the process of eruption (fig. 21), the tooth and its formative organs gradually move forward as a whole, and the apex of the dentine forming the anterior end of the tooth pierces the surface epithelium. This procedure is accompanied by new changes in the tooth-forming organs. For while the same process of cell-division continues at the basal end of the dental papilla and enamel-organ, these structures are subjected to

Fig. 21 Longitudinal section of the upper tooth of an 8-day albino rat, showing the apex of the tooth piercing the surface epithelium. X 10.

Fig. 22 Longitudinal section of the upper tooth of a 12-day albino rat, showing the increased size and curvature of the tooth, the basal end directed more towards the palatal surface and the progression of the apex of the tooth through the epithelium. X 10.

Fig. 23 Longitudinal section of the upper incisor of a 26-day albino rat, showing the well-established occlusal surface, the approximation of the basal end towards the palatal surface, abundant blood-vessels in the pulp, and the position of the granular osteodentine filling in apex of the pulp chamber. X 10.

new conditions at the erupting end of the tooth. Before detailing these changes, it may be advisable to state, in a general way, the changing circumstances attendant upon eruption. Up to this time the anterior end of the tooth has been nearly stationary, but there has been continued growth backward of the posterior extremity. At this time the rate of progression foi-ward is greatly increased, and the rate of progression backward much reduced. As suggested before, the process of eruption may depend largely upon the fact of increasing calcification in the bones, rendering them more resistant to the backward growth of the developing tooth. Whatever may be the causal factors, from now on the tooth continues to grow out at a regular rate, through the development of new cells at the basal end of the formative organs, these cells in turn giving rise to the hard parts of the tooth. Within a few days after eruption, the use of the tooth involves the process of attrition by which, in spite of the regular rate of growth, the exposed length is kept nearly constant for any age.

It is generally agreed that, by reason of the protoplasmic processes which extend into enamel and dentine from ameloblasts and odontoblasts respectively, these cells must be carried along with the tooth as it moves. Thus, as there is constantly a regeneration of these cells at the basal end of the tooth, there must be an opposite process of some nature by which these cells are eventually lost at the apical end, when carried thither by the outward progress of the tooth. First we may follow the history of the ameloblasts in this locality. Before eruption, the enamel-organ is continuous with the stratified epithelium forming the sheath around the gingival margin, and this relation continues at and after eruption. As the tooth moves forward during eruption the ameloblasts must move along with it and, when those at the anterior end approach the gingival margin, they must either be held there, or be carried out on the enamel until detached. On examining longitudinal sections at 12 days (fig. 22) it is seen that the ameloblasts, as they approach the gingival margin, become shorter and shorter, until, beneath the thickened sheath of epithelium forming the gingival margin they acquire a flattened form. As a continuation of these flattened cells next the tooth is seen, extending out into the space between the erupted tip of the tooth and the epithelial gingival margin, a thin layer of tissue, which must be looked upon as the portion of the enamelorgan which has been carried out during eruption. At later stages this same appearance occurs — a thin layer of flattened cells continuous with the enamel-organ lying in the space between the tooth and the epithelium of the gingival border. It may be that some of the cells are added to the epithelium of the gingival margin, but the majority appear to be continually carried out, and eventually detached.

The mesenchymal tissues of the pulp at the anterior end are little afl'ected by the mere act of eruption and not until some days later when attrition begins, do we see definite changes. At eruption the anterior conical extremity of the tooth is formed of osteodentine, containing within its matrix the remains of scattered cells and blood-vessels. Immediately posterior to this begins the true fine-tubed dentine with a central pulp-chamber. The cells at the anterior end of the pulp-chamber are irregularly arranged, but following backwards one soon sees the odontoblasts in parallel arrangement at the periphery of the chamber. At 10 days, when the apex of the tooth has pierced the epithelium and is easily seen from the outside, the measurements of the upper and lower teeth are 7 and 11 mm, respectively. At 12 days, they have increased to 7.5 and 11.8 mm.

CHANGES IN APEX OF TOOTH BY USE

Already at 12 days, when one examines the exposed ends of the teeth, they show little pits, which have been caused by the pressure of the opposing teeth. At 14 days, the ends are flattened, and at 16 days, because of the increased obliquity of this flattened surface due to the wearing away of the lingual side of the dentine, they are acquiring a cutting edge. The length of these occlusal surfaces continues to increase so that by 19 or 21 days (fig. 24), they have nearly the appearance typically seen in the fully developed teeth. The osteodentine of the tip of the tooth is softer than is true dentine, for when the young tooth is dried this end shrivels and darkens in color. This cap of osteodentine on the end of the tooth may be useful, as suggested by Sachse ('94), because of its softness, in allowing the early formation of the functional occlusal surface. When this soft substance begins to wear away the tissues of the pulp would soon become exposed were there not a provision for the filling in of the apex of the pulp-chamber. This is effected by the formation of an irregular hard matrix, which may also be called osteodentine, within the extremity of the pulp-chamber. As



Fig. 24 Isolated upper and lower incisors of several ages of young albino rats. The pointed shape just before eruption is seen at 7 days. At 12 days, there is yet very slight change in the apices. At 21 days the occlusal surfaces are concave, and at 26 days they have nearly the typical mature appearance. X 2.

the outer surface of the tooth wears away, this formation is constantly taking place a short distance from the occlusal -surface.

Thus in examining a longitudinal section of the tooth at an age when the process of attrition has begun, and the typical occlusal surface has been formed (e.g., 26 days, fig. 23), we find this form of secondary dentine or osteodentine filling in the distal extremity of the pulp-chamber. As one approaches the anterior end of the pulp-chamber, the pulp becomes more and more restricted and the blood-vessels appear congested. Proceeding distally, the irregular matrix formation is seen between the cells and blood-vessels and finally near the occlusal worn surface is a granular mass of osteodentine with no circulating blood in it, but spaces are still seen containing the remains of the pulp elements. Here the living elements have disappeared, but by staining (e.g., with acid fuchsin), the remains of these may be made out in less calcified spots in the matrix. Evidently the odontoblasts and other tissues of the pulp which move with the dentine, become more and more compressed at the narrowing apex of the pulp-chamber, and finally there is this irregular deposit of secondary dentine between them, which serves to obliterate the pulp-chamber. As the tooth moves out, this process is constantly going on, just in advance of the occlusal surface, and keeps pace with the process of attrition.

It is interesting to note the rate at which the teeth are increased in length during their formative period and prior to attrition.

Up per Lower mm. mm.

1 day old 2.3 3

4 days old 3.6 5

7 days old 5 7-8

10 days old 7 11

Average growth 0.52 nun. and 0.88 mm per day

As will be seen later, this exceeds the rate at which the mature tooth continues to grow out.

DESCRIPTION OF MATURE TOOTH AND TOOTH-FORMING ORGANS, IN FIVE-MONTH ANIMAL

In the mature tooth, the general relations are shown in figure 25, made from a photograph of a decalcified section of the upper tooth of a 5-month animal. The regular curved outline is seen, with the greater proportion of the length imbedded within the jaw, and only a small part projecting. The formative end lies witliin an investment of bone belonging to the maxilla. At this end the dentine is very thin and the pulp greatest in amount. As one goes forward, the dentine increases regularly in thickness while the pulp-chamber becomes smaller and smaller. The vacuolated appearance at the anterior end of the chamber is due to shrinkage of the pulp tissue during fixation. The enamel has been lost in the process of decalcification except over the basal third. Numerous blood-vessels are seen within the pulp.


Fig. 25 Longitudinal section of upper incisor of a 5-month albino rat. The letter a shows where the next illustration (fig. 26) is taken. X 6.

Fig. 26 Small portion of the preceding, more highly magnified, to show the enamel-organ and the enamel, and the odontoblasts and dentine. X 135.

The enamel-organ is continuous over the convex labial surface of the imbedded portion of the tooth but is restricted to the most posterior region of the other surfaces, extending only 1 mm. forward from the basal margin. The enamel-organ differs in its structure in three regions of the labial side, and may be described separately in these three parts: (1) at the basal formative end, (2) near the gingival margin and (3) in the long intervening region. In (1) the enamel-organ is being constantly regenerated by the addition and growth of new cells. In (2) the enamel-organ is undergoing a retrograde process, while (3) represents the region where the enamel-organ is at its highest functional development, although its activity in increasing the thickness of the enamel is restricted, as noted before, to the basal third or less in the upper and to the basal half in the lower tooth.

Considering first the region (3), as shown in figure 26, the enamel-organ is conspicuous by reason of its tall ameloblasts and the high, narrow papillae. The enamel-organ is described in three layers — inner, middle and outer. The inner layer consists of the ameloblasts, which measure about 40 /x in height, with nuclei situated towards the outer end of the cells. The middle is composed of two strata (a) stratum intermedium, and (b) enamel pulp. The stratum intermedium is formed of 1 or 2 rows of fairly regular cuboida cells resting upon the outer ends of the ameloblasts, but the enamel pulp is not now recognizable as a distinct layer and exists principally within the papillae. The outer layer of the enamel-organ consisted originally of a single layer of cells, but these are no longer regular in form or arrangement. Together with the remains of the enamel pulp, the outer layer forms the papillary elevations, 60 to 70 M in height. These papillae are surrounded by an abundant capillary blood supply for the nourishment of the cells engaged in the formation of the enamel, and the purpose of the elevations is apparently to increase the surface area through which absorption may take place from the blood-stream.

The enamel is in two layers (fig. 26), the rods while traversing the inner layer being very distinct, and inclining towards the apex of the tooth at an angle of from 50 to 54° with the dentine surface. The continuations of these rods in the outer layer are not so distinctly seen, but the inclination, as made out in thin sections, is still greater towards the apex, forming an angle of from 20 to 25° with the plane of the surface of the dentine. As noted before in the study of enamel, the rods in the inner layer, when observed in cross-sections of the tooth, decussate at an angle of from 70 to 90°, but when they reach the outer layer all run parallel. The fact that the rods run in these various directions seems incontrovertible, but in the light of our present knowledge of enamel formation it is difficult to understand how this condition is arrived at. If each ameloblast is responsible for an enamel-rod, then it follows that because the alternate layers of rods are oblique to one another, the ameloblasts responsible for these series of rods must have changed their relative positions during the process of formation of these rods. No such phenomenon has been observed, or even suggested. The other possibility is that the matrix of the rods is formed in a regular manner, but that afterwards, before calcification is complete, the rods become re-arranged owing to pressure strains.

The plane of cUrection of the rods is suggestive of the importance of the enamel-organ in the persistent growth. For always the general plane of the rods, as they leave the enameldentine junction, is towards the outer end of the tooth, as if the ameloblasts, while engaged in enamel-formation were always held back by the enamel, in which their processes were imbedded.

The basal formative end of the enamel-organ (region 1) in the adult animal corresponds very closely in structure with what has already been described for earlier ages, e.g., 4 days. This is the region where the enamel-organ is constantly being renewed, and it retains the same embryonal character at all stages of development.

At the anterior end where the enamel-organ is continuous with the surface epithelium (region 2), a gradual transition occurs between the typical enamel-organ and the stratified squamous epithelium (fig, 27). As one follows the innermost layer of the enamel-organ forward, the cells become shorter, until they are cubical and finally flattened in shape. Here the other layers also lose their regular arrangement, and form, with the preceding, a thin layer of stratified cells. This layer can be followed directly into contact with the epithelium of the gingivus.



Fig. 27 Longitudinal section of the apex of the tooth of a 5-month albino rat, showing at a the position of the outward prolongation of the remains of the enamel-organ, and at h the more granular osteodentine filling in the apex of the pulp chamber. X 10.


The cells, however, do not lose their identity in the surface epithelium but remain separate as a thin layer lying against the enamel (fig. 27, a). This thin layer of epithelium, therefore, represents the ultimate fate of the enamel-organ after it has completed its functional activity. It is being continually pushed out and its most anterior part must be continually being lost.


RATE OF GROWTH OF THE INCISOR TEETH

Two methods were used for determining the rate of growth of the incisor teeth (a) cutting off one or more teeth at the gingival margin and (b) making marks upon the enamel. The results here given are based on the latter method, as giving more nearlj^ the normal rate of growth. By means of a dental engine, the animal always having been anesthetized, a fine transverse notch was made on the enamel of the incisors a short distance from the gingival margin. The interval between this mark and the tip of the tooth was then measured. At the end of about a week the distance between these two limits was again taken, and the difference between the two measurements showed the amount of wearing away. Two series of experiments were made by this method upon adult animals. In the first series six animals were used and in the second four animals, and measurements were made for several consecutive weeks. The longest period that one individual was studied was six weeks.

The two series gave very similar results. The lower tooth was always found to grow more rapidly than the upper. The upper tooth averaged 0.31 to 0.32 mm. per day, or 2.2 mm. per week, while the lower tooth averaged 0.4 mm. per day, or 2.8 mm. per week. No doubt there are many variations of the rate of growth under different circumstances, so that these figures must be taken as representing the average rate under one particular set of conditions. The food of these animals was the mixed diet now in use in the rat colony of The Wistar Institute. A short series of trials was made with animals kept in a large glass jar and given only soft food. In these animals the rate of wearing away corresponded very closely to that seen in animals which have also hard food and have the opportunity of gnawing. In these, therefore, the interaction of the opposing teeth must have caused the attrition.

For assistance with these experiments we wish to thank Dr. Stotsenburg, who greatly aided us in carrying out our observations.


It is interesting to compare these results with those obtained on the rabbit — the only other rodent which has been carefully studied in respect to the growth of its incisors. MacGillavry ('75), using a young adult rabbit, made marks upon its lower incisors 2.5 mm. and 3 mm. from the tip. After five to seven days the marks had disappeared. Evidently the rate of growth was about 2.5 to 3 mm. per week. Noe ('02) used a rabbit which happened to possess overgrown teeth. The animal accidentally broke off the lower incisors in the bars of its cage, and Noe made observations upon the rate of their growing out. This he found to be .615 mm. per day, or 4.3 mm. per week. This is larger than MacGillavry's results and may have been due to the unopposed growth and to the other abnormal conditions which may have been present in the formative organs.

Using MacGillavry's figures for comparison, it would seem that the lower teeth of the albino rat and of the rabbit grow out at about the same rate.

OVERGROWTH OF INCISORS

Examples of overgrowth of the incisors of rodents, especially in rabbits and hares, which were hunted as food, must have been observed from early times. In the older literature, they are referred to principally as curiosities, which have excited the interest of whoever has found them. later the causes of the malformations were also considered. Thus Jenyns ('29), to cite only one observer, found several examples in wild rabbits, and has given a good illustration of the curved aspect of the teeth. He also clearly states the several causes which, in his opinion, may give rise to the condition. In addition to the one usually accepted at his time — accidental breaking off of one tooth — he considered also as causes (a) too soft food, (b) morbid or too rapid secretion of the osseous matter of the teeth, and (c) dislocation of one of the condyles.

Wiedersheim ('02-'03) has reported a case occurring in a rat, where he found an associated assymmetry of the cranium. He is in doubt as to which was cause and which was effect — the overgrowth of the teeth or the assymmetry of the cranium.


Fig. 28 Cranium of albino rat, showing the overgrown upper incisors recurving to the left side. The left incisor passes to the outer side of the skull, while the apex of the right incisor has penetrated the bone of the maxilla in the region of the basal end of the left incisor. X 1.

Fig. 29 Cranium of the same albino rat shown in the preceding figure, viewed from the right side. It shows the overgrown lower incisors recurving to the right side, and the cavity which the right incisor has worn in the palate bone.

Figures 28 and 29 show a skull obtained some years ago from the rat colony of The Wistar Institute by Dr. Stotsenburg, and prepared in the Histological Laboratory by Miss E. F. Brooks. The upper teeth curved to the left side of the head and the lower to the right side. As seen in figure 29, the right lower has penetrated through the bone of the palate into the nasal chamber, while the right upper (fig. 28) has recurved and grown into the maxilla.

In The Wistar Institute rat colony, at the time when the animals were fed on bread and milk, frequent examples of this and similar conditions were found, but now under a more varied mixed diet they practically never occur.

Beretta ('13) has recently made an analysis of these abnormalities and has classified them in three groups.

(1) Overgrowth of the upper and lower incisors through lack of an opposing tooth.

(2) Overgrowth of the incisors of the upper and lower jaws through deviation of the jaws.

(3) Prognathism of the lower jaw, and as a result, overgrowth of the incisor of the lower jaw.

In the present instance, diet seemed to be the controlling factor, probably by reason of its influence on the hardness of the bone of the alveoli from which the teeth grew out.

SUMMARY

The rate of growth of the upper and lower incisor teeth of Mus norvegicus albinus, in the mature animal, averages 2.2 and 2.8 mm. per week, or 12.5 cm. and 14.5 cm. per year, respectively.

Growth is due primarily to the proliferation and growth of cells at the basal end of the enamel-organ, where new enamelforming cells arise, and at the basal end of the dental papilla where new dentine-forming cells develop.

The enamel-organ of the adult forms a narrow circular band around the basal end of the tooth, and extends forward from this on the labial side only. It coincides in its lateral boundaries with the enamel, and extends along the entire imbedded portion of the tooth. Anteriorly, it comes in contact with the epithelium of the gingival margin, and is carried out continually as a narrow band of cells lying on the enamel, between the latter and the gingival epithelial tissue.

The first indication of the anlage of the incisors appears in 14-day-old fetuses. In fetuses, 21 daj^s of age (just before birth), enamel and dentine formation is beginning. In animals 1 day old the upper and lower teeth measure 2.3 and 3 nrni. At 8 to 10 days the teeth erupt, and at 10 days measure 7 and 11 mm. respectively. This period is therefore characterized by the rapid elongation of the teeth.

The process of attrition begins within a few days after eruption, so that by 19 or 21 days of age, the typical occlusal surface is formed. Up to the time of eruption the anterior end or apex of the tooth is immediately under the oral epithelium, while the basal or growing end is continually progressing posteriorly. After eruption, the basal end becomes nearly stationary in position, while the whole tooth structure is continually moving forward. The extra-gingival length of the tooth is kept constant, however, by the attrition of the occlusal surface, either through use in gnawing or by the action of the opposing teeth.

The histogenesis of the enamel-organ is practically completed by the 4th day after birth, although it does not attain its final relations to the tooth as a whole, until after eruption. In the 18-day fetus the enamel-organ is similar in all parts, and the cells of the inner layer measure the same, both lingually and labially. From this period forwards, however, the labial portion continues to progress towards its fully differentiated functional structure, while the lingual portion retrogresses, until at 4 days after birth the latter is disrupted, by the ingrowth of the surrounding connective tissue. Contrasting the cells of the inner layer — the potential ameloblasts — on the labial and lingual sides, thej^ are practically the same in the 18-day fetus, but at 19 days they are found to measure 24 and 20 m respectively. In the 21-day fetus, they measure 30 to 34 and 12 fx, and 1 day after birth the true ameloblasts on the labial side have increased to 40 n, while the non-functional cells of the lingual side are only 10 fx in height. At 4 days, the latter cease to form a continuous layer, by reason of the dispersion of the cells by the surrounding connective tissue, except at the basal formative region.

Characteristic of the permanently-growing enamel-organ are the epithelial papillae, formed by the elevations of the outer layer of the enamel-organ, and the cells of the enamel pulp. Between these elevations are numerous capillaries which insure a rich blood supply to the enamel-forming cells.

There are three layers in the functional enamel-organ — inner, middle and outer. The inner is constituted of the tall ameloblasts, and the middle is made up of two divisions, (a) stratum intermedium and (b) enamel pulp. The latter unites with the single layer of cuboidal cells which compose the outer layer, to form the epithelial papillae (fig. 26).

The apex of the primitive tooth is formed of a variety of secondary dentine — 'osteo-dentine' of Tomes — which is softer than true dentine, and differs in its structural arrangement (fig. 20). After eruption, this terminal portion of osteodentine is soon worn away by attrition, and the typical occlusal surface is developed, as seen at 19 or 21 days. At 21 and 23 days the first two molars erupt in both upper and lower jaws, and from now on the animal is able to secure food for itself, and if necessary can maintain an independent existence.

As the tooth continues to be worn away there is a provision for the continual filling in of the apex of the pulp-chamber by the formation of what may also be called osteodentine. This is a form of secondary dentine, containing, when first formed, cells and blood-vessels. This is always at a little distance, about 1 mm., from the occlusal surface, but as any part of the tooth, in its outward progression, approaches the occlusal surface, the soft elements disappear within the osteodentine, and the latter forms a hard continuous surface with the adjoining true dentine. The position of this osteodentine is marked as a line on the occlusal surface of the teeth (fig. 5).

Prior to eruption there develops around the apex of the tooth, as it lies in contact with the surface epithelium, a thickened ring of stratified epithelium. This ring of tissue is pierced by the apex of the tooth at eruption, and would seem to have the function of serving as a resistant margin for the soft tissues, and of preventing other tissues being carried along with the erupting tooth.


The length of the teeth varies with the size of the cranium (table 1) so that the persistent growth is not only sufficient to offset the continual attrition, but also serves to keep the length of the teeth in a definite relation to the length of the skull, as the latter increases in size.

The lower tooth is always longer than the upper, and this difference manifests itself even in the anlagen of these structures in the 19-day fetus (figs. 11 and 12).

The contour of the enamel, as seen in cross-sections, is characteristically different in the upper and lower teeth (fig. 4).

The enamel of the tooth is composed of two layers which are different in appearance. The enamel rods run in two sets which decussate with each other in the inner or plexiform layer, but they change their direction as they continue into the outer layer, so that in it they are all parallel. In longitudinal sections, the general direction of the rods (fig. 26), is to incline towards the apex of the tooth, as they run from the enamel-dentine boundary to the outer surface of the enamel.

In conclusion, we wish to thank Professor Piersol for generous assistance in many ways, and Professor Donaldson for his constant interest in the study. We also wish to acknowledge the kind assistance of Mr. E. F. Faber in the preparation of the drawings.


LITERATURE CITED

Adloff, p. 1898 Zur Entwicklungsgeschichte des Nagetiergebisses. Jena. Zeitschr. fiir Naturwissenschaft, Bd. 32, ss. 347-410.

Beretta, a. 1913 La normala dentatura dei roditori in rapporto alle anomalie dentali in questi osservate. La Stomatologia, t. 10. Abstract in Deutsche Monatsschrift fiir Zahnheilkunde, April, s. 287.

VON Brunn, a. 1887 Ueber die Ausdehnung des Schmelzorganes und seine Bedeutung fiir die Zahnbildung. Arch. f. mikr. Anat.. Bd. 29, ss. 367-383.

BuRCKHARDT, R. 1906 In Hertwig's Handbuch der Entwickelungslehre der Wirbeltiere, Bd. 2, Teil 1, Kapitel 4, ss. 349-456. "Die Entwickelungsgeschichte der Verknockerungen des Integuments und der Mundhohle der Wirbeltiere."

Cope, E. D. 1888 The mechanical causes of the origin of the dentition of the Rodentia. Amer. Nat., vol. 22, pp. 3 11.

Donaldson, H. H. 1912 The history and zoological position of the albino rat. Proceed. Acad. Nat. Sci., Philadelphia.

Freund, p. 1892 Beitriige zur Entwicklungsgeschichte der Zahnanlagen bei Nagethieren. Arch. f. mikr. Anat., Bd. 39, ss. 525-5c6.

Fritsch, C. 1914 Untersuchungen iiber den Bau und die Innervierung des Dentins. Arch, fur mikr. Anat., Bd. 84, ss. 307-20.

Hatai, S. 1907 On the zoological position of the albino rat. Biol. Bull., vol. 12, pp. 266-273.

Jenyns, L. 1829 Observations on a preternatural growth of the incisor teeth occasionally observed in certain of the Mammalia Rodentia. Loudon's Magazine Nat. Hist., London, vol. 2, pp. 134-137.

Jolyet et Chaker 1875 De I'acte de ronger, etudie chez les rats. Comptes Rendus et Memoires de la Soc. de Biol., pp. 73-74.

Lowe, L. 1881 Beitrage zur Kenntniss des Zahnes und seiner Befestigungsweise im Kiefer. Arch. f. mikr. Anat., Bd. 19, ss. 703-719.

MacGillavry, T. H. 1875 Les dents incisives du Mus decumanus. Arch. Neerl. Sc. exact, et nat., Haarlem.

Meyerheim, M. 1898 Beitrage zur Kenntnis der Entwicklung der Schneideziihne bei Mus decumanus. Dissertation. Leipzig.

Mummery, J. H. 1912 On the distribution of the nerves of the dental pulp. Philos. Trans. Roy. Soc, London, vol. 202, B., pp. 337-349.

NoE, J. 1902 Vitesse de croissance des incisives chez les Leporides. Comptes Rendus, hebd. des Seances et Memoires de la Soc. de Biol., pp. 531-532.

OuDET, J. E. 1823 Experiences sur I'accroissement continue et la reproduction des dents chez les lapins. Jour, de Physiol. Exper. et Patholog., Tomes 3 et 4.

Owen, R. 1840-45 Odontography. London.

Reichert, E. T., and Brown, A. P. 1910 The crystallography of hemoglobin. Pub. Carnegie Inst, of Washington.

Retzius, a. 1838 Bemerkungen iiber den inneren Bau der Zahne mit besonderer Rlicksicht auf den in Zahn vorkommenden Rohrenbau. Mliller's Archiv.

Roetter, F. 1889 TJeber Entwicklung und Wachstum der Schneidezahne bei jNIus musculus. Morphol. Jahrb., Bd. 15, ss. 457-477.

Ryder, J. A. 1877 The significance of the diameters of the incisors in Rodentia. Proc. Acad. Nat. Sci., Philadelphia, vol. 29, pp. 314-318.

1878 On the mechanical genesis of tooth-forms. Proc. Acad. Nat. Sci., Philadelphia, vol. 30, pp. 45-80.

Sachse, B. 1894 Entwicklung der Schneidezahne bei Mus musculus. Dissertation. Leipzig.

Stach, J. 1910 Die Ontogenie der Schneidezahne bei Lepus cuniculus. Extrait. Bui. d'Acad. Sc, Cracovie.

Tomes, C. S. 1914 A manual of dental anatomy; human and comparative. 7th Ed. Edited by H. W. Marett-Tims and A. Hopewell-Smith.

Tomes, J. 1850 Structure of the dental tissues of the order Rodentia. Phil. Trans. Royal Society of London, pp. 529-567.

Tullberg, T. 1898-99 Ueber das System der Nagethiere. Nova Acta Reg. Soc. Sc. Upsaliensis, Series 3, ss. 1-514.

Weber, M. 1904 Die Saugetiere.

Wiedersheim, R. 1902-03 Ein abnormes Rattengebiss. Anat. Anz., Bd. 22, ss. 569-573.

Williams, J. L. 1896 The formation and structure of dental enamel. Dental Cosmos, vol. 38.

Woodward, M. J. 1894 On the milk dentition of the Rodentia with a description of a vestigial milk incisor in the mouse (Alus musculus). Anat. Anz., Bd. 9, ss. 619-631.


A Peculiar Structure In The Electroplax Of The Stargazer, Astroscopus Guttatus

James G. Hughes, Jr.

From the Histological Laboratory of Princeton University, U. S. A.

THREE FIGURES

The purpose of this paper is to determine the function and composition of the pecuhar pointed fibers and long pointed rods lying in the electric layer of the electroplaxes of the stargazer, Astroscopus guttatus.

Before proceeding with a discussion of these rods, a brief description of the electric organ of this fish (according to Dahlgren)^ will be given.

The electric apparatus is composed of two organs, which form two vertical columns roughly oval in horizontal section, and placed behind and somewhat under each eye. Each organ extends from the peculiar bare spot on the top of the head down to the tissues which form the roof of the oval cavity; and is composed of about 200 thin layers of electric tissue, which extend horizontally all the way across the organ. These layers of tissue are flat, and alwaj^s at the same distance from one another. Each layer contains about 20 electroplaxes, the outlines of which present a very irregular or scalloped appearance. The electric tissue in which the electroplaxes are imbedded is in appearance a jelly-like or mucous-like tissue, usually known as electric connective tissue, and which I have shown in the course of my work to be of the same composition as white fibrous connective tissue. The nerve and blood supply runs in the above tissue. The general form of a vertical section of an electroplax is shown by figure 1, which is a drawing of part of a section of a single electroplax.


1 Anat. Anz., Bd. 29, S. 387, 1906.


Each electroplax is composed of three principal layers, a nervous or electric layer which forms the upper surface, a middle layer, and a lower or nutritive layer which along with the middle layer is evaginated into a large number of long papillae. All three layers are deeply marked with a dense series of fine striations, which are peculiar to the electroplaxes of several other fishes. The upper or electric surface is flat and smooth and receives the nerve endings. The current of electricity runs downward through the organ which produces it, and thus the nerve endings in accordance with Pacini's law are found on the negative pole of the electroplax.

Proceeding directly to the subject of this paper, we may say that one of the most interesting of the points noted in the electroplaxes, when properly fixed and stained with iron hematoxylin, is a series of rod-like or thread-like objects running horizontally in the electric layer, among, above and below the nuclei and without any apparent connection with them (figs. 1 and 2).^ These rods are of various sizes and shapes, and in form are said to resemble the classic thunderbolts seen in the hand of representations of Jove. They usually taper slowly and branch extensively at one or both ends. Some of these branches sometimes seem to be mere lines, while others are wide and heavily pointed; at their other ends the rods are usually rounded; this latter appearance may be due, however, to the cut ends of the rods, for as noted above they sometimes branch at both ends. Some are short and heavy in appearance while others are long and thread-like. Peculiar looping, twisting, or knot-like bends are sometimes found at points on the longer rods. The outlines and contour of these rods are always smooth. Their size may vary from thick or thin rods of over 300 /x in length down to small ones that do not exceed 1 m- In those electroplaxes where the rods are few they sometimes lie parallel and point in a definite parallel direction, while in others where the rods are very numerous they do not seem to have any definite arrangement. In this latter condition the rods present a very wavy appearance. Their form may be seen in figures 1 and 2, which are drawings of the electric layer of an electroplax when

^ All the figures are drawings of sections of electroplaxes of Astroscopus guttatus.


Fig. 2 Horizontal section through an electroplax. Only the electric layer is seen; a, an extensively branching rod; h, fine branches of the above rod; c, a large characteristic loop in a rod; d, a cut end of a rod; e, nuclei of the electric layer. X 1200.


stained with iron hematoxylin. These drawings show the electric layer in which the rods are found in horizontal section (fig. 2) and in vertical section (fig. 1).

The purpose, function, and chemical composition of these rods have been previously unknown to histologists. In order to determine anything in respect to their function or purpose, a knowledge as to the class of organic substance to which they belong, whether muscle, connective tissue, nervous, or chitinous, and also a rough knowledge of their chemical composition is imperative. The contour and form of these rods as they appear under the microscope resemble both smooth muscle fibers and fibers of elastic connective tissue. The belief that the function of these rods was somewhat of the nature of support for the delicate substance of the electroplax, and the fact that their form resembled connective tissue fibers led the writer to take for one of his first hypotheses, that they were of some form of connective tissue, and to perform accordingly the following series of experiments. As the most logical and best way for determining the kind of connective tissue, if any, of which the rods might be composed, a number of stains used by other investigators to identify similar substances were applied and the results noted. Controls were used on known tissues.

Before taking up the connective tissue stains, however, a description of the results from the iron hematoxylin staining is now noteworthy; the material used being fixed in pure corrosive sublimate.

The jelly connective tissue stained a very light gray. The nutritive, striated, and electric layers stained a much darker gray. The nuclei in all the layers stained somewhat black. The pointed fibers, and rods found in the electric layer stained a deep black, thus being clearly differentiated from the surrounding cytoplasm. In some electroplaxes they were very numerous, while in others the number was rather small.

The connective tissue stains applied as follows:

(1) Mallory's connective tissue stain, using the modification given in Lewis' Text-book of histology." White fibrous connective tissue should stain blue in this medium.

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 10 to 12 minutes in a 1 per cent aqueous solution of acid fuchsin. They were then transferred directly to a stain consisting of 0.05 grain of aniline blue (soluble in water) and 0.2 grain of orange G dissolved in 100 cc. of a 110 per cent aqueous solution of phospho-molybdic acid. In this they remained from 2 to 3 minutes. They were then rinsed in distilled water, dehydrated rapidly, cleared and mounted.


102 JAMES G. HUGHES, JR.

A description of an electroplax as seen under the 2 mm. oil immersion lens is as follows: The white fibrous or electric connective tissue stained a light blue or purple. The electric nutritive and middle layers or the electroplax proper stained a reddish purple, and the nuclei as a whole in all of the layers, stained somewhat lighter than their surrounding cytoplasm.

The peculiar rods and fibers stained a deep red, and were thus clearly differentiated from the other elements of the electroplax. They were very numerous and as noted above their outlines were always smooth. Blood corpuscles lying in the jelly connective tissue, of which there were only a few, stained a brilliant red, much the same as the rods.

The white fibrous or jelly electric connective tissue (between the electroplaxes) , as noted above, stained a light blue. The pointed fibers and rods stained a deep red. This would indicate therefore, that these rods are not composed of white fibrous connective tissue. It would seem also that they are not muscle for they stained a different shade of color from the rest of the electroplaxes, which I have found in the course of my work to stain much the same as muscle.

(2) Vcn Gieson's connective tissue stain, in which white fibrous connective tissue should stain red:

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 4 to 5 minutes in a 1 per cent aqueous solution of hematoxylin. They were rinsed in distilled water and transferred to a stain consisting of a saturated aqueous solution of picric acid containing 20 per cent acid-fuchsin. They remained in here 15 to 20 minutes and were then rinsed, cleared, and mounted. The white fibrous connective tissue layer stained pink. The three layers of the electroplax stained brown. The nuclei and rods stained the same color, that is, brown; and, while the nuclei could be seen with difficulty, the rods were scarcely visible owing probably to their similar refractive index. The fact therefore that the rods did not stain the same color as the white fibrous connective tissue, which stained pink, indicates again that they are not composed of white fibrous connective tissue. By Van Gieson's stain, therefore, I have


ELECTROPLAX OF ASTROSCOPUS 103

confirmed the evidence as presented by Mallory's stain in respect to the composition of the rods; that is, they are not white fibrous connective tissue.

I now undertook to apply stains which were tests for elastic fibers, and the first under this head is Weigert's resorcin-fuchsin stain in which connective tissue according to Weigert and other writers stains dark blue. The stain was made up as follow^s:

One per cent of basic fuchsin and 2 per cent of resorcin were dissolved in water; 50 cc. of the solution were raised to the boiling point, and 25 cc. of liquor ferri sesquichlorate P.G. were added and the whole boiled with stirring from 3 to 5 minutes; a precipitate was formed. After cooling, the liquid was filtered, and the precipitate which remained on the filter was boiled with 50 cc. of 95 per cent alcohol. It was then allowed to cool, filtered and the filtrate made up to 50 cc. with alcohol, and 1 cc. of hydrochloric acid added.

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 6 hours in the above alcoholic solution of the precipitate. They were then washed in 95 per cent alcohol, dehydrated quickly, cleared and mounted. A description of the results are as follows: The electroplax proper, with its three layers, did not take the stain at all. The jelly electric tissue around the electroplax stained a brilliant blue. The rods contained in the electric layer of the electroplax were invisible. The fact therefore that the rods did not take the stain at all shows they are not composed of elastic connective tissue. The control used in this case for elastic tissue was a section of the ligamentum nuchae of a horse, which, when put in the stain for exactly the same time as the electroplax, came out a deep blue. The ligamentum nuchae was chosen as a control since it is, perhaps, the best known and best representative of elastic tissue found in the animal kingdom.

Not desiring to rely solely on the above stain to prove that the rods are not composed of elastic tissue, the electroplaxes were treated with the digesting fluid, pepsin, and the results noted. Vertical sections of electroplaxes, having been freed from paraffin, were put for 3 minutes in a very weak solution


104 JAMES G. HUGHES, JR.

of pepsin in 0.2 per cent HCL (the concentration being 0.5 gram of commercial pepsin to 100 cc. of 0.2 per cent aqueous HCL). The electroplaxes were then treated with the following stains: (1) Iron hematoxylin. The result was that all the rods and part of the nuclei, and less dense portions of the electroplax had been digested. (2) Mallory's connective tissue stain was applied, the electroplaxes being stained according to the directions given in the first part of the paper. The results were the same as with the iron hematoxylin, namely, the less dense portions of the electroplaxes and the rods had disappeared. The fact therefore that these rods were disgested in 3 minutes by a weak solution of pepsin discredits absolutely the hypothesis that the rods are composed of elastic connective tissue, for elastic fibers are digested only very slowly by pepsin, the time required being several hours; the control in this case being the same as that noted above — the ligamentum nuchae of a horse.

Having shown that the rods are not composed of connective tissue of any sort, the next most logical hypothesis was that they were of some form of keratin, chitin, or chondrin, and the following test was performed accordingly.

Vertical sections of the electroplaxes of Astroscopus, after the paraffin had been removed, were put for 24 hours in a 72 per cent solution of hydrochloric acid. The acid was then washed out and the sections stained with iron hematoxylin and then examined with the microscope. The rods could not be found, but hollow spaces corresponding to the shapes of the rods were found in the electric layer of the electroplax. Figure 3, a drawing of a slide after treatment with hydrochloric acid, shows these spaces clearly. The spaces are seen to be a little wider and larger than the rods, showing that the rods must have swelled to a certain extent before being dissolved by the acid. This latter statement was also confirmed by treating the rods with the acid for only a short time, and then examining with the microscope; the result being that the rods had swelled to a considerable amount. The other elements of the electroplax, nuclei, etc., in their general form remained intact. There


ELECTROPLAX OF ASTROSCOPUS 105






,;.^










•,"~^









f


.w) ^


'^







l^



« 




Q


%



?^



t



« 


«?







' «. '




^


94,


\


A


c;}



/^


£■


»



D


-^


1


f


'•N,




a>



(D


^







'^


%



'


.-) ^


^



e



'^>


■5


i'

3 (>y



^


(^


>1



,f)


£)


V)


'^^


(?)


Fig. 3 Oblique section through an electrophix treated with 73 per cent hydrochloric acid. The portion of the electric layer shows the electric nuclei, and also the spaces which were occupied by the rods before they were dissolved. The spaces have a greater diameter than that of the rods because the rods swelled before being dissolved. X 1200.

fore this indicates clearly that the rods are not composed of chitin or keratin, for these substances are not attacked by hydrochloric acid of the above strength. To strengthen this statement we have only to quote the results from the pepsin treatment noted above, in which the rods were digested in 3 minutes. They could not therefore have been composed of chitin or keratin, for according to Encycl. mikr. Technik, these substances are not attacked at all by pepsin. From the abo\'e facts therefore we may with a good degree of certainty conclude that the rods are not composed of any kind of chitinous or keratinous substance. Having found that the rods do not consist of any kind of connective tissue or of chitin or keratin the only reasonable hypothesis left was that they were some form of muscle fiber, not having however the same chemical com


106 JAMES G. HUGHES, JR.

position as the ordinary striated fiber, from which the electroplax is derived. Accordingly the following stains were apphed :

(1) Van Gieson's picro-nigrosine, which stains muscle a yellowish-green and connective tissue blue, mixed as follows: To 45 cc. of saturated aqueous solution of picric acid, 5 cc. of 1 per cent aqueous solution of nigrosine were added, the whole mixed thoroughly. Paraffin sections of the electroplaxes (vertical) fixed in pure corrosive sublimate were stained for 12 hours in this mixture; then washed in picric alcohol, dehydrated rapidly, cleared and mounted. A description of an electroplax is as follows: The jelly electric tissue stained blue. The electroplax proper with its three layers stained a yellowishgreen. The rods were just visible, and were stained the same color and to the same degree as the electroplax. The fact therefore that the rods stained the same color as the electroplax and as muscle shows that they are very probably composed of some muscle-like substance. It may be noted here that the electroplax proper always stains about the same as voluntary muscle, a control consisting of voluntary muscle having proved the truth of this statement several times. The reason for this is apparent when we recognize the fact that the electroplax is derived from a striated muscle fiber. This stain also confirms the results of Weigert's resorcin-fuchsin stain that the rods are not composed of any kind of connective tissue, and also indicates decidedly that the jelly electric tissue is white fibrous connective tissue. The controls used were the adductor muscle from an oyster, the ligamentum nuchae of a horse, and the white fibrous connective tissue in the umbilical cord of a sheep, all being stained exactly the same period of time as the electroplax.

(2) Van Gieson's picro-fuchsin, which stains muscle yellow and connective tissue red, was mixed as follows: To a saturated solution of picric acid were added a few drops of a saturated aqueous solution of acid fuchsin, until the mixture became a deep red. The stain was then ready for use. (It may be noted that, if too much acid fuchsin be added, muscle as well as connective tissue will stain red).


ELECTROPLAX OF ASTROSCOPUS 107

Paraffin sections of the electroplaxes (vertical) fixed in pure corrosive sublimate were stained for 6 hours in the above mixture. The results were as follows: The jelly connective tissue stained red, the electroplax proper with its three layers stained yellow. The rods were scarcely visible, staining the same color (yellow) as the electroplax. The fact that the rods stained yellow would indicate again that they are composed of muscle tissue, and this in connection with the picro-nigrosine stain in which the rods stained the same color as muscle gives us strong ground for beheving that the rods are composed of muscle tissue; probably involuntary muscle fibers as the rods are not striated. The fact also that the jelly electric tissue stained red shows again that it must be of the same composition as ordinary white fibrous connective tissue. The controls were the same as those for Van Gieson's picro-nigrosine.

It may be objected that the rods did not stain the same color as the electroplax proper with Mallory's connective tissue stain. To explain this we may say that the rods stained a brilhant red, which is the color that muscle tissue should take in the above stain. The fact that only the electroplax stained a reddish-purple shows that the rods are more strictly composed of a muscle substance than the electroplax itself.

The only tissue now left of which the rods might be composed is nervous; that is, the rods might be nerve endings of some kind, which they resemble a little. In order to test this Paton's silver nitrate stain for demonstrating nerve fibers and endings was apphed and the results carefully noted. The stain and fixation were as follows:

The electric organ was fixed in 10 per cent formalin solution, neutrahzed with magnesium carbonate. It was then cut in small strips about 4 mm. thick. These were washed with running tap water for 12 hours and then in three changes of distilled water for about 30 minutes. The tissue was then put in 1 per cent silver nitrate solution for 6 days in the dark. The tissue became a reddish-brown in color. (It may be noted here that if the tissue becomes a yellowish-brown the stain has not been applied correctly, and it is advisable to throw the whole speci JOURNAL OP MORPHOLOGY, VOL. 26, NO. 1


108 JAMES G. HUGHES, JR.

men away.) It was then placed in a freshly prepared solution of silver nitrate, made as follows:

To 20 cc. of 1 per cent silver nitrate solution, two drops of a 40 per cent solution of caustic soda were added. A gray precipitate was formed. Twenty to thirty drops of strong ammonia were then added — just enough to dissolve the precipitate. The tissue was allowed to remain in this for 45 minutes; then washed in distilled water containing a few drops of glacial acetic acid (25 drops of acid to 100 cc. of water). It was left in this for about 10 to 15 minutes, until the reddish-brown color had changed to yellowish-brown. It was then washed in distilled water, and placed for 12 hours in 1 per cent hydroquinone containing 5 per cent neutral formal. It was again washed with distilled water, dehydrated, and imbedded in paraffin through chloroform. The tissue was then cut and sections mounted with balsam.

A description of a vertical section of an electroplax so stained is as follows: The electroplax proper stained a very light brown. The nuclei, staining a little deeper, could be clearly seen. The electric connective tissue did not stain at all, and was not even visible. The nerve endings, which were very abundant, stained dark brown or black, thus being clearly differentiated from the rest of the electroplax. The rods could not be found. If present, they must have stained light brown, the same color as the electroplax, for they certainly were not present as nerve endings, which as I noted above, stained a dark brown or black. This conclusion therefore is evident, that the rods are not nerve endings of any kind.

The fact therefore that these fibers and rods have been shown to be composed of a muscle-like substance brings up a new kind of muscle fiber to the attention of histologists. These fibers have not been shown to be contractile, are non-striated, dense and exist without any apparent connection with each other. In view of the above facts we cannot give to these elements of the electroplax any other function except that of support, and this will consequently have to suffice until some other investigator presents a better interpretation.


CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT

HARVARD COLLEGE. NO. 256.


CHROMOSOME STUDIES

iit. inequalities and deficiencies in homologous chromosomes: their bearing upon synapsis

AND the loss of UNIT CHARACTERS W. REES BREMNER ROBERTSON

FOURTEEN FIGURES (THREE PLATES)

In November, 1910, while preparing a larger paper upon the problem of synapsis in the germ cells of certain grasshoppers belonging to the subfamily Tettigidae (Robertson '15), I found some individuals in which there occurred a pair of unequal homologous chromosomes. I made a series of drawings of these pairs at the time, intending to incorporate them later into my larger paper. That paper is not yet completed, owing to the duties of teaching during the last year at Kansas University. The importance of these inequalities in members of a chromosome pair, however, seems to warrant publishing the results in brief form. In this account I wish to point especially to a possible relation between these unequal chromosomes and the behavior of certain unit characters in breeding; also to the bearing which the permanency of such chromosomes has upon the problem of parasynapsis.

In the Tettigidae, a subfamily of the short-horn grasshopper family Acrididae, I have found, for all the species of at least four different genera which I have examined, the number of chromosomes to be uniformly 14 in the female (figs. 1, 11) and 13 in the male (figs. 4, 9). I have also found all to have, with limited variation, among the autosome (ordinary chromosome) series, two extremely long pairs of chromosomes, the 7's and 6's (figs. 1, 3, 4), two intermediate pairs, the 4's and 3's (figs. 1-3) or o's and 4's (figs. 9, 10), and two very small pairs, the 2's and I's (figs. 1-3, Tettigidea) or the 3's and 2's (figs. 9-10, Acridium).

109


110 W. E. B. ROBERTSON

The sex chromosome — single in the male and paired in the female — may rank in size as No. 5 (Tettigidea), No. 3 (Paratettix) or No. 1 (Acridium), depending upon the genus, but this variation between different genera does not seem to be accompanied by any very considerable difference in the relative sizes of the ordinary chromosomes.

Of the constancy of these size relations, I am very certain, having examined a large number of individuals in each species. Being certain of the size relations, I was very ready to recognize any abnormal variations in this respect when they appeared. I found such variations in two genera: two cases in Tettigidea parvipennis, only one of which is given (figs. 4-8), and two in Acridium granulatus (figs. 11-13).

1. A DEFICIENT NO. 4 CHROMOSOME IN TETTIGIDEA PARVIPENNIS

To understand the abnormal chromosomes we must first examine the normal ones. Figures 1 to 3 are of cells from normal individuals. Figure 1 is from the wall of an egg tube. It shows 14 chromosomes, the number characteristic of the female. The chromosomes are numbered and paired according to size. Two of the chromosomes (nos. 1 and 2) have been drawn at one side for convenience. This figure is also typical for male 2 x cells, with this exception, that the sex-chromosome. No. 5, is always unpaked in the male. Figures 2 and 3 are lateral views of the first maturation division in the male germ cells. These show the members of pairs about to separate from each other. The members are still in contact at the distal ends in most cases. The No. 7 pair in figure 2 has formed a cross and is still paired through a greater extent than in the other pairs. The sex chromosome ranks fifth in size in Tettigidea and may be seen passing undivided to one pole in these cells. Among the autosomes (ordinary chromosomes) of figures 2 and 3 there can be seen a very slight difference in size between the I's and 2's, a considerable difference between the 3's and 4's, and a much greater difference between the 6's and 7's. We are concerned at present with the relative sizes of the 3's and 4's only.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 111

Since the diameters of the chromosomes are uniform throughout the series in any given cell, the relative sizes can be learned best by measurement of lengths. My measurements have been made from drawings outlined by means of a camera lucida, the image being projected at the level of the base of the microscope. A 2 mm. Leitzoil-immersion objective and an 18 X Zeiss compensating ocular were used for outlining. I feel reasonably certain that my drawings show approximately the correct relative sizes of the chromosomes in each cell.

The average length (doubled) of the No. 3 chromosomes in the figures ( X 2600) of six cells from two normal individuals of Tettigidea parvipennis is 15 mm. The average length of the No. 4 chromosomes under the same conditions in the same six cells is 17.05 mm. The ratio of the No. 3 to the No. 4 chromosomes is therefore as 1 to 1.14. This may be taken as approximately the normal size relation of the No. 3 and No. 4 chromosomes; that is, No. 4 is about one-seventh longer than No. 3.

In figures 4 to 8 are cells from a male individual of the same species, in which one member of the No. 4 pair of chromosomes (4 — ) is abnormally small. The average length of the No. 3 tetrad is here 13.1 mm. This is shorter than in the preceding case of six cells, but in this instance all chromosomes are affected similarly, the reduction in size being due probably to the cells having been in slightly different stages when killed. This does not affect the relative lengths of the chromosomes, however. The average (doubled) length of the larger (no. 4) diad (figs. 5-8) in the same cells is 15.5 mm. The ratio therefore of the No. 3 tetrad in these cells to the larger diad of the abnormal No. 4 tetrad, or to a No. 4 tetrad made up of two such diads, would be as 1 to 1.18; i.e., the ratio of the No. 3 chromosomes to the normal member of the No. 4 pair here is as 1 to 1.18. This is not far from the normal ratio, 1.14, and the difference is quite within the range of the probable error due to inaccurate measurement, etc. But the average (doubled) length of the smaller diad of the abnormal No. 4 tetrad is 12.7 mm., instead of 15.5 mm. The ratio therefore of the No. 3 tetrad to a tetrad made up of two such No. 4 diads would be as 1 to 0.97; i.e., the ratio of the


112 W. R. B. ROBERTSON

No. 3 chromosomes to the smaller No. 4 chromosome is as 1 to 0.97. This shows the smaller No. 4 diad to be even smaller than the No. 3 chromosomes. I believe that the larger diad of this abnormal No. 4 tetrad is the normal No. 4 chromosome, since its ratio, 1.18, is so near to the normal ratio, 1.14, and that the smaller diad is abnormal and has lost a part of its distal end ; (my reason for thinking the distal end deficient will appear later) . Its ratio, 0.97, instead of 1.14, gives it a shortage of 0.17, or nearly one-sixth of 1.14. It seems therefore to have lost one-sixth of its normal length.

The size of this deficient No. 4 chromosome is constant for this individual. All division figures which occurred in lateral view showed the members of this unequal pair in practically the same relative sizes. Not only the germ cells but also somatic cells (fat body) exhibited the same proportions. The fact that the size ratio is constant in all germ cells and even in body cells seems to point to its germinal origin; i.e., it must have been present in the fertilized egg from which this animal developed.

There is no constant relation between the members of this unequal pair of chromosomes and the sex chromosome, as the cells of the maturation division show (figs. 5, 6, 8), for the sex chromosome passes as often to the pole which receives the small No. 4 chromosome as to the pole which receives the large member.

Another very noticeable evidence of the abnormality and defectiveness of this No. 4 chromosome is shown in its manner of contact with its larger normal mate in the first maturation spindle (figs. 5, 6, 7 c). In these three figures its attachment is not strictly terminal, as it should be. This, it seems to me, is evidence of a deficiency at the distal end of this chromosome. This will be better understood if I describe briefly the method of chromosome pairing in the Tettigidae (Robertson '15). During the period of synapsis, which occurs in the early part of the growth period of the first spermatocyte, homologous chromosomes pair side to side (parasynapsis) , as shown in figures Ai, A2 (pi. 3) . After this period of synapsis, during the growth period which follows, a separation of the pairing chromosomes takes


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 113

place. This separation begins at the proximal ends of the pair and' gradually moves along toward the distal ends (figs. A3 to A5) . By the proximal end I mean that from which the spindle fiber springs and that which travels in advance toward the pole. The opposite, blunt end is the distal end. The proximal ends of the pair diverge, each through an angle of 90°; i.e., until they are 180° apart (figs. A3 to A5). The proximal ends now point in opposite directions, and the pair, thus attached at their distal ends, form a rod. In this condition the pair (tetrad) enters the first maturation spindle (fig. A5).

The defective tetrad, like the others, has gone through this process. But a portion of the distal end of one of the chromosomes being gone, the chromosome cannot conjugate properly with its mate and so has a tendency to slip to one side when the tetrad reaches this stage, as figures 5 to 7, and E2 to E5 show. The normal conjugant (diad) finds at its distal end no corresponding portion in the defective mate with which to pair. The chromosomes were probably paired normally at the proximal ends and in all parts which are present, leaving the distal part of the larger normal member extending beyond the shortened distal end of the defective mate (fig. Eo) . In 'the process of separation which follows conjugation the defective chromosome (diad) had its shortened distal end rotating on the side of the distal end of the longer, normal mate. When the diads had rotated apart 180° this shortened end was as a result out of line with the end of its longer mate (figs. E2 to E5). This is the condition in which the defective chromosome of figures 5, 6 and 7 c is seen.

2. AN UNEQUAL PAIR OF ANOTHER TYPE OCCURRING IN ACRIDIUM GRANULATUS

While working out the chromosomes in dividing ovarian follicle cells of the female individuals of Acridium granulatus, I found one animal which showed among its 14 chromosomes five long members (fig. 11) instead of four (the two 6's and the two 7's). This puzzled me in my pairing of the chromosomes. The material was laid aside for the time. A few weeks later I found a male individual which showed in its first maturation divisions


114 W. R. B. ROBERTSON

the same five long chromosomes. My problem was solved at once when I saw that this extra long chromosome paired in maturation with one of the ordinary chromosomes of the complex, the No. 1 (figs. 12-13).

This unequal pair appears to be of a type different from the deficient tetrad just described, and so must be treated separately. It is of primary importance in its bearing on questions of synapsis and of maturation division. Only two animals having it were found, a male and a female. These, and most others of this species worked upon, were collected from a spot 75 feet square near Waverly, Massachusetts, and are probably related individuals.

Figures 9 and 10 show cells from normal individuals exhibiting the typical condition of the chromosomes in Acridium granulatus. There are two long pairs (6's and 7's), two intermediate pairs (5's and 4's), two short pairs (I's and 3's), and the sex chromosome, which in size ranks No. 2 in this genus. A small, faintly staining, fragmentary body is shown (dotted) in figure 9. It is not present in all cells, and is probably a nucleolar structm-e of some sort. The normal chromosomes paired for the first maturation division may be seen in figure 10. Their size relations are clearly shown there: two large pairs (6, 7), two intermediate pairs (4, 5) and two small pairs (1, 3), as well as the small 'accessory' chromosome (2).

Figures 11 to 13 are of the abnormal male and female. In figure 11 (female) the five, instead of four, long chromosomes are clearly shown. The fifth long chromosome (1) appears odd because it has no equal chromosome with which to pair. The first maturation division in the male (figs. 12-13), however, shows it pairing with the small (no. 1) chromosome. In the male cells, moreover, it shows a constriction at a region about as far from its distal end as the length of the small (no. 1) chromosome with which it is paired.

The other chromosomes are quite normal in these animals (figs. 11-13). In the female (fig. 11) there are the two long pairs (7's and 6's), the two intermediate pairs (5's and 4's), the short pair (3's), the 2's (which are the sex chromosomes and are


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 115

paired in the female), one normal No. 1 chromosome, and the abnormal (long) No. 1. In the male cells (figs. 12-13) there are two large pairs (7 and 6), two intermediate pairs (5 and 4), one small pair (3), and the sex chromosome (2) unpaired; the other small tetrad is represented by one small No. 1 chromosome paired with the abnormal long chromosome. It is evident from these two figures that this long structure is connected with one of the small (No. 1) chromosomes.

The long abnormal diad bears no constant relation to the sex chromosome in its distribution to the second spermatocyte cells, as figures 12 and 13 show. A large number of dividing cells examined showed that in the reduction division it passed as often to the pole which did not receive the sex chromosome as to that which did. I feel certain, therefore, that this chromosome bears no relation to the sex-determining chromosome.

2a. Discussion

What is the origin of the long chromosome?

The partially constricted off distal portion of this long body (figs. 12-13) is probably a No. 1 chromosome and in synapsis it was possibly paired side to side with the opposite normal No. 1, leaving the remainder of the long chromosome, the proximal end, to extend beyond it (figs. Bi and B2). In the Tettigidae, as I have said above, side-to-side pairing in synapsis occurs during the bouquet stage. Following this period the chromosomes of a pair separate from each other, beginning at the proximal ends. Remaining in contact at the distal ends, they continue to rotate apart until the pair appears upon the first maturation spindle as a long rod frequently constricted in the middle (figs. 10, 12, 13 and figs. Ai to A5). That is exactly what has occurred in the case of this abnormal tetrad, but to the end of one of the No. 1 members is attached this extra 1^ portion, which increases the length of this member to two and one-half times its normal dimension (figs. Bi to B4). Unfortunately no satisfactory prophase stages are present to show the behavior of this element in synapsis, but, judging from the constriction


116 W. R. B. ROBERTSON

at one end and knowing the behavior of the unequal tetrads of other individuals (figs, 4-8), I believe that here we probably have the result of an unequal division of some preceding generation where a No. 1 tetrad failed to divide at the proper place, in the middle between the two members, but instead so divided as to give a ^ portion of a No. 1 pair to one pole and a halfportion to the other pole (Ci and C2). As a result of this, it might be imagined that there would be some generations of individuals with a sesqui-valent (l|-valent) No. 1 chromosome present pairing with the normal No. 1 (figs. D], D2). The normal No. 1 might pair with the normal part, or with the fragment. In order to get a 2^-valent No. 1 chromosome the parts of which are largely oriented in one direction it will now be necessary to suppose that, at some reduction division of the germ cells in succeeding generations of animals, a normal No. 1 chromosome became attached during the synapsis period to this sesqui-valent No. 1 chromosome. It might do this by becoming fused with, or by failing to separate from, the fragmentary (cut off) end of the sesqui-valent member after some method as is indicated in figures Di to De, giving as a result a 2|-valent chromosome (fig. De), such as we have in figures 10 to 13. All portions of this chromosome, except the sesqui-valent fragment in the middle, would then be oriented in one direction (see arrows in figs. Ci, Co, and Di to De). My reason for thinking that at least the terminal parts, which make up the greater part of the long chromosome are oriented in the same direction, is based upon the fact that I always see the chromosome arranged on the spindle in perfectly normal position, pointing directly toward the pole to which it is about to go, and upon the fact that the mantle fiber of this chromosome always springs from the longer (1^) portion of the chromosome (figs. 12, 13, and Bi to B5). The chromosome always travels with the larger, sesqui-valent portion in advance (figs. 12, 13, B4 and B5).

In this animal evidently the normal No. 1 chromosome paired during synapsis with the distal No. 1 portion of the 2^-valent chromosome (figs. 12, 13, and Bi to B5). It should pair also


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 117

with the sesqui-valent portion, but I have seen no evidence that this occurs. Possibly the sesqui-valent portion has become so non-functional that it fails properly to attract the normal No, 1 chromosome in the synapsis period.

This explanation is the best I can propose, in the light I now have, to account for the origin of this long chromosome. The facts are that it is approximately two and one-half times the length of a normal No. 1 chromosome, all or most of its parts are evidently oriented in one direction, and a constriction near the distal end marks off a portion which evidently pairs in synapsis in the normal manner with the No. 1 chromosome and separates from this chromosome in the normal manner in the first maturation division (figs. 12, 13, and Bi to B5).

An essential fact is that we have here cases of an abnormally large chromosome, which is constant in size in individuals of both sexes. Its relative size is the same in all the dividing germ cells found in the male, and hkewise in the somatic cells (follicle) of the female. I believe that it is a permanent structure, so far as these two individual animals are concerned.

A second important fact is that this abnormally large chromosome alternates with a normal chromosome and it may or may not be present in either sex. Theoretically, we may have, depending on the presence or absence of this chromosome, three sorts of male individuals and three sorts of female individuals; those containing two normal No. 1 chromosomes, those containing one normal and one abnormal No. 1 chromosome and those containing two abnormal No. I's. I have found the first two cases in both sexes. The latter case has not yet been found in either sex. We have therefore a basis for a Mendelian ratio. The presence of one long and one short chromosome might be considered the cell condition of the heterozygote, or hybrid; the presence of two short chromosomes or of two long ones would give the homozygotes, recessive and dominant, if such relations may be imagined in so far as these chromosomes are concerned.


118 W. R. B. ROBERTSON

3. ON SYNAPSIS AND REDUCTION

In their behavior these unequal tetrads throw a good deal of hght on the nature of the synapsis of chromosomes and their separation from each other in the reduction division. In regard to the beginning of this process, the individuals furnishing these abnormal chromosomes do not have much to show, as I have said above. This beginning, however, I determined from normal spermatogenesis, and have worked it out in my second paper.

I have found the pairing process in the Tettigidae to start as a parasynapsis and to end in a telosynapsis (end-to-end pairing). In the synizesis period following the last spermatogonial division, the members of each pair of chromosomes, six pairs in all, having assumed the thread condition, are seen to arrange themselves in side-by-side fashion. The number of threads is at first twelve, in addition to the accessory chromosome. The twelve threads are seen to become six double and finally six single threads. During this period the sex chromosome usually lies at one side of the nucleus. This condition of pairing continues into the later growth period, when the members of each pair move apart, the separation beginning first at the proximal ends (figs. Ai to A4), the chromosomes of a pair remaining attached to each other at the distal ends (figs. A4, A5). Each rotates through 90° and the two then form a rod (fig. A5), which may show, by a slight constriction, the point of contact of the two members. In this condition the paired chromosomes (tetrads) enter upon the first maturation division.

In the behavior of these unequal pairs I feel perfectly certain that the same thing must have- taken place, for T see no reason to think that these abnormal pairs should behave differently from the normal chromosomes during this process. Their behavior on the maturation spindle and in the anaphase following is in every way similar to that of the normal chromosomes, and we have every reason to believe that a parasynapsis ending in a telosynapsis has taken place. The unequal pairs appear on the maturation spindle in the telosj^napsis condition.

Attention, first of all, should be called to the permanency of size of these abnormal pairs, especially of the abnormal members


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 119

of each paii-. In the first type of inequality which I described (figs. 4-8), all dividing cells examined showed the members of the No. 4 pair unequal and constantly of the two respective sizes. The normal No. 4 chromosome was always of the size normal for No. 4 chromosomes and its deficient (No. 4) mate was hkewise of a constant size but uniformly one-sixth less than the normal No. 4 in all cells, both somatic and germinal. The constancy of this relative inequality was very clearly shown, especially in those figures of the first spermatocyte division which appeared in lateral view (figs. 5-8) . A similar uniformity of sizes I found in the unequal pair of the second type (figs. 12, 13) in both male and female animals. Abundant evidence of the relative sizes was given, especially in the tetrads of the first spermatocyte divisions. Every cell showed the No. 1 portion of the abnormal tetrad to be of the normal No. 1 size (figs. 10-13) and the longer 2|-valent portion to be likewise of a constant size. It was not only of uniform size in all the cells of the male individual, both somatic and germ cells, but in another individual, a female, it was also found to be of the same length (fig. 11).

These facts indicate that in the latter case this abnormally long chromosome must have been handed on as such, not only through many generations of cell division, but also through many generations of individual animals. In the former case the deficient No. 4 chromosome — which was of practically the same size, not only in a large number of spermatocyte cells, the germ cells, but also in the body cells — indicates again that such an abnormal chromosome maintains its identity from generation to generation, from fertilized egg to fertilized egg.

The permanency of these abnormal chromosomes has an important bearing upon one problem of synapsis; upon the question as to whether or not a complete side-to-side fusion of homologous chromosomes takes place during this period. I have attempted to illustrate my ideas by diagrams (figs. Fi to F5, Gi to Ge, and Hi to He). If we suppose that a fusion of the chromosomes takes place, we should expect to find in the case of the unequal No. 4 pair something like figures Fi to F3 occurring. The shortened No. 4, pairing with that portion of the normal


120 W. R. B. ROBERTSON

No, 4 to which it corresponds, or with which it is homologous, and leaving the distal end of the normal No. 4 projecting beyond (fig. Fi), would, on fusion with it, form a single cylindrical body having one end, the distal, smaller in diameter than the remainder of the cylinder. If a splitting occur at the end of synapsis in a plane formed regardless of the old plane of fusion, having merely for its object the splitting of this single fused body into symmetrical halves, there would result two daughter chromosomes of equal size but with smaller diameter at the distal end than throughout the remainder of the chromosome (figs. Fs to F5). There would then be no such uniform inequality of the No. 4 chromosomes preserved in the first spermatocyte divisions of this animal as we have found. ' Instead of having a normal sized 'No. 4 and a (say) five-sixth-sized No. 4 in every cell after the first maturation division, therp would be two No. 4 chromosomes of equal length but shorter than the normal (fig. F5). On the contrary every first spermatocyte metaphase and anaphase (figs. 5-7) shows one normal sized No. 4 chromosome of the same fixed relative size and likewise one deficient No. 4 chromosome of a definitely fixed relative size.

If we turn to the abnormally long tetrad (figs. 12, 13), we have even more definite proof that fusion longitudinally and splitting along a new plane in all probability does not occur. The normal No. 1 chromosome evidently has paired with the distal portion of the long No. 1 chromosome in a manner similar to the diagram shown in figures Bi and B2. If in such pairing it fuse completely with its longer mate, its would form a cylinder again, with one end of larger diameter, the opposite of smaller diameter (figs. Gi to G4). If, on the splitting of the fused thread, the new split be formed, not along the old plane or fusion, but upon any plane giving two equal daughter threads, we would get two long chromosomes of the same length with diameters large at the distal end instead of a long chromosome and short chromosome, of the same relative lengths and equal diameter, with which we started. Again, if the new split appeared in the fused chromosome in a plane at right angles to that of fusion (figs. Hi to He), we should


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 121,

get a similar result, which is also contrary to the facts in the case. The chromosomes, when they come out of the pairing process, are evidently of the same size they were on entering the process. The abnormal sized 2^-valent No. 1 chromosome is of the same size in all the germ cells after the process as in the germ cells of the same individual before the process, and even in the body cells of another individual (fig. 11). The same may be said of its normal mate. No tags or projections were seen in any case to indicate that a new longitudinal plane of division had occurred.

In both types of unequal tetrads we have very strong evidence that homologous chromosomes, on entering the side-to-side pairing process of synapsis, remain as distinct individuals, retain their identity throughout the period and come out of it with at least the same size they had on entering it. Each pairing chromosome maintains its distinct individuality during this period.

This is opposed to the ideas of Jannsens ('09) and Morgan ('11) as expressed in the theory of the 'chiasma type.' In their theory they assume that homologous chromosomes in parasynapsis twist about each other and fuse. On splitting, a plane passes down the fused body, regardless of the previous spiral fusionplane, resulting in two daughter chromosomes which may not be identical with the two chromosomes which entered the process. Each new one may contain parts of both original chromosomes. If such had been the case, the separation or formation of a short and a long chromosome out of the fused chromosome (Bo to B5), with such regularity of size, etc., as we have shown, could not have occurred. Or, supposing that spiral twisting and fusion had occurred and that splitting again was limited to the larger end of the fused chromosome (figs. G3 to G4), we should expect that the shorter member resulting (figs. B2 to B5) would at least show fragments of the side of the long chromosome attached to its proximal point in those cases where the splitting plane did not coincide with the old plane of fusion between the short and long chromosomes. No such attached fragments were found. All short or normal No. 1 chromosomes in the metaphase and anaphase of the first spermatocyte divisions were of uniform length


122 W. R. B. ROBERTSON

and showed that they retained the same size and were as free from terminally attached fragments as they were on entering the pairing process.

In regard to the question of pre- and post-reduction, I have evidence here that the first maturation division is the reduction division so far as regards three of the seven pairs of chromosomes of the Tettigidae subfamily of grasshoppers. These pairs are the Ts, the 4's and the sex chromosomes. The abnormally long No. 1 chromosome is seen in Tettigidea separating from its normal mate (figs. 12, 13), the deficient No. 4 is seen separating from its normal mate (figs. 5-8), and the unpaired sex chromosome (paired in the female, figs. 1, 11) is seen passing over whole to one of the poles in the male cells in smiilar manner, just as if it had had a mate from which to part at this division, where such parting is being carried out by the members of the ordinary chromosome pairs (figs. 2, 3, 5, 6, 8, 10, 12, 13). The other chromosome pairs (the 2's, 3's, 6's, and 7's) behave similarly to the I's and 4's in every respect during the synapsis, prophase, metaphase, and anaphase periods of the first spermatocyte divisions. This leads one to think that probably the first spermatocyte division is the reduction division for all pairs of chromosomes, in the Tettigidae subfamily of grasshoppers at least.

4. THE DEFICIENT NO. 4 CHROMOSOME AND THE LOSS OF UNIT CHARACTERS

The most important of these unequal tetrads are those in which one member of the pair is of less than the normal size (figs. 4-8) . The importance lies in this, that such a deficiency may be the result of the dropping of a' part of a chromosome and in this way may furnish the basis in the germ cell for the loss of unit factors in heredity.

In animal and plant breeding there are many unit characters which might be considered to have resulted from the loss of something from the germ plasm, whether this something be an enzyme of some sort or a substance upon which an enzyme might act.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 123

It has been shown by Castle ('11) and others that in mice, rabbits, and guinea-pigs the ordinary gray color of the wild rodent is not a simple character, but on the contrary, is very complex. It depends — for example, in mice — upon the simultaneous presence of at least seven or eight color factors. The complete dropping of any one of these from the germ plasm modifies the color of the animal.

The factor most commonly dropped is that for the production of color, the animal — whether potentially gray, black, or brown, etc. — being an albino, actually without color. The next factor very commonly dropjDed is that for barring of the fur. This does not show unless black or brown pigment is present in the hair. The barring of the hair is due to the fact that some factor prevents the development of black and brown pigment granules in a jDortion of the individual hair immediately below the tip, leaving a black tip and a dark base with a light yellow band between them. When this barring factor is dropped, we get a black or a brown animal instead of a gray. The next most commonly dropped factor is that for the production of black, giving cinnainon or brown animals. By dropping the factor for self color, spotting results, spotting of two sorts, white upon ai^olored coat, or yellow upon a black, brown, or gray coat,^giving in the extreme cases white with black or brown eyes ^nd yellows with ])lack or brown eyes. By dropping the dark-^ed character we obtain pink-eyed animals with a scarcity of pigment in the fur. By dropping the intensity-of-pigmentation factor we get diluteness of pigment, giving dilute-pigmented gray, black ('blue'), brown ('cream'), or yellow animals. When all of these factors are present we have the wild gray type. By the dropping of any one of them entirely the color may be modified accordingly.

It is evident that these animals lack something. The dropping of the black pigment, for example, gives animals which are cinnamon or brown. No black can be produced in the race until this factor is again added to the mixture. It is entirely absent. In this case black cannot be considered a latent character which has been restricted in some way from attaining its development, as is the case in albinos of gray or black animals;

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 1


124 W. R. B. ROBERTSON

these on being crossed with brown, or any animal that contains the color factor, will give gray or black, etc., showing the gray or black characters to have been present in substance though not developed. If the black be absent, nothing can restore it except the addition of black again. There is evidently a complete absence of the black pigment material from the fur of the animal.

In the varieties of domestic plants many similar cases may be given. But before mentioning the color varieties I wish to call attention to the cross between the 'cupid' and the 'bush' variety of sweet pea as one of the best examples. The cupid is a dwarf plant whose internodes are very short, making the stem correspondingly short, about 9 to 10 inches. It has the prostrate habit, that of lying on the ground, due to the diverging habit of the branches. The 'bush' variety produces branches which do not diverge but grow upright, making a tall bush growing 42 to 48 inches in height. These varieties, on being crossed, produce in the Fi generation plants which show a reversion to the habit and size of the wild sweet pea of Sicily (Punnett '11). They have the long internodes and the long stem of the bush variety combined with the prone, prostrate habit of the cupid variety. By inbreeding the Fi individuals the Fo show the tall 'bush' variety, the tall procumbent, the short procumbent or original 'cupid,' and the short 'cupid' bush-like variety. The factors concerned are the long internodes versus the short, and the procumbent versus the erect habit. The two varieties evidently owe their origin to the dropping of one or the other of these factors from the germ plasm of the wild type. The bringing of them together in the hybrid supplies the lack in both races (the 'cupid' and the 'bush' varieties), the result being the wild type.

In flower color of sweet peas we have a >similar case. Most white sweet peas breed true to white, but there are two varieties of white which on being crossed produce a purple colored variety, like the wild Sicilian species. On being inbred, the F2 generation shows nine of the colored variety and seven of the white. Of the whites some breed true, some give a 3.1 ratio, i.e., three whites


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 125

to one colored, while some give all colored again. This shows that there are two factors concerned. The presence of both is needed to produce the colored flowers, the absence of either one giving white. It is the r2 result of an ordinary dihybrid Mendelian ratio, in which there are nine cases where both factors are present and colored results, three cases where only one factor is present and white results, three cases where the other factor is present and w^hite results, and one case where neither factor is present and a white which breeds true results.

The colored individuals in the F2 generation are found to belong to six classes depending upon the presence or absence of a purple factor, a light-wing factor giving a bi-colored flower, and a factor for intensity of pigmentation, in the absence of which a diiutely colored flower results. The wild type is intensely colored, and bi-colored, i.e., purple with blue wings. By dropping the purple a red bi-colored or uniformly colored flower is obtained in both the dilute and intense series, by dropping the bi-color factor uniformly purple or red are obtained, by dropping the intensity factor both varieties of the red are obtained in dilute form, and, finally, by dropping either the color-producing base or the color developer we get any of these color varieties in the albino form. The wild purple Sicilian species contains all these factors. By the dropping of these factors one by one and by inbreeding, all the color varieties of our domestic sweet peas have been obtained.

Now, it seems to me that it would be quite possible to account for phenomena of this kind in plants and animals as the result of an unequal division of the chromosomes in the reduction division, similar to what I have found evidence of in Tettigidea parvipennis. If but one member of the pair of chromosomes showed the deficiency it might not give a result in the organism. When both chromosomes of the pair show such a deficiency, a condition w^hich would result only when inbreeding occurs, then such a deficiency might be shown in the somaplasm by some such defect as albinism, blackness, dwarfishness, etc. It is true that such a chromosome would evidently be an abnormality of a deficient kind, but are not all such traits as albinism in plants


126 W. R. B. ROBERTSON

and animals, melanism in rodents, retarded mental development, feeblemindedness in man, dwarfisms, etc., abnormal deviations from the type of the species which may be classed as deficient characters?

The amount which may be dropped from one chromosome, such as I have shown in Tettigidea, may be considered too much to allow of the existence of the organism in the homozygous condition, i.e., where both members of the pair lack it. It is conceivable that a limit in the amount that may be absent might occur and that, going beyond that limit, the homozygous condition might be lethal, as in yellow mice. Here the homozygote always dies, or may never be formed, as has been shown by the results obtained from breeding yellows together. The litters are three-quarters the normal size and the young are found to be two-thirds yellow and one-third gray or other colors, never breeding true to yellow. This indicates that the yellow parents were heterozygotes and that the one-fourth pure yellows, which we would expect in F2, have never been formed. Baur ('07) found, on crossing two varieties of snapdragon — the green leaved with the golden leaved — that the offspring were 50 per cent green and 50 per cent golden leaved. The greens bred true but the golden variety produced 25 per cent greens, 50 per cent golden and 25 per cent that were almost white. The latter died at the end of germination, when the food in the seed was used up, since they possessed no chlorophyll. The golden variety was variegated with green patches (chlorophyll-containing cells) and so could manufacture its own starch}^ food.

The yellow condition in mice and in the snapdragon may be due to a greatly deficient chromosome, so greatly deficient that a zygote having two such deficient chromosomes might have too great a deficiency to be able to develop, or, after having developed, may lack some substance, such as the chlorophyll in the snapdragon, necessary for carrying on one of the vital life processes. In addition, this lack might be so great that it would over-ride the normal chromosome in the heterozygous zygote, giving a yellow mouse instead of a gray, black, or brown, as the case may be; or in the snapdragon a golden variety instead of a green


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 127

variety. That something of this nature has occurred, we are inclined to believe from the fact that the golden variety is simply a plant which lacks chlorophyll in small variegated patches over its surface, whose color is therefore due to this condition. In the cases here given possibly there is a law at work correlating the amount of material that may be lacking from a chromosome with the recessiveness or dominance of the trait resulting in the organism. A defective chromosome may continue to give a trait which is recessive to the normal condition until the defectiveness of the chromosome reaches that point where its deficiency becomes so great that the homozygous zygote cannot develop. At that point the defect becomes dominant to the normal condition and individuals can exist only in the heterozygous or normal condition.

It is strange that yellow should be dominant in mice while in most other species of domestic animals it is recessive. This may be due to the position of the yellow determinant along the chromosome. In most species it may be thought of as lying near the end of the chromosome and accordingly could be dropped very easily, causing little disturbance and giving a recessive trait. In the mouse it may be conceived of as lying farther from the end of the chromosome. On dropping enough of the chromosome to cause yellow, a greater disturbance would be created and the defective trait resulting would accordingly be dominant to the normal trait. I make this as a suggestion merely.

The chances for such abnormal divisions are limited by the number of pairs of chromosomes in the species and by the varying amounts which may be dropped from each chromosome in each case. In guinea-pigs the number of pairs is twentyeight (Stevens '11). The chances for abnormal divisions in guinea-pigs are therefore large. Where the number of chromosomes is small the chances are smaller. The amount that may be cut off from each individual chromosome might vary enough to give several varieties due to the variation in this respect in one pair of chromosomes. The same might be said of each of the twenty-eight pairs in guinea-pigs. Any of these varying conditions in a single pair of chromosomes might combine with


128 W. R. B. ROBERTSON

all possible combinations of similar variations in the twentyseven other pairs. Such might be the basis of all of our variations in guinea-pig inheritance.

It is noticeable in any animal or plant which becomes domesticated that very soon there appear whites, blacks, browns, spotted, yellows and others of the color varieties common to domesticated species. The same may be said of other characters of the species, size for instance. On domestication inbreeding occurs. This gives rise to homozygous strains, which maj^ be isolated. In the wild state inbreeding is not so prevalent. Promiscuous mixing occurs. A summing up of the characters results and all normals, which are usually dominant, are present and show. The weaker recessive characters, if present, are covered up. They exist in the single or heterozygous condition and so do not show. If they exist in the homozygous condition they may show, as in albinos, but the organisms may be killed off by natural selection. Under domestication man preserves these recessives.

The loss of parts of chromosomes may explain very easily the appearance of such phenomena in the domestication of species. The fact that there appear always the same or nearly the same color varieties in each species may be due to this, that theiichromosomes are more or less similarly organized, that there are approximately the same number and in many respects the same individual chromosomes to be dealt with in each species, and finally, that these chromosome pairs are subject to the same vicissitudes of fortune in division at the maturation period in each species. True, there are minor variations, in chromosomes as we pass from species to species. There are also minor variations in color inheritance as we pass from species to species. But these variations are small when we think of the similarities. All species of rodents show grays, all show albinos, all show blacks, all show some form of brown, and yellow^s. The method of inheritance of the yellows, for instance, might vary from species to species, but they are yellow, nevertheless.

The same spontaneous variations, such as albinism, probably occur not once but over and over again in the same species in various parts of the world entirely independently of each other.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 129

If the basis of albinism lie in an abnormal reduction division of a certain pair of chromosomes, we should expect it to do just that sort of thing. So long as the same number of pairs of chromosomes occurs in a species, so long will the same variations continue to occur. In this way we shall continue to have white animals produced anew by 'mutation' — blacks, yellows, spotted, and all the varieties not only of color but in respect to other properties of the body as well. In the same way we might always expect to have produced a certain percentage of defective human beings, such as the classes of feeble-minded, imbeciles, epileptics, etc., each of which seems to be due, in many cases at least, to something lacking in the germ plasm (Davenport '11). Germinal variations of this kind, it seems to me, might be at the basis of De Vries' 'eversporting varieties,' which gave such abnormalities as striped flowers, five-leaved clovers and monstrosities of various sorts, such as pistoUody, twisted and flattened stems, etc. Again, it seems possible that germinal variations of this sort might lie at the basis of those of De Vries' mutations which he distinguished as retrogressive in character; i.e., which were characterized by the dropping out of some character from the parent species. They might lie at the basis of some mutants which he considered progressive, but which showed some retrogressive traits, such as the brittleness of the stem in Oenothera rubrinervis. My reasons for supposing this are as follows: De Vries found his 'eversporting varieties' producing their abnormal individuals continually. He found his parent species, Oenothera lamarckiana, continually throwing off the same mutants in certain proportions. No single parent plant proved ever to be wholly destitute of mutability." In his parent species, lamarckiana, he has probably a constant fundamental number of chromosomes to deal with. He has a reduction division taking place every time a germ cell is formed. He has the same possibility of abnormal, unequal, divisions of tetrads at the time of this division, giving a deficient homologous chromosome. He has self fertilization (inbreeding), which would tend to bring such defective chromosomes together. He has frequent cases of sterility in the inbred offspring of his mutants,.


130 W. R. B. ROBERTSON

as one would expect in instances where a vital factor had been dropped. He has mutations which, with one or possibly two exceptions, are of a retrogressive nature; i.e., lacking something necessary which was present in the parent spe.^ies. The gigas variety was due evidently to a doubling of the number of chromosomes (Gates '09). It seemed to lack nothing, but the other mutants seemed all to lack traits, some more useful, some less useful, which were present in the parent species. These phenomena, it seems to me, point to something for their basis like the abnormal variations in reduction divisions, such as I have described in Tettigidea parvipennis.

It is interesting to compare the number of mutations De Vries obtained from lamarckiana with the number of chromosomes. His number of chromosomes was 14, seven pairs. He obtained seven mutations from his plants. One of these, gigas, was evidently due to a doubling of the number of chromosomes. It, however, was not a defective mutant, and so may be left out of account here. The other mutants seemed to have something lacking, and there were six of them. Of these scintillans seems to have been heterozygous, producing on inbreeding, lamarckiana and scintillans. It also produced, in a small percentage, some of the mutants most frequently produced by lamarckiana. Possibly scintillans had one over-deficient chromosome, such as I have postulated for yellow mice or the golden snapdragon. Many sterile pollen grains were found. Possibly the cause of sterility lies here. On this hypothesis, the fact that scintillans can produce oblonga, lata, and nannella is not surprising. The other chromosome pairs are just as liable to accidents in germ cells of scintillans as of lamarckiana. Lamarckianas may be produced by scintillans; why may not the mutants of lamarckiana be produced also? Each of the other five mutations might be based respectively upon deficiencies in one of the remaining five pairs of chromosomes. Since these remaining mutants breed true in each case, it would be supposable that, in order to show, they must be in the homozygous condition. Thus we may possibly account for the five remaining mutants. This hypothesis, it seems to me, is worthy of consideration here. I


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 131

see many reasons to suppose that De Vries was dealing, in part of his mutants at least, with something similar to what T have described in this paper as deficient homologous chromosomes.

Deficient chromosomes, such as I have found paired with their normal sized mates in Tettigidea parvipennis, it seems to me, furnish a sufficient explanation for the loss of unit factors from the germ plasm. Looked at in the light we now have of the behavior of unit characters which belong to this 'loss' group, the hypothesis appears very probable. It seems to me that Professor Morgan and his students, who are working upon Drosophila, should also take into consideration the possibility of such deficient chromosomes. It is a good working hypothesis, and I am going ahead with breeding experiments upon this species, Tettigidea parvipennis, in the hope of getting some results. As to what connections there may be between these deficient chromosomes and the greater problem — the origin of new characters — it is difficult to imagine. This matter had better be left until we have more knowledge of the behavior of these chromosomes.

The observational work upon which this paper is based was done at Harvard University under the direction of Prof. E. L. Mark. The writing was completed at Kansas University. I wish to express here my gratitude to Dr. Mark for the help and criticism he has so kindly given to me from time to time.


132 W. II. B. ROBERTSON

ADDENDUM

After this paper was worked out, a treatise by Miss Carothers ('13) appeared, describing unequal tetrads in three species of the 23-chromosome grasshoppers, Brachystola magna, Arphia simplex, and Dissosteira Carolina. I wish to consider her paper briefly here.

In the twenty specimens of the three species examined she finds the members of one of the three pairs of small chromosomes always unequal in size. The unequal pair occurs in spermatogonial, in first spermatocyte and (separated) in second spermatocyte cells. The members of the unequal pair agree with those I have found, in that they become separated from each other during the first and not during the second maturation division. This is evidence again that the first maturation division is the reducing division. In their passage to the second spermatocytes these unequal meml^ers (diads) agree with those of my material in that they are distributed to these cells irrespective of the presence or absence of the sex chromosome. In this respect our unequal pairs differ from those of Gryllotalpa, described by Payne ('12), where the longer chromosome was always accompanied b}^ the sex chromosome in the anaphase of the reduction division. Payne may have been dealing with a group of sex chromosomes similar to what he has already worked out in another order of insects ('09).

Miss Carothers' material differs from mine, however, in that she finds the unequal pair present in every animal. I cannot agree with her in this respect, in Acridium or Tettigidea, as my drawings have shown. Possibly further search will show that the unequal pair is not always present in the species upon which she has worked. If this should be the case, the hypothesis of selective fertilization which she advances would be unnecessary. So far as Tettigidea and Acridium are concerned, it is not necessary to postulate selective fertilization.

In thinking that the great number of combinations of chromosomes possible is sufficient to account for all variations, I fear Miss Carothers may be mistaken, (iates ('09) has shown that in


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 133

Pisum the number of pairs of chromosomes (seven) is not large enough to account for the number of pairs of allelomorphic characters which beha^'e independently of each other in breeding experiments, if we assume that the basis of each member of one allelomorphic pair must be permanently located in one member of a single pair of chromosomes:

In Pisum eleven or more pairs of allelomorphs have been observed and the reduced number of chromosomes is only seven; which shows that in this case at least, several characters must reside in one chromosome. The characters must then be confined to separate particles or corpuscles of the chromosomes, and an interchange of homologous particles according to chance during maturation would give the Mendelian combinations.

I am not quite willing to believe that the basis of an allelomorph may slip from one chromosome to another. Yet it is very evident, so far as I can see, that the number of chromosome pairs behaving independently of each other is too small to allow them to be the basis for the number of allelomorphic pairs of characters behaving likewise independently of each other. Possibly some of these extra pairs of allelomorphs may be accounted for by the deficient chromosome hypothesis which I have advanced, or possibly by the 'chiasma' theory of Morgan, though I have evidence against the latter in these unequal chromosomes and in the V-chaped chromosomes of Chorthippus and Jamaicana (Robertson '15).

Additional instances of unequal chromosomes, so far as I have been able to find in the literature, have been reported by Baumgartner (Science '11), Hartman ('13), and, I have been informed, by Voinov ('12). The last mentioned paper is published in a European journal to which I have been unable to get access. Baumgartner reported an unequal pair in Gryllotalpa, but he gave no drawings and no description. Since that time, Payne ('12) has shown that the unequal pair in Gryllotalpa is related to the sex chromosome, the larger member of the pair going with it in the reduction division.

A short time ago Mr. F. A. Hartman called my attention to the fact that he had already described unequal divisions of some


134 W. R. B. ROBERTSON

of the small chromosome tetrads in the first spermatocyte cells of Schistocerca in his paper (March '13) on Variations in the size of chromosomes. " His paper deals chiefly with variations in size of chromosomes due to, what he believes to be, their unequal growth in the cell. Thinking that the whole paper was devoted to 'variation due to unequal growth,' I overlooked the latter part in which he illustrates and describes briefly a few cases of what he considers unequal division in the first spermatocyte. One of these cases (his fig. 86) may possibly be due to faulty conditions of sectioning. The other cases (his figs. 83, 85 and 87 to 91) are probably variations similar to those Carothers has since described in Brachystola, Arphia, etc. That "size variations may be due to unequal growth" I am inclined to doubt, but Hartman is to be given credit for recognizing the importance of variation in chromosome size in its relation to 'variation' in 'animals,' since his work was done while teaching in a high school away from any university contact and especially since he was entirely ignorant of the Mendelian laws and their relation to variation. Had he known of these and the related work, he would likely have come to the same conclusions that I have.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 135

BIBLIOGRAPHY

Baur, E. 1907 Untersuchungen fiber die Erblichkeitsverhaltnisse einer nur in Bastardform lebensfiihigen Sippe von Antirrhinum majiis. Bericht Deutsch. Bot. Ges., Bd. 25, pp. 442-454.

Carothers, E. Eleanor 1913 The Mendelian ratio in relation to certain Orthopteran chromosomes. Jour. Morph., vol. 24, no. 4, December, pp. 487-504.

Castle, W. E. 1911 Heredity. D. Appleton & Company; 184 pp.

Davenport, C. B. 1911 Heredity in relation to eugenics. Holt & Company; 298 pp.

DeVries, H. 1904 Species and varieties. Open Court Publishing Company; 847 pp.

Gates, R. R. 1909 a The stature and chromosomes of Oenothera gigas De Vries. Archiv fiir Zellf., Band 3, Heft 4, pp. 525-552.

1909 b Cytological basis of Mendelism. Bot. Gaz., vol. 47, pp. 79 81.

Hartman, F. a. 1913 Variations in size of chromosomes. Biol. Bull., vol. 24, no. 4, March, pp. 223-238, 4 pis.

JanNsens, F. a. 1909 La theorie de la chiasmatypc. La Cellule, tome 25, fasc. 2, pp. 387-411, 2 pis.

LuTZ, Anne M. 1907 A preliminary note on the chromosomes of Oenothera lamarckiana and one of its mutants, O. gigas. Science, N. S., vol. 20, pp. 151-152.

Morgan, T. H. 1911 Random segregation versus coupling in iNlendelian inheritance. Science, N. S., vol. 34, no. 873, p. 384.

Payne, F. 1912 The chromosomes of Gryllotalpa borealis Burm. Archiv fiir Zellf., Band 9, Heft 1, pp. 141-148.

Punnett, R. C. 1911 Mendelism. Third edition. INIacmillan Comjiany; 192 ])p.

Robertson, W. R. B. 1915 Chromosome studies. I and II. (Not j-et pubJishcd.)

Stevens, N. M. 1911 Heterochi'omosomes in the guinea-pig. Biol. Bull., vol. 21, no. 3, pp. 155-167.

VoiNOv, D. X. 1912 La spermatogcnese chez Gryllotalpa vulgaris, C. R. Soc. Biol. Paris, tome 72, pp. 621-623. (This reference is taken from Carothers' ('13) Bibliography).


EXPLAXATIOX OF PLATES

The drawings of plates 1 and 2 were outlined with an Abbe camera lucida at a nuignification of 3900 diameters obtained with a Leitz 2 mm. oil-immersion objective and a Zeiss X 18 compensating ocTilar. with draw-tube set at 150 mm. and drawing made at the level of the base of the microscope. In the process of rei)roduction, they have been reduced one-third, and therefore appear at a magnification of 2600 diameters. The numerals affixed to the chromosomes indicate their relative sizes, the smallest being numbered '1."

PLATE 1

EXPLANATION OF FIGURES

' 1 to S Tettigidea parvipennis

1 Chromosomes of an oogonial cell; female.

2 First spermatocyte; tetrad No. 4 is normal; No. 5 is the sex-chroniosoine; male.

3 Chromosomes of a first spermatocyte of a third animal (male) showing a normal No. 4 tetrad; No. 5 and No. 2 seen somewhat foreshortened.

4 to 8 From a fourth animal, male.

4 Chromosomes of a spermatogonium; all chrome somes are s})lit and in luetaphase; one of the chromosomes of pair No. 4 is deficient.

5 First spermatocyte; the deficient No. 4 (4 — ) separating from its mate (4).

6 First sijermatocyte; deficient No. 4 ('4 — ) in abnormal, oblifjue contact with its mate (4).

7 Deficient No. 4 tetrads taken from three other first spermatocyte di\iding cells in the same animal, showing uniform size l)ut variation in manner of contact of the conjugating chromosomes.


13(3


rNEQUALITIES IN HOMOLOGOUS CHROMOSOMES

W. R. B. EOBERTSOX


PLATE 1




7 G



4- i- 4


Virtr I 8 {


i a, i u i



137


PLATE 2


EXPLANATION OF FIGURES


8 Deficient chromosome (4 — ) in the anaphase of the first spermatocyte.

9 to 13 Acridium granulatmn. 9, 10 From two normal males.

9 Spermatogonium; No. 1 chromosomes normal.

10 First spermatocyte, No. 1 tetrad normal.

11 Follicle cell of female. One abnormally long (2^-valent) No. 1 chromosome. 12, 13 From abnormal male.

12 First spermatocyte, the 2^-valent, abnormal, No. 1 (1) separating from its normal mate (1) ; going with the sex chromosome.

13 First spermatocyte, the 2i-valent No. 1 chromosome not going with the sex chromosome.


138


INKQUALITIE8 IN HOMOLOGOUS CHROMOSOMKS

\V. R. B. ROBERTSON


PLATE 2



139


JOUBNAI, OF MORPHOLOGV, VOL. 26, NO. 1


PLATE 3

EXPLANATION OF FIGURKS

Ai to He, schematic

Ai to Ae Illustrating pairing in synapsis and separation at first maturation division of homologous chromosomes in Tettigidae. Arrows indicate direction of orientation of the chromosomes; lines and dots indicate respectivelj' maternal and paternal origin of the pairing chromosomes.

Bi to B5 Illustrating manner in which the 2|-valent abnormal No. 1 chromosome pairs with and separates from its normal mate; arrows indicate orientation of parts.

Ci to De A supposed method of origin for the 2|-valent No. 1 chromosome.

Ci Co Accidental unequal division in the reduction mitosis, giving a sesquivalent No. 1 chromosome; arrows indicate orientation of parts.

Di Pairing in synapsis of the sesqui-valent No. 1 chromosome of Co with a normal No. 1.

D2 D3 Separation in late synapsis.

D3 to Do Imagined revolution of the normal No. 1 about the fragmentary end of the sesqui-valent No. 1, giving a 2|-valent No. 1 chromosome having the two end portions oriented in the same direction.

De Carried over whole in a reduction division giving a 2§-valent No. 1 chromosome. The distal No. 1 portion corresponds to that part of the long chromosome in Bi to Bo, which pairs with the normal No. 1.

El to E5 Evident method of pairing of the deficient No. 4 of figures 4 to 8. Normal No. 4 projects beyond its deficient mate (fig. E2) at its distal end. In separation the deficient mate evidently has rotated on the side of this projecting end, and has come into position on the metaphase spindle out of line with its normal mate; compare figures 5, 6, 7 b, and 7 c.

Fi to F5 Showing result expected if in parasynapsis on the pairing of the deficient No. 4 with its normal No. 4 mate there occurred a complete fusion followed by a splitting of the fused body into two symmetrical parts. The chromosomes would be alike in size in the anaphase of reduction (contrary to fact).

Gi to Ge Showing expected result if the same as above took place in the pairing of the 2|-valent No. 1 with its normal No. 1 mate. There would be two long chromosomes of equal length having clubshaped distal and slender proximal ends, the short No. 1 getting a portion of the long No. 1 (contrary to fact).

Hi to He Showing the result if the 2^-valent and 1-valent chromosomes paired and separated in a plane at right angles to the above split (figs. Gi to Ge), the plane of splitting not agreeing with the plane of fusion of the short and long chromosome (fig. Go). Two equal chromosomes would result each composed similarly of like parts of both (contrary to fact).


140


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES

\V. R. B. ROBERTSO>f


PLATE 3



D



m^^



tSw^ E ^^E^


/s


I


E, E


V


iiiMMAm^^m>


M)MMv m ' >mwM/f %


^&Mm mm/u/m//>


G.


G G,


I



i ;


F^


->• »•


G



345


^


R


^


m


^


H


H.


141


THE EMBRYOLOGY OF BDELLODRILUS PHILADELPHICUS

GEORGE W. TANNREUTHER

Zoological Laboratory, University of Missouri

TWENTY-SIX TEXT FIGURES AND EIGHT PLATES

CONTENTS

Introduction 144,

Natural history 146

Brief outline of development 147

Cleavage 149

1 . Designation of cleavage cells 149

2 . Types of cleavage 149

a. Oblique period of cleavage 149

b. Transitional period of cleavage 161

c. Bilateral period of cleavage 163

3 . The first somatoblast 1 63

4. The second somatoblast 169

5 . The entoblast 170

General history of the germ bands 172

1 . Inner stratum of the germ bands 173

2 . Middle stratum of the germ bands 175

3 . Outer stratum of the germ bands 176

The ectoderm and its products 177

1 . The nervous system 177

2. The excretory system 184

Growth " 186

A comparative study of different forms 187

1 . The first somatoblast 190

2 . The second somatoblast 193

3 . Variations in the method of mesodermal formation 199

a. Ecto-mesoblast 199

b. Coelo-mesoblast 199

4. Variations in the source of entoderm 205

General adaptation and interpretation of cleavage 207

1 . In the cleavage of Bdellodrilus 208

2 . In the cleavage of other forms 212

General summary 213

Literature cited 215

143

JODRNAT. OF MORPHOLOGY, VOL. 20, NT. 2 JUNE, 1915


144 GEORGE W. TANNREUTHER

INTRODUCTION

The Discodrilidae are, in many ways, extremely favorable for the study of annelid development. The material can be readily obtained at almost any season of the year. The development of any one egg can be followed from the time of fertilization to its complete development. The smallness of the eggs and the chitinous-like cocoon are the most objectionable features to contend with. Notwithstanding these facts, this group of annelids has been almost completely neglected by investigators in the study of cell lineage.

Moore, in his paper on The anatomy of an American Discodrilid (Bdellodrilus illuminatus)," refers incidently to his 'embryological studies' in the course of his investigations, but has published no account of the Discodrilid development.

Salensky, in his paper on "The development of Branchiobdella an European Discodrilid, parasitic on Astacus fluvitalis," gives an account of the cleavage, axial relation, origin of the germ layers and the formation of the adult structures. But the points of chief importance are so inadequately described and imperfectly worked out, that his results have no special significance in the study of Discodrilid development.

The development of Bdellodrilus philadelphicus, one of the Discodrilidae, has, so far as I can learn, never been worked out. It is in many respects a very important form, not only from the standpoint of development, but from its adult anatomical structure as well.

The more important points in the following paper may be briefly summarized as follows:

1. The cleavage is unequal and regular, but may be variable in some eggs. A very small cleavage cavity is present. The gastrula is formed by the epibolic process. The blastopore occupies a very small area on the ventral surface. Its point of closure corresponds to the median ventral side of the adult worm.

2. The early cleavage planes are definitely related to the future organs of the adult worm; i.e., every cleavage foreshadows


EMBRYOLOGY OF BDELLODRILUS 145

the position of some definite future formation. The large macromere D after the formation of d^ divides very unequally, the smaller cell becomes the entomere D and the larger cell becomes the mesomere d^ (M). The entire mesoblast is derived from the large cell M after its equal cleavage. The primary mesoblasts M, M, or meso-teloblasts, are completely grown over by the derivatives of X and the cleavage cells of the third generation of ectomeres. The descendants of the primary mesoblasts are differentiated into two distinct groups of cells. The first group becomes the dorsal mesoderm of the adult worm. The second the mesodermal germ bands, becomes the ventral and lateral mesoderm. The cells of the first group remain undifferentiated until late development. The latter becomes differentiated into muscle tissue much earlier than the former.

3. When completely formed, the germ bands consist of three distinct strata of cells: (a) An outer stratum, ectoblast from one to two cells thick, which is produced by the three generations of ectomeres. This layer persists as the definitive hypodermis and secretes the cuticle; (b) A middle stratum, which gives rise to the nervous system and the nephridia; (c) An inner stratum, mesoblast which gives rise to all of the mesodermal elements, blood vessels, septa, reproductive organs, etc.

4. The middle stratum is composed of eight distinct longitudinal rows of cells, which at first lie at the surface and form part of the general ectoderm (ectoblast), but afterwards become completely covered over by the ectoderm. There are four rows in each germ band, terminating at the posterior end in a large cell or teloblast. The inner or ventral neural row on each side gives rise to the corresponding half of the nervous system. The three remaining rows of cells (nephridial) on either side, give rise to the nephridia. The outer nephro-teloblast often proliferates but few cells.

5. The brain or cephalic ganglia take their origin from the extreme anterior ends of the neural rows and are distinctly independent of the ectoderm.

6. The cleavage of the entomeres A, B, C, and D is continued to the end without delay. The entire digestive tract,


146 GEORGE W. TANNREUTHER

with the exception of the very short stomodaeum and proctodaeum is derived from the entomeres. The proctodaeum is on the dorsal side of the tenth segment. The stomodaeum is formed at the apical pole. The embryo is completely tm'ned on itself, i.e., the extreme anterior and posterior ends are in immediate contact. The outer or curved surface, becomes the ventral side of the future adult worm.

NATURAL HISTORY

The Discodrilids occupy rather a unique position in the annelid group. They resemble the Hirudinea in their parasitism, in their general shape, in the presence of an anterior and posterior sucker and in the existence of chitinous jaws. The last character is not found in any other oligochaet, but occurs in a large number of leeches. These facts, perhaps not important in themselves, are indications of a very close relationship between the Discodrilids and the Hirudinea, a group which they approach, not merely in such habits as the formation of the cocoon in which the eggs are enclosed, but in many other points of internal and external structures. The fundamental differences between the two groups are not numerous and are not of such importance as has been assigned them by different writers. The Discodrilids are classified as a distinct family of the Ohgochaeta.

Bdellodrilus philadelphicus occurs very abundantly on Cambarus virilis, especially in the early spring and summer months. A few may persist throughout the entire winter in their natural habitat.

For convenience, the animal may be divided into three distinct regions; the head (pharynx), the body proper, and the posterior sucker. The head is much broader than the anterior body segments. The head is composed of four distinct annuli, which perhaps represent distinct segments. The first or peristomal annulus is divided into very mobile dorsal and ventral lobes or lips, which exhibit slight median emarginations, but are otherwise entire. It has sensory hairs and papillae, which are common in this family. The fourth annulus is very narrow.


EMBRYOLOGY OF BDELLODRILUS 147

The middle two appear as muscular rings. The chitinous jaws are triangular, the dorsal with a single tooth, the ventral jaw with a pair of smaller teeth. No lateral mucous glands which are very common in some of the species are present.

The body proper consists of eight strongly bi-annulate somites or rings. The anterior somites are longer and broader than the posterior. When contracted, the minor annuli of the somites are telescoped within the major annuli. The fifth, sixth, and seventh somites are sexual. The first, second, third, fourth and eighth somites are nephridial. The spermatheca is broad, thin walled, and nearly cylindrical. The penis is carried to the exterior by the eversible bursa, into which its projecting end is received. There is a conspicuous prostate in addition to the large glandular sperm sac. These parasitic forms remain attached to the ventral surface of the host throughout their entire life history. The eggs are deposited on the ventral surface of the host, more abundantly where the water is kept in constant motion by the movement of the appendages. Each egg is enclosed in a distinct separate stalked cocoon. The base of the stalk is firmly attached to the host. The deposition of eggs occurs during the entire year, if the parasites be kept in aquaria at room temperature. In their natural habitat eggs are not deposited during the severe winter months.

BRIEF OUTLINE OF DEVELOPMENT

The cleavage of the ovum takes place with considerable precision and regularity. Especially is one impressed with this striking phenomenon, after following the cleavage of many ova. The only perceptible variations being (a) slight differences in the time at which the individual cells divide; (b) slight variations in the size of the same cells in different ova. The rate of cleavage varies somewhat with temperature. Occasionally all the cleavage cells of an individual egg are nearly equal and it is impossible to orient the embryo before the germ bands begin their formation. This, however, is an exception, rather than a usual occurrence.


148 GEORGE W. TANNREUTHER

As development progresses the variations between individual embryos become less apparent and as far as can be recognized, do not affect the final result.

The history of the cleavage is distinguished by three well marked periods, namely: oblique, transitional, and bilateral. In the first period, which extends to the twenty-four-cell stage, the germ layers are differentiated, and the parent cells, which give rise to the future organs are definitely marked out.

The first cleavage is nearly transverse to the median longitudinal axis of the adult worm. The second cleavage plane occurs at an angle of forty-five degrees to the first. The third cleavage plane is horizontal and separates the four ectomeres above from the four macromeres below.

Three generations of four ectomeres each are successively separated, from the macromeres A, B, C and D. The first generation of ectomeres (a^, b', c^ and d^, are formed in a right handed direction. The second generation of ectomeres (a-,b^ c- and d-), are formed in a left handed direction. The third generation of ectomeres (a^*, b^, c^ and d/*), are formed, in a right handed direction. From these twelve ectomeres the entire ectoderm is formed.

The ectomere d- gives rise to all or nearly all the ectoderm of the trunk, to the nervous system and to the nephridia.

The oblique type of cleavage is maintained in the division of macromeres. At the close of the oblique period the embryo consists of twenty-four cells (text fig. 6 and fig. 36). The relation of the cleavage cells to the germ layers is as follows:

4 macromeres entpderm

1 mesomere mesoderm

„„ , fl9 ectoderm

20 ectomeres. < , ,,,. , , , , . ,.

1^ 1 (d^). .ectoderm, nervous system, nephridia

Bilateral division now occurs in some of the ectomeres, while others may continue to divide obliquely. The transitional period shows both types of cleavage. Oblique cleavage persists in some of the cells until the fiftieth or more cell stage.

In the third period, the cleavage becomes essentially bilateral


EMBRYOLOGY OF BDELLODRILUS 149

in most of the cells and the teloblasts of the right and left halves of the embryo are formed. Bilateral symmetry now becomes definitely established and the animal increases in length very rapidly.

CLEAVAGE 1. DESIGNATION OF CLEAVAGE CELLS

In the designation of the cleavage cells, for the sake of uniformity and convenience, I have for the most part adopted the system followed by Wilson in his "Cell lineage of Nereis," and Lillie in his study on "The embryology of the Unionidae." The first four cells (macromeres) are designated by the capital letters A, B, C and D. The generations of micromeres (ectomeres) by the small letters a, b, c, and d. The first index number indicates the generation to which the ectomere belongs. Thus a\ b^" or c^'i'- or d^'^ all belong to the first generation; c^, b',d2'* belong to the second generation, etc. A, B, C and D correspond to the vegetal pole; a, b, c and d to the apical pole. When a cell divides the products receive the designation of the parent


cell with the addition of a further index number; thus b


2 ,1


lb2.


Exceptions to this rule are made only in the case of special cells, which, for convenience, receive special designations: thus d^ of the second generation of ectomeres becomes the 'first somatoblast' and is designated by (X), and its small derivatives by x^, x2, x^ etc.; d^ the 'second somatoblast' is designated by (M).

2. TYPES OF CLEAVAGE

a. The oblique period of cleavage: one to twenty-four cells

First cleavage: The first cleavage occurs about five to ten hours after the deposition of the egg. The time varies somewhat with external conditions. The plane of division passes through the area where the polar bodies are formed (fig. 1) and divides the egg into two very unequal parts, AB and CD (text fig. 1 and fig. 2). The smaller of the two cells AB is anterior, and


150


GEORGE W. TANNREUTHER




EMBRYOLOGY OF BDELLODRILUS


151



1 b


2 '^



Fig. 1 Two-cell stage from apical pole view. Fig. 2 Early three-cell stage from apical pole Fig. 3 Four-cell stage from apical pole view. Fig. 4 Four-cell stage from vegetal pole.

a-h, median longitudinal plane of future adult; 1-1, first cleavage plane; 2-2 second cleavage plane; a, anterior; h, posterior; r, right side; I, left side.

the larger cell CD is posterior. The cleavage at first is very deep and the cells are rounded, but soon they begin to press against each other and flatten at their point of contact. Before the second cleavage begins the egg assumes its original elliptical shape and the point of contact externally, between the two cells is represented by a mere line or shallow groove. No actual fusion of the two cells ever takes place; sections always show a distinct line of separation between them.

The deutoplasm is equally distributed in both cells. The cytoplasm surrounding the nucleus contains very little yolk


152 GEORGE W. TANNREUTHER

material. This makes it possible, not only to recognize the position of the nucleus, but to be able to make out the exact position of the cleavage spindle in the living egg.

Second cleavage: The second cleavage is meridional and takes place at an angle of forty-five degrees to the median plane of the future adult. The two cells divide at different times (occasionally both cells divide simultaneously). These two cleavages taken together represent the second cleavage in other annelids. CD divides first into two very unequal parts (text fig. 2 and figs. 3, 78). The division of AB is nearly equal (figs. 5 and 6). The largest cell, D, is posterior. B is anterior, inclined a little to the right. C is right (text figs. 3-4) and A is left with reference to the median axis of the future worm. The large cell D has a tendency always to divide first. The exact formation of the four macromeres must be carefully worked out, and correctly understood, since their position largely determines the orientation of the future organs.

For descriptive conveniences the region of the first generation of ectomeres will be considered as the upper or apical pole and the point directly opposite, as the lower or vegetal pole. The centers of the upper and lower poles of the dividing ovum, coincide with the median longitudinal plane of the adult worm. The poles however may be shifted somewhat anteriorly or posteriorly, with reference to the macromeres, more especially to D in the formation of d-. When viewed from the upper pole A and C are in contact, while B and D are separated. But when viewed from the ventral pole A and C are separated and B and D are in contact (figs. 6-7). This extensive cross furrow found at the vegetal pole is also present in forms like Nereis, Clepsine and Crepidula; while in those forms like Unio, in which the greatest mass of the four macromeres is concerned in the formation of ectoderm instead of endoderm, the cross furrow is greatest at the animal pole. These cross furrows ('Brechungslinie' of Rauber) have no special significance in the development of Bdellodrilus, as the cleavage of the macromeres is carried to the end, immediately after the three generations of ectomeres are formed. In those forms like Nereis, where the cross furrow


DESCRIPTION OF PLATES

All drawings were made with a camera lucida under a magnification of about 125 diameters. All whole amount drawings, with one or two exceptions, were made from the living egg. The variation in size of the surface views is due to a difference in the size of the eggs. The sections were not uniformly magnified. Stippling has been adopted for the sake of clearness.


REFERENCE LETTERS

a., anterior mi. a., minor annulus

bl., blastopore mj.a., major annulus

c, ciliated cells mo., mouth

C.C., cleavage cavity p., posterior

C.I., cerebral lobes pb^~', polar bodies

coe., coelom ph., pharynx

e.cp., egg capsule pr., proctodaeum

ec, ectoderm so.mes., somatic mesoderm

en., entoderm sp.mes., splanchnic mesoderm

gn., ganglia st., stomodaeum

7nes., mesoderm v., ventral

A, left macromere

B, anterior macromere

C, right macromere

D, posterior macromere

aS b^, cS dS a'S etc., first generation of ectomeres a^ b^, c^, d^, a^-^', etc., second generation of ectomeres a^, b^, c', d^, a^'\ etc., third generation of ectomeres a"*, b*, c"*, d, etc., fourth generation of micromeres X = d^ first somatoblast X, X, right and left proteloblasts X('), neuroblast Z(2), X(3), X(4), nephroblasts M = d'* second somatoblast , 7n, secondary mesoblast x^-x', small derivatives from X N, posterior end of nephric rows Nc, posterior end of neural rows nc, neural rows np, nephric rows


153


PLATE 1

EXPLANATION OF FIGURES

1 Surface view of an unsegmented ovum, to show the polar bodies and the cleavage nucleus.

2 Two-cell stage from the upper pole.

3 Two-cell stage; cell CD dividing.

4 Three-cell stage from the upper pole; division of CD is complete.

5 Same stage as preceding; cell AB dividing.

6 Four-cell stage from the upper pole; the cleavage spindle for the first ectomere forming.

7 Four-cell stage, ventral view.

8 Four-cell stage viewed from the left side.

9 Same as the preceding, viewed from the right side.

10 Four-cell stage from the upper pole, showing the formation of the first ectomere.

11 Five-cell stage from the upper pole, d^ formed.

12 Stage showing the cleavage spindle of the second ectomere.

13 Six-cell stage from the upper pole; cleavage spindles for third and fourth ectomere forming.

14 Eight-cell stage from the upper pole; the macromeres are considerably flattened.

15 Same as the preceding, from the ventral pole; similar to the same view of the four-cell stage.

16 Eight-cell stage from the left side.

17 Same as the last showing the behavior of the macromere D in the formation of the first somatoblast.

18 Stage a little later than the preceding.

19 Nine-cell stage from the left side after the formation of the first somatoblast.

20 Xine-cell stage turned a little to the left, so that all the cells are visible.


154


EMBRYOI.OGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 1



Kline, del


156 GEORGE W. TANNREUTHER

persists until late development, it serves as an unmistakable point of orientation.

Figures 8 and 9 show the four-cell stage from the left and right sides. The dorsal ventral axis of A, B and C is about the same as that of D, but immediately after the formation of the first generation of ectomeres, the cells A, B and C shorten and become more rounded (f g. 16). In later stages of development these cells often become very much flattened and cause the developing embryo to appear unusually large, when viewed from the upper or lower poles.

Third cleavage (eight-cell stage): In the formation of the first generation of ectomeres (d^, c^, b^ and a^, each of the four macromeres divide obliquely. The ectomere end of the cleavage spindle is uppermost. The macromere D divides first; d^ is budded off from D towards the upper pole, in the direction of the hands of a watch (dexiotropic), (figs. 10-11). We have thus a fivecell stage. Each of the macromeres C, B and A next bud off a small cell towards the upper pole. These are not formed simultaneously, but in the invariable order g\ h^ and a\ Thus there occurs successively, a six, a seven and an eight-cell stage (figs. 11-14). In figure 13, an upper pole view, D and C have divided and A and B are preparing to divide. In both A and B the asters of the ectomere end of the spindles are visible. The position of the opposite end of the spindles are indicated by circles. This figure shows the oblique nature of the cleavage spindles. The spindle in A points to the space between A and B. The spindle in B points to the space between B and C. In figure 14, an eight-cell stage, the exact relation of the ectomeres and macromeres are shown as they normally appear from the apical pole. The position of the first generation of ectomeres is obvious. They suggest a possible rotation, after their formation, through an angle of about forty-five degrees in the direction of the hands of a watch. If actual rotation did occur there would be no difficulty in explaining their final position. But the fact that the cleavage spindles are oblique and the position of the completely divided nucleus can be definitely determined, before there is any indication of the cytoplasmic division of the


EMBRYOLOGY OF BDELLODRILUS 157

parent cell in the formation of the daughter cells, suggest that the apparent rotation process is not mechanical or even produced by pressure of the macromeres. The formation of d^ in figure 10 shows how the process takes place; d^ is budded off obliquely from the macromere D over the inner posterior edge of A and becomes partly imbedded in A. Its final position is determined by the direction of the cleavage spindle. This characteristic method in the formation of the ectomeres is quite a prevalent one. It occurs not only in the eggs of annelids, but in those of the molluscs and polyclads as well.

Fourth cleavage: A nine-cell stage is reached in Bdellodrilus by the division of the macromere D in an oblique direction. Figure 16, an eight-cell stage viewed from the left side, shows the position of the macromeres A, B and C with reference to the macromere D, before the formation of the ectomere d^. The large macromere D contains about two-thirds of the volume of the dividing ovum. In preparation for the formation of d^, D elongates in an obhque direction at an angle of about forty-five degrees to the horizontal plane of the developing embryo. The ventral anterior portion of D shifts forward beneath A, B and C (fig. 17). After the formation of d-, D takes a position directly beneath the first generation of ectomeres, and completely covers the inner ends of A, B and C (figs. 19-20). In some instances D is shifted more anterior and completely covers the ventral surface of the other macromeres (fig. 20) ; but in most cases, as in the nine-cell stage, D occupies the region of the ventral pole, directly beneath the first generation of ectomeres (figs. 22-23). The formation of d^ is shown in figures 16 to 19. The division is equal in most cases. When unequal, d'^ is the larger cell. In figure 20, a nine-cell stage turned to the observers left so that all the cells are visible, A and B are preparing for the formation of a- and b-. In most the succeeding stages d-, the 'first somatoblast' will be designated by the capital letter X. It is also the first cell of the second generation of ectomeres. The formation of a-, b' and c- is shown in figures 20, 21, 22 and 24 in side and top views respectively. The second generation of ectomeres, with the exception of d^ (X), is about the same size as those of the


PLATE 2

EXPLANATION OF FIGURES

21 Nine-cell stage, viewed from the right side.

22 Same stage as the preceding, from the upper pole; spindles for the second generation of ectomere are forming.

23 The same stage from the ventral pole.

24 Twelve-cell stage from the upper pole; spindles for the third generation of ectomeres are forming.

25 Thirteen-cell stage from the left side; the cell d^ nearly formed.

26 Same stage as the preceding from the right side; the embryo is considerably elongated.

27 Fourteen-cell stage from the right side; X dividing to form x^.

28 Same stage as the preceding, ventral view.

29 Fifteen-cell stage from the upper pole; c' budded off symmetrical with d^.

30 Fifteen-cell stage from the left side; upper pole turned considerably to the left.

31 Same stage as the preceding from the right side; the small cell x^ is drawn out between c^ and X.

32 Fifteen-cell stage, ventral view, as a transparent object, with the position of all the cells indicated; drawn from a prepared specimen, cleared in xylol.


158


EMBRYOLOGY OF BDELLODRILUS


PLATE 2


■G. W. TANNREUTHER





C-/



. A R




. c


H


d'


>.::&,:■;?




jill^d'




-p^


3 ■■"• -^'


X


3/


Kline and Tannreuther,


deL



-;r'-~ ^


y^


vX


-D




e' 32



159


JOUBNAL OF MORPHOLOGY, VOL. 26, NO. 2


160 GEORGE W. TANNREUTHER

first. The order of their formation is d- (X), c-, b-, a^, the same as the first generation. Figure 24 illustrates the twelve-cell stage from the upper pole. The cells are somewhat flattened. The macromere D is located a little to the left of the median longitudinal plane, while X is symmetrical with reference to the median axis of the future worm.

The cleavage spindles of the third generation of ectomeres form in an oblique direction. The thirteen-cell stage is reached by the formation of d. The manner in which d is formed, is rather unique when we take into consideration the size and position of D with reference to the other macromeres A, B and C (figs. 23-25). It is budded off from the outer surface of D and takes up a position symmetrical with c^ The fourteencell stage is reached, by the formation of x^ (fig. 27), it is budded off from the median right side of X. Its final position is between X and C. Figure 28 shows the same stage as the preceding in ventral view, turned a little to the observer's right.

Immediately after the formation of x^ the macromere C buds off c', thus making a distinct fifteen-cell stage (fig. 29). The order of formation of the third generation of ectomeres is the same as the first and. second. Figures 29, 30, 31 and 32 show the fifteencell stage in dorsal, left, right and ventral views respectively. In figure 33, a'^ and b^ are formed and. the first generation of ectomeres are preparing to divide; d' and c^ divide first; at the same time x^ is budded off from X, symmetrical with x^, between D and d'^ thus making a twenty-cell stage (fig. 34). Figure 35 is a side view of a twenty-two-cell stage after a^ and b^ have divided. This figure represents an anterior posterior elongation of the embryo, which is a very common occurrence. The cells, taken as a mass, are very plastic and may assume different shapes. This peculiarity is only secondary and has no special significance. The cells become more spherical before division and flatten out somewhat after the division is complete.

The twenty-three-cell stage is reached by the formation of x^ from the upper posterior side of X between c^ and d^ (fig. 36).

The division of the first generation of ectomeres is unequal and. radial rather than oblique. From the twenty- to the thirty


EMBRYOLOGY OF BDELLODRILUS 161

cell stage several types of cleavage are present; oblique, radial and bilateral. This period of variable cleavage will be designated as the transitional period.

b. The transitional period of cleavage: twenty- to thirty-cell stage

After the formation of x'^ there is a short inactive period and in many of the developing embryos, the cleavage furrows become very indistinct. Cleavage is again initiated by the formation of d^ from the large macromere D. The cleavage is oblique and very unequal (text fig. 5 and fig. 85).

The smaller cell is almost completely hidden when first formed. It is budded off directly between A and B, near the ventral anterior surface (fig. 37). The smaller cell persists as D (entomere) and the larger cell d^ or M becomes the 'second somatoblast.' After the formation of M the entire endoderm is contained within the entomeres A, B, C and D (figs. 37-38).

The germ layers are now distinctly separated and the embryo at this stage of development is composed of twenty-four cells (text figs. 6-7). Nereis at the same period of differentiation, shows thirty-eight cells. Unio (Lillie) at the time of the separation of the germ layers contains thirty-two cells. This difference is due, in case of Bdellodrilus to the lagging of division in the cells of the upper pole.

The composition of the embryo at the twenty-four-cell stage is as follows:

Entomeres A, B, C, D 4

Ectomeres of first generation 8

Ectomeres of second generation 4

Ectomeres of third generation 4

Mesoblast M 1

First somatoblast derivatives 3

24

Many of the cells during the transitional period have a definite shape and if separated from the cell complex, they could be readily recognized. The embryo at this stage of development is somewhat spherical (figs. 36-38). Immediately after


162


GEORGE W. TANNREUTHER

D



Fig. 5 Twenty-four-cell stage in ventral view, to show the division of the large macromere D. The larger of the two cells d^ (M) becomes the 'second somatoblast.' The smaller cell D, becomes one of the four entomeres; D is scarcely visible from the exterior.

Fig. 6 Same as the preceding, in an apical pole view.

Fig. 7 Twenty-four-eell stage, ventral view, shows the bilateral division of M. D after the bilateral division of M becomes more distinct from surface view.

Fig. 8 Horizontal section of an early embryo to show spindles in the formation of x^ from either proteloblast X, X.

Fig. 9 Horizontal section little later than preceding, to show the small cell x^-x^; apical pole view.


EMBRYOLOGY OF BDELLODRILUS 163

the establishment of the germ layers, the bilateral division of the 'first and second somatoblasts' occurs. The bilateral division of the 'second somatoblast' usually precedes that of the first; occasionally they divide simultaneously.

c. The bilateral period of cleavage: twenty-five-cell stage

The first bilateral cleavages occur in the first and second somatoblasts (text fig. 7 and figs. 37-43). The small superficial cells of the lower pole are derived from the second and third generation of ectomeres and from the derivatives of X. The arrangement of these cells with reference to the blastopore is shown in figure 42. The entomeres A, B, C and D are partly grown over by the other cells and the open space becomes the blastopore. It is bounded anteriorly and laterally by small cells from the second and third quartettes, and posteriorly by the primary mesoblasts M,M. Its hinder lip, which is formed by the primary mesoblasts, lies anterior to the center of the lower pole. The closure of the blastopore takes place by a convergence of the cells from all sides. The principal growth of cells is from in front backwards, formed by the derivatives of the second and third generation of ectomeres (figs. 42, 43, 51). The entomeres now divide very rapidly and the cells soon become smaller than those of the ectomeres, which grow over them (figs. 43, 51).

3. THE FIRST SOMATOBLAST

The history of the 'first somatoblast' in Bdellodrilus is of considerable interest when considered from the standpoint of its origin and its derivatives. When first formed from the posterior macromere D, it contains one- third of the entire bulk of the developing embryo. As already described, it first buds off the small cell x^ on the right, x- symmetrically on the left and a third cell, x^, on the median posterior upper side. These three small cells are symmetrical with reference to the median longitudinal axis. The fourth cleavage divides the somato


PLATE 3

EXPLANATION OF FIGURES

33 Seventeen-cell stage from the upper pole; spindles in the first generation of ectomeres forming.

34 Twenty-cell stage from the upper pole; cells c^~\ cU~' and x- just formed.

35 Twenty-one-cell stage from the right side; b^~\ new cell; this embryo is unusually elongated.

36 Twenty-three-cell stage from the upper pole; d'~' and x^, two new cells formed; embryo considerably flattened.

37 Twenty-four-cell stage from the ventral side; the unecjual division of D has just occurred; D partially visible; the cleavage spindle of M forming.

38 Twenty-five-cell stage, ventral view; division of M complete and the spindle for the first bilateral division of X is forming.

39 Twenty-five-cell stage from the left side.

40 Twenty-five-cell stage from the upper pole; embryo is nearly spherical.

41 Twenty-six-cell stage, ventral view.

42 Same as the preceding, ventro-anterior view.

43 Twenty-nine-cell stage, same aspect as preceding; a"*, b"* and c* are the three new cells formed.

44 Forty-two-cell stage from the upper pole; increase in number of cells due to the rapid division of the ectomeres.


164


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 3



42 X'

Kline and Tannreuther, del.


43 X/


165


PLATE 4

EXPLANATION OF FIGURES

45 Sixty-three-cell stage from the upper pole.

46 Same stage as the preceding, from the ventral pole; very few of the ectomeres are visible.

47 Seventy-cell-stage, ventral view. Increase in number due to division of small cells. Consult table of cleavage. First nearly equal division of the proteloblasts, X, X. This division separates the neural and nephridial elements.

48 Seventy-two-cell stage, ventral view.

49 Little later than the preceding stage, to show x^ and x".

50 Embryo, ventral view, to show the first division of the nephroblasts; transverse axis greater than the longitudinal.

51 Embryo, ventral view, turned anteriorly to show the blastopore.

52 Embryo to show the lengthening of the anterios-posterior axis. The small cells, x", x", are good points to mark the orientation of the different figures. All figures on plate are similarly orientiated with reference to the right and left sides of the embryo.

53 Stage a little later than the preceding.

54 Embryo from upper pole, to show derivatives of x^ between the nephroblasts.

55 Embryo with upper surface turned posteriorly, to show the rapid division of the ectoblast cells.

56 and 57 Show that either nephroblast X^-^) or X(') may divide, to form the three nephroblasts on either side.


166


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 4



X-X

Kline and Tannreuther, del


167


168 GEORGE W. TANNEEUTHER

blast into two equal parts, right and left (figs. 40-41). These two cells, for convenience in description, will be called the posterior right and left proteloblasts. At the fifth division each proteloblast buds off a small cell, x^, external to x^ and x- respectively (fig. 44). At the sixth division each of the proteloblasts buds forth a small cell, x^ on either side of x, beneath the derivatives of c^ and d^ (text fig. 8). At the seventh division each of the proteloblasts buds off a small cell, x" on the ventroanterior edge at the junction of the two cells (fig. 46).

At the eighth division each proteloblast, on either side of the median plane of the embryo, divided into two equal parts (figs. 47, 48, 93). The four cells formed become the posterior teloblasts, X<i^ and X*, right and left of the median axis (fig. 47). X'l' without any further division, becomes the neuroblast on either side, and X'^' becomes the nephroblast on either side of the median axis (figs. 47-48). Next X'-', right and left, divides equally, giving X<2* and X^' (figs. 50, 52). Next either X'-" or X*^' divides equally. If X*^ divide, which is the common occurrence, we get X'3' and X'^'. But if X<-' divide instead of X^^\ the final result is the same. In either case, we get four teloblasts on each side (one neuroblast and three nephroblasts) as shown in figures 56 and 57. Next X'-^ on either side divides very unequally and gives rise to x^ on the anterior ventral outer surface (fig. 49).

The progeny of the 'first somatoblast,' when the teloblasts are completely formed, is twenty cells. Table 2 shows the derivatives of the 'first somatoblast.'

TABLE 2

x'x'x*x^V' x'x'x'x'


1/ /^


X'


(') x^


^^^'^ i Son'iQloblast \<Xx)*

^ Proteloblast ProteloWest


EMBRYOLOGY OF BDELLODRILUS 169

4. THE SECOND SOMATOBLAST

Immediately after the formation of X from the posterior macromere D, d^ is budded off from D (figs. 24-25). Next D divides very unequally in an oblique direction and gives rise to d^ (M) the 'second somatoblast,' as previously described. Figures 37 and 85 show the position of D and M after the cleavage of the macromere D. The twenty-five-cell stage is reached by the bilateral division of M (figs. 37-38). The cells M, M at first are a little to the left of the median plane, but later in course of development they become symmetrical to the longitudinal axis of the adult worm.

Soon after the bilateral division of M, the 'second somatoblast,' each cell M,M right and left buds off five or more small cells directly beneath the first generation of ectomeres (figs. 86-87). It is impossible to detect these cells except by means of sections, hence the uncertainty as to their exact number. They are characterized by their large nuclei with homogeneous staining chromatin and they contain but little yolk material. These small cells divide once or twice soon after their formation from the primary mesoblasts and then remain inactive until late embryological development, at least until after the germ bands are completely formed and the embryo has undergone considerable differentiation (as the formation of the lumen of the digestive system, etc.).

These undifferentiated mesodermal cells occupy the region which becomes the central dorsal side of the embryo, at the point where the developing worm is completely turned on itself (figs. 92, 98-99). The history of these cells can be readily followed through their different stages of development, so that there can be no question as to their exact origin and history. When the embryo begins to straighten, the progeny of these small cells extend toward either end and form the splanchnic and somatic mesoderm on the dorsal side of the worm. This secondary mesoderm later becomes continuous with the primary mesoderm, which forms directly from the mesoblasts M,M.


170 GEORGE W. TANNREUTHER

5. THE ENTOBLAST

The formation of the entoblast in Bdellodrilus represents an unusual type of development among the annelid worms. The macromeres A, B, C and D, after the formation of d\ give rise to the entire entoderm, D is greatly reduced after the formation of d^ The position of the entomeres is shown in figures 40 and 42. In figure 40 A, B and C appear rather large, because of the flattened condition of the cells. In figure 42 (from the ventral pole) the cells are rounded and appear more normal. The position of the entomeres and their boundary cells are distinctly shown. This figure shows more clearly the bulk of the entoderm, when compared with the mass of the entire egg. In figure 43 (a twenty-nine-cell stage) A, B and C have divided nearly equally. This division is considered by some investigators as the formation of the fourth generation of micromeres; d^ of the D quadrant has formed earlier. Figures 44 and 45 (apical pole views) show the upper outer edge of the entodermal cells. In figure 46 (the same stage as preceding from the ventral pole) a very small part of the entodermal and ectodermal cells are visible. This figure shows the prominence of the four large cells, which later form the ten teloblasts. These four large cells, from their position, resemble the four large entomeres, which are so prominent in many other forms. These cells (X, X, M, M), according to Selensky, share equally in the formation of the germ layers, i.e., ectoderm, endoderm and mesoderm are produced by each of them.

In forms like Clepsine, Crepidula and others, at a similar or later stage of development, the entomeres are very prominent and the ectomeres with the first and second somatoblasts, form a cap of cells on their upper surface. In Bdellodrilus the conditions are different. The ectomeres and the entodermal cells form a cap of cells on the upper anterior surface of X, X, M and M. This difference is due to the prominence of the first and second somatoblasts, which constitute the greatest bulk of the embryo. At about the seventy-cell stage the ectodermal and endodermal cells are nearly uniform in size (figs. 47-49). In


EMBRYOLOGY OF BDELLODRILUS 171

figure 51 (a little later stage) the blastopore is nearly closed. This early closure of the blastopore in Bdellodrilus, is due largely, to the ventro-anterior shifting of the macromere D over A, B and C in the formation of the somatoblasts (figs. 18, 85).

The closure of the blastopore, in some of the annelids, occurs at a very late stage of development. In Clepsine the teloblasts give rise to rows of cells, which pass anteriorly around the entomeres A, B and C beneath the edge of the blastodisc or cap of cells. The blastodisc with these rows of cell cover about half of the entomeres. By the downward growth of the blasdodisc and the concrescence of the germ bands, the closure of the blastopore is completed. The closure of the blastopore in Clepsine occurs on the ventral side, nearer the anterior end. In Bdellodrilus, the germ bands are not formed until later and take no part in the closure of the blastopore. Text figures 10 to 13 show the position of the ectoderm, entoderm, and the first and second somatoblasts, at different stages in the closure of the blastopore. The region of closure is similar to that of Clepsine.

At the time of the formation of the secondary mesoblast just beneath the first generation of ectomeres, the entire entoderm is situated in the anterior half of the embryo. But soon after the formation of the m cells (text fig. 12 and fig. 86), the entodermal cells by a rapid proliferation extend posteriorly between the m cells and the primary mesoblast. During the formation of the primary mesoblast, the meso-teloblasts themselves are carried posteriorly, ahead of the entoderm. The entoderm, thus becomes situated between the m cells or secondary mesoderm above and the mesoblast bands or primary mesoblast below. The entoderm in reality never reaches the posterior limit of the meso-teloblasts, as shown in figures 98 and 99.

The interior of the developing embryo, now consists of a solid mass of small entodermal cleavage cells (figs. 95-99), heavily laden with yolk. These cells are readily distinguished from the surrounding mesodermal cells, by their deeper cytoplasmic stain. Figure 99 (a vertical longitudinal section near the median plane) shows the position of the entodermal cells in the embryo.


172 GEORGE W. TANNREUTHER

As the embryo elongates, the entodermal cells increase in number. This process of growth is continued until the digestive tract is completely formed. Figure 99 shows the anterior and posterior limits of the digestive tract, which is formed from the four entomeres. The anterior end shows a distinct lumen, while the posterior end is yet a solid mass of cells. The entire digestive tract, except the insignificant stomodaeum and proctodaeum, is entodermal in origin. The proctodaeum is not formed until the time of hatching. It occurs on the dorsal side of the tenth segment. The embryo is completely turned on itself (fig. 99) and brings the anterior and posterior ends of the digestive tract in close proximity. The differentiation of the digestive tract begins anteriorly and progresses posteriorly. As growth continues, the outer cells of the entodermal mass form an epithelial layer. At first the cells are somewhat flattened, but soon take a columnar position, and form the columnar epithelium of the digestive tract. The cells within soon lose their staining properties, break up and. serve as food for the developing embryo.

The digestive tract in its course of development, passes through the following stages: the first stage is represented by the four entomeres A, B, C and. D; the second, stage by a solid mass of cleavage cells (the cell boundaries are often very indistinct) within the center of the embryo; the third stage by an elongation of the entodermal mass as the larva lengthens, and. by the establishment of a lumen; the fourth stage by a thin layer of flattened epithelium and. later a columnar epithelium; the fifth stage, the cells within the epithelial layer serve as food ; and sixth the formation of the stomodaeum and. proctodaeum.

GENERAL HISTORY OF THE GERM BANDS

The term 'germ bands' has been variously interpreted by different investigators on cell lineage. The term germ bands, or the German equivalent 'Keim Steifen,' is usually restricted to the strata derived, from the teloblasts, the ectoblastic layer being excluded. It is held by others that the germ bands of annelids are purely mesoblastic.


EMBEYOLOGY OF BDELLODRILUS 173

Balfour, Hatschek, Goette, Kowalevsky and many others made use of the term 'mesoblastic bands' as the equivalent of the germ bands. In Hirudinea, according to Whitman, the germ bands are composed, of three distinct layers; the ectoblastic, mesoblastic and the neuroblastic elements. Wilson gave the same interpretation in his studies on The embryology of Lumbricus." In Bdellodrilus the term 'germ bands' includes the three strata of cells as in Hirudinea and Lumbricus.

1. INNER STRATUM OF THE GERM BANDS

After the formation of the teloblasts, five on either side of the median axis (one neuroblast, one mesoblast and three nephroblasts), the mesoblasts or meso-teloblasts are the first to begin the formation of the germ bands by a forward proliferation of cells near the posterior hp of the blastopore (text figs. 12, 13, 17). The plane of division, is nearly at right angles to the formation of cells in the secondary mesoblast (text figs. 10, 13). The cells of the mesoblast bands are considerably smaller than the teloblasts from which they originate. They grow forward between the entoderm and the ectoderm and finally meet at the anterior end of the larva. As these bands grow forward they become several cells broad, but seldom more than two cells deep. Their differentiation begins anteriorly and progresses backward. The first cells of the mesoblast bands, when formed, are on the surface, but soon become covered by the ectodermal cells. As the mesoblast band, extends forward below and around the entoderm, it forces its way to the extreme anterior end of the embryo beneath the ectoderm. It finally encloses the digestive tract on the ventrcl and lateral sides and becomes continuous with the secondary mesoblast on the dorsal side. The two mesoblast bands fuse first at the anterior end along the median ventral side and subsequently with the dorsal secondary mesoderm. In figure 99 (a longitudinal section) the mesoblast is differentiated into splanchnic and somatic layers, with the coelom between. The longitudinal muscles become differentiated before the circular. At the extreme posterior end the


174


GEORGE W. TANNREUTHER



Vertical section to the left of the median plane.

Vertical section of a ninety-cell stage.

Vertical section of an embryo at the time of closing of the blasto


Figs. 10-13 Diagrammatic figures to show the ventro-posterior extension of the ectomeres, in the closure of the blastopore.

Fig. 10 Thirty-three cell stage, taken a little to the right of the median plane.

Fig. 11

Fig. 12

Fig. 13 pore. The mesoblast bands have just begun.

The heavy stippling represent endoderm; the light stippling mesoderm, and the unstippled the ectoderm or ectomeres. m, secondary mesoblast; M, mesoblasts; hi, blastopore; hlc, point where the blastopore closes; A', derivatives of the 'first somatoblast;' mcs, mesoblast bands.

meso-teloblasts are represented by an undifferentiated mass of cells, which later give rise to the musculature of the last three segments of the worm, and are directly concerned in the formation and movements of the posterior sucker.


EMBRYOLOGY OF BDELLODRILUS


175


2. MIDDLE STRATUM OF THE GERM BANDS

The middle stratum of the germ bands can readily be distinguished while the embryo is still nearly spherical. Upon close examination it is seen that the ectoblast cells are arranged into four distinct rows, on either side of the median ventral axis (figs. 65-66). Each row terminates posteriorly in a large cell or teloblast.

Text figures 15 and 17 and figure 58 show the early formation of these rows of cells. Sections of these various stages show that



Fig. 14 Surface view from upper pole, to show the position of the ten teloblasts. The meso-teloblasts or mesoblasts have budded off eight or ten cells in the formation of the mesoblast bands. Their position is indicated by dotted outline. The broken outline represents the region of the entoderm. The position of the ectoderm is indicated by a continuous line.

Fig. 15 Third horizontal section from the top, passing through four of the large nephroblasts. The spindles represent the beginning of the first division in the formation of the middle germ band. The anterior end and the right side of the section are a little below the horizontal plane.

Fig. 16 Seventh horizontal section from the top. It passes through the upper portion of the entoderm and the secondary mesoblast (m).


JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


176


GEORGE W. TANNREUTHER


these rows of cells form a part of the general ectoderm, being partly covered here and there by adjoining cells. In later stages of development, these rows of cells become completely covered as they gradually sink beneath the surface, and thus come to lie between the mesoblast and the ectoderm or ectoblast.



Fig. 17 Twelfth section from the top, to show the anterior extension of the mesoblast bands below and around the entoderm.

Fig. 18 Nineteenth section from the top, to show the lower side of the mesoblast. The section passes below the entoderm. The spindles represent the beginning of the first division of the neuroblasts to form the neural rows.

Heavy stippling represents entoderm; light stippling mesoderm and the unstippled portion the ectoderm. The sections of figures 15-18 were eight micra thick. There were 21 sections in all. a, anterior, and p, posterior, represent the respective ends of the cleavage cells, but not the future ends of the embryo; r, right; I, left; ent, entoderm and ect, ecoderm.


3, OUTER STRATUM OF THE GERM BANDS

This stratum forms the definitive ectoderm and needs no further description at this point of development.

The embryo now elgongates very rapidly, and the general shape of the adult worm becomes recognizable. The teloblasts become less and less distinct, until finally the cell rows terminate posteriorly in a group of small cells. The mesoteloblasts extend farther posteriorly than the neuro-teloblasts. New cells are always formed from the anterior surface of the teloblasts. There can be no question as to the origin of the germ bands from corresponding teloblasts, as their formation


EMBRYOLOGY OF BDELLODRILUS 177

can be followed step by step. The mesoblastic and neuroblastic portions of the germ bands can be traced to the anterior end of the embryo. The meso-teloblasts are the last to disappear. They are distinct until after the formation of the stomodaeum and its connection with the pharynx. The concrescence of the germ bands begins anteriorly and progresses posteriorly.

THE ECTODERM AND ITS PRODUCTS

The three generations of ectomeres are given entirely to the formation of the ectoderm, which later becomes differentiated into the definitive hypodermis, with its glands, the cuticle and the anterior and posterior ends of the digestive tract. The ectoderm includes, in addition to the above, all of the teloblasts, except the two larger and deeper ones which represent the mesoblasts. The reason for regarding the eight teloblasts and their derivatives as a part of the general ectoderm, is on account of their origin and position. In position, they are superficial at first and can not be distinguished from the general ectoderm, except by their arrangement in rows. Small cells are budded off from the teloblasts, which form the trunk ectoderm. In Clepsine these teloblasts are at first superficial at the posterior end of the embryo. In Lumbricus they are found directly in the general ectoderm, and beyond question form a part of it.

1. THE NERVOUS SYSTEM

The nerve chain in Bdellodrilus first appears as a double row of cells, nearly uniform in size. Each row of cells originates from a single cell, the neuroblast. The neuroblasts, when first formed, are widely separated, but symmetrical to the median axis of the body. Figures 47 and 48 (in ventral view) show their position when first formed by an equal division of the proteloblasts X and X. They take up their position on either side of the mesoblasts (figs. 47, 48, 93). When first formed the neuroblasts are turned somewhat anteriorly as shown in the horizontal section of figure 93. This movement of the cells to their final position, independent of the former position of the cleavage


178 GEORGE W. TANNREUTHER

spindle, is a common occurrence in Bdellodrilus. In some instances it is necessary to employ sections, in order to determine the origin of cells. The transverse axis of the embryo at this stage is often greater than the longitudinal (figs. 49-50). This condition persists for a brief period only, during the formation of the teloblasts. As the embryo increases in length the neuroblasts are carried more and more posteriorly (figs. 56-57).

In order to get a better understanding of the origin and orientation of the neuroblasts — X^^^ right and X* left — with reference to the other teloblasts, the figures of plate 4 are so arranged that the left side of the developing embryo corresponds to the reader's left. In figure 45 the upper pole is turned a little posteriorly, to show the upper outer edges of the entodermal cells. Figure 47 (from ventral pole) shows some of the ectodermal cells. The remaining figures are either turned forward or backward on their transverse axes. The ectomeres x** and x*^ right and left serve as good points for orientation (figs. 46-53) . After the formation of the teloblasts, bilateral symmetry is fully established. The meso-teloblasts, however in some instances, are still a little to the left of the median axis. This variation in the symmetry of the mesoblast does not in any way change the end result. In the early history of the germ band formation the teloblasts X^^^ and X^^^ are slightly separated, while X^^' and X^-^ are widely separated from the corresponding teloblast on the opposite side (figs. 56, 58). The neuroblasts and the nephroblasts begin their proliferation of cells to form the germ bands, about the same time (fig. 58). At this stage of development, the exact orientation of the embryo is distinct. Since the embryo is completely turned on itself, the further use of the terms, apical and ventral poles, is significant only as being convenient in description. The mouth, as stated above, is formed in the center of the apical pole and the anus in close proximity on the dorsal side of the tenth segment. Figure 59 (upper pole view) shows the complete curvature of the embryo. The heavily shaded portion represents approximately, the boundary between the anterior and posterior ends. This figure shows that the teloblasts are coming more and more in a straight line. Since the two ends of


EMBRYOLOGY OF BDELLODRILUS 179

the embryo are in immediate contact, it is impossible, except by longitudinal sections, to determine the exact point of separation. The ectoderm of the anterior end of the embryo, which is derived from the three generations of ectomeres is continuous with the ectoderm derived from the 'first somatoblast.'

The separation of the two ends of the embryo becomes recognizable in the early formation of the germ bands, as shown in figures 59 and 60. The posterior and ventral shifting of the neuroblasts (figs. 58-60) continues until all of the teloblasts are in a direct line. The small cells between the teloblasts are derived from the first somatoblast. In viewing the embryo from the upper pole (which now corresponds more to the anterior and posterior ends of the future animal) the germ bands extend laterally, downward and forward, being curved somewhat posteriorly as they pass from the upper to the lower pole (fig. 59). The meso-teloblasts in figure 58 are still visible from the exterior. In figure 59 they are almost grown over, while in figure 60 they are completely covered. This is due to the posterior shifting of the neuroblasts and the growth of the ectomeres from above and below. In an embryo viewed from the right side (fig. 61, a little older than fig. 60), the position of the neural and nephric rows of the germ band are shown. As the rows extend anteriorly they are more difficult to distinguish from the ectoderm. The neural rows alone can be followed to the extreme anterior end. The posterior end of the embryo is widely blunt, while the anterior end is more rounded. The heavily shaded portion represents the point of separation between the two ends.

Figure 62 represents the same embryo from the upper pole, with the ends of the embryo rotated or turned a little posteriorly. In figures 63 and 64 (from right and left sides respectively) the embryo is more elongated and the point of separation between the two ends is more distinct. The neuroblasts are lagging in their posterior extension. Their position is median ventro-posterior, as shown in figure 65. Their concrescence is not yet complete at the posterior end. In the following stages of development the cells of the neural and nephric rows divide


PLATE 5

EXPLANATION OF FIGURES

58 Embryo from upper pole, tilted a little to the right. The position of the ten teloblasts are shown; the small cells between the teloblasts on the surface are derived from x® and x^ on either side.

59 Same view as the preceding; the neuroblasts have migrated a little posteriorly and are approaching each other.

60 The ecto-teloblasts are nearly in a direct line; the germ bands have begun to form; the two primary mesoblasts M, M are no longer visible from the exterior; the transverse heavily shaded joortion shows the approximate point of separation between the two ends.

61 Embryo viewed from the left side; the posterior end is extremely blunt.

62 Same embryo as preceding, from the upper pole (upper pole corresponds to the anterior and posterior ends). Shows very strikingly the close proximity of the two ends.

63-65 Represent the same embryo from the right, left and ventral sides respectively. The ectoderm which partially covers the germ bands is not shown.

66 Embryo from upper pole; bilateral symmetry is well marked; the teloblasts are considerably reduced by the time they come in contact with their fellows on the opposite side.


180


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 5



6^ )(

Tannreuther, del.


181


PLATE 6

EXPLANATION OF FIGURES

67 Embryo turned slightly to the left to show the anterior and the posterior ends; the embryo at this stage begins to rotate within the egg membrane.

68 The same as the preceding from the ventral pole, turned a little to the left.

69 Embryo viewed from the right side; condition before the posterior end becomes drawn out or pointed.

70 Embryo from the upper pole; shows compressed condition of the two ends; at this stage the embryo rotates very rapidly.

71 Embryo viewed from the right side; the teloblasts are partially visible at the posterior end; the tapering of the posterior end is well marked.

72 Embryo to show the overlapping of the ends ; indications of segments are visible anteriorly; the stomadaeum is distinct.

73 Unusual condition, where the two ends remain in immediate contact until after the form of the worm is distinct; this occurs in eggs with an unusually large cocoon.

74-6 Different stages in the final growth of the embryo.

77 Condition of embryo at the time of emergence from the egg.


182


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 6



75

Kline and Tannreuther, del


183


184 GEORGE W. TANNREUTHER

very rapidly and gradually become covered by the ectodermal cells as they sink beneath the surface (figs. 66-72), and form the middle stratum of the germ bands. In a nearly median longitudinal section (fig. 99), the relation of the parts are shown. The neural plate at different points shows the formation of ganglia. The anterior end of the section passes through the exact median plane and does not show any gangha. The neuroblasts still persist at the posterior end.

The entire nervous system arises as two simple longitudinal rows of cells, and each row is produced by the continued proliferation of cells from a single cell, 'the neuroblast.' This proof is established by the study of surface preparations in connection with sections taken in different planes through the developing neural elements. The neural rows can be followed to the extreme anterior end, where they pass up around the pharynx and give rise to the cerebral ganglia on either side (fig. 96) by a thickening of the anterior extremity of the corresponding neural rows. There are exactly four rows of cells in the middle stratum of each germ band. The outline of the rows can be easily seen in surface views of the living embryo (figs. 63-66). They are more distinctly marked at the posterior ends, and become less distinct anteriorly, which is due to the more advanced development; that is, each row becomes double, then triple, etc. (figs. 68-69) and at the same time, its boundary lines become less distinct.

2. THE EXCRETORY SYSTEM •

After the bilateral division of the 'first somatoblast,' each proteloblast contains the neural and nephric elements of their respective sides. According to Whitman, these two cells are called the neuro-nephroblasts. But when each proteloblast X, X divides equally the neural and nephric elements become separated, X*^^ neural and X*-' nephridial (figs. 47-48). The nephroblast X'-' on either side next buds off a very small cell x^, which becomes ectodermal (fig. 49) . Immediately after the formation of this small cell, X'-' divides nearly equally, and forms


EMBRYOLOGY OF BDELLODRILUS 185

X'-' andX*^' on either side (figs. 50-54). Both cells are nephridial. This fact perhaps is made more suggestive by the behavior of X*"' and X'^\ Either of these cells may divide equally, but never both in the same embryo. In either case we have three teloblasts derived from the nephroblast X'^* on either side. The cells of the nephridial rows are somewhat smaller and narrower than those of the neural rows. In some cases the outer nephridial row of cells is very short. In other embryos it is composed of but one or two cells and its presence is hard to verify, suggesting a possible disappearance in the group. As stated above, the nephridia arise in connection with a continuous nephric cord of ectoblastic origin, which forms a part of the middle stratum of the germ band and lies along side of the neural row. Each nephric cord terminates at the posterior end in three teloblasts. Thus the entire nephric cord of cells is formed by the continued division of the 'nephroblasts,' which agree precisely with the neuroblasts in structure, action and mode of origin. The nephric cord at first is composed of three rows of cells posteriorly, but passing forward the rows are no longer definitely separated and. the nephric cord or plate consists of an irregular series of cells which extend anteriorly to the posterior end of the pharynx. The formation of the nephridia progresses from in front backwards and keeps pace with the formation of new segments in the embryo. The beginnings of a pair of nephridia are found in each of the main segments. Only two pairs of nephridia are retained in the adult worm. The details of the formation of these segmental organs have not been worked out.

Berg considers the entire nephridia in Criodrilus as mesodermal in origin; Whitman held the extreme opposite view, that the entire nephridium was ectodermal in origin; while Wilson regarded the nephridia as being part mesodermal and part ectodermal in origin. In Bdellodrilus the nephridia are ectodermal. The anterior pair occurs in the first, second, third and fourth body segments. The left nephridia of the anterior pair, extends from the first to the third segments inclusive, while the right extends from the second to the fourth segments inclusive. Both


186 GEORGE W. TANNREUTHER

have a common opening on the dorsal side of the third segment. The funnel of the left occurs in the second and. that of the right in the third segment. The posterior pair is found in the eighth segment. Each nephridium has a separate opening to the exterior on the dorsal side of the eighth segment.

GROWTH

The developing embryo does not increase appreciably in bulk until after the teloblasts are formed. Up to this period it is merely a division of the egg content into the various cell complexes. Even at this stage the increase in the long axis of the embryo is brought about by a decrease in the transverse diameter. Figures 50 and 55 show the transverse axis greater than the longitudinal, while in figure 56 and 57 the longitudinal axis is greater, due more to a change in shape than to growth. The egg content is very plastic and when removed from the cocoon the egg membrane, in most cases, is not of sufficient tenacity to retain the embryo intact. The ten teloblasts are shown in figures 56 and 57.

The first increase in length is due to the formation of the mesoblastic portion of the germ bands (text figs. 17-18). The neuroblastic and nephroblastic portions of the germ bands begin simultaneously after the meso-teloblasts have formed eight or ten cells (text figs. 15, 18 and fig. 58). Figures 58-71 show the various stages in the formation of the germ bands. Figure 71 is about the last stage when the germ bands can be detected externally. A longitudinal section of figure 71 near the median axis shows a differentiation of the germ bands into their incipient organs (fig. 99). From this point of development, growth is very rapid, and the embryo begins to rotate on its transverse axis. The movement is produced by the action of cilia, which occur on the large ectodermal cells in the median ventral half of the anterior end of the embryo (figs. 96-99). These ciha disappear before hatching, but the cells from which they are produced persist as a part of the ectoderm. The anterior and posterior ends are no longer in immediate contact, as in figure 71, but begin to overlap. The ends of the embryo


EMBKYOLOGY OF BDELLODRILUS 187

now take the position within the egg membrane of the least resistance to their further growth. Figure 74 shows the overlapping of the ends. The stomodaeum is completely formed and the annuh of the pharynx are visible. Figure 73 shows an unusual condition in the position of the ends. At this stage of development the animal often turns on its longitudinal axis, largely on account of the action of the muscles, and, instead of the convex side being ventral, it now becomes dorsal. This rotation on its longitudinal axis has no significance, as has been thought by previous investigators, in the later stages of development. The animal is extremely plastic and may assume any position or shape, as shown in figures 74 and 76. Figures 77 shows the completely developed animal at the time of emergence from the cocoon. The number of the segments is distinct. This peculiar form of growth within the cocoon is merely adaptive. Occasionally, when the cocoon is of an unusual size, the developing worm is less bent on itself.

A COMPARATIVE STUDY OF DIFFERENT FORMS

In following the cleavage cells of annelids, molluscs and polyclades, one is impressed with the striking resemblances in their different stages of development. If this marked similarity alone were a sufficient criterion for a basis of classification, some of the most widely separated forms, when considered from the standpoint of their early development, would be grouped as closely related species. How can such resemblances in development be explained in animals which are so unlike in their late stages of growth? Are they merely the result of such mechanical principles as surface tension, alternation of cleavage, and pressure, or is the nature and structure of the protoplasm the common cause? According to Driesch, 'the striking similarity' between the types of cleavage in annelids, molluscs and polyclades does not appear startling and is easy to understand, since cleavage is of no systematic worth. However, the more recent investigators on cell lineage, according to Heath, look upon the early cleavage stages as something more than a mere


188 GEORGE W. TANNREUTHER

manifestation of simple mechanical forces. Rather are the blastomeres the expression of the active intrinsic forces, which control development from the earliest stages unto the end. Gravity, surface tension, cohesion and pressure no doubt are effective, but not to the extent that they become the controlling or coordinating agents in development. The early cleavages are as important as those occurring in later life, and may even be considered more so. Also the long continued resemblances which exist in the development of these different forms from the earliest segmentation of the eggs are as fundamental and deep seated as are the homologies existing in the adults."

The number of these resemblances in the annelids and molluscs is surprisingly great. In all forms accurately studied, the first three generations of ectomeres give rise to the entire ectoderm. The mesoblast arises at the fourth division of the posterior macromere D. The remaining members of this quartette and the macromeres produce the entoderm. The division and position of the cells up to the twenty-four or thirty-cell stage are identical in many different species. Beyond this point Wilson believes a divergence between the two classes ensues, and that development proceeds upon two entirely different lines. However, subsequent investigators have shown that the supposed differences are more superficial, and that the points of resemblances become more numerous and extend throughout longer periods of development. Lillie ('95) showed that points of resemblance existed in the lamellibranchs and the annelids, and that in both classes there is an essential similarity between the development of the 'first somatoblast.' In annelids this structure develops to a greater extent than in Unio, but the two have many points in common.

Mead ('97) and Conklin ('97) showed that the rosette series had the same origin and position in aimeUds and molluscs, and that in both it probably gave rise to the apical sense organ. According to Conklin, it also gave rise to the cerebral ganglia, while Mead considered this particular point doubtful. Furthermore, Mead in his annelid studies demonstrated that the same cells in five different annelids gave rise to the entoderm; that


EMBRYOLOGY OF BDELLODRILUS 189

the head kidney in Amphritrite and Nereis developed from the same cells. Conklin further states that the axial relation of all the blastomeres, with the possible exception of the macromeres, are the same in both the annelids and molluscs, and that the larval mesoblast in Crepidula and Unio arises from the same group of ectodermal cells.

Heath ('99) found that the prototroch in anneUds and molluscs was homologous, and that the twenty-two to twenty-five cells concerned have exactly the same origin, direction of cleavage, and destiny. Also that the remainder of the first quartette, forming the head vesicle with its rosette series and moUuscan cross cells or intermediate girdle cells, has in all probability, the same fate in both. He found many other resemblances and concludes :

Thus it is seen that not only in the origin and position of the various quartettes do resemblances appear, but that the early cleavage of these are in many cases cell for cell the same. In later stages close cell homologies cease, but the relation of the cell groups and their development in giving rise to larval or adult structures follow along much the same path. After passing these facts in review and considering the various structures in detail and modifications which they undergo, one fact presents itself with greatest clearness, that between Ischnochiton and the annelids the resemblances are moref undamental and closer than are the differences.

For a more direct comparative study of Bdellodrilus with the annelids and molluscs, special references will be made to Clepsine (Hirudinea), Dinophilus (Polychaete) , and Unio (Lamellibranch). In all these forms the first and second cleavages are meridional and divide the eggs into four unequal macromeres (text figs. 19-22). In Dinophilus C and D are approximately posterior and A and B are anterior. In the other three forms B is anterior, D posterior, C right and A left. In each case D is the largest cell; A, B and C are nearly equal; B is usually the smallest when variation occurs. The eight-cell stage has the same structure, and in all probability arises in the same manner in the four forms, the only apparent difference being the much greater relative size of the ectomeres in Dinophilus than in the three remaining forms. The first cleavage plane in Bdellodrilus


190


GEORGE W. TANNREUTHER




Fig. 19 Four-cell stage of Unio, upper pole (after Lillie).

Fig. 20 Four-cell stage of Bdellodrilus, upper pole.

Fig. 21 Four-cell stage of Dinophilus, upper pole (after Nelson).

Fig. 22 Four-cell stage of Clepsine, upper pole (after Whitman).

occurs at nearly right angles, while in Unio and Clepsine it is inclined at an angle of about forty-five degrees to the sagittal plane of the future adult. In Dinophilus the direction of the first cleavage is in doubt. The second cleavage plane in Unio, Clepsine and Bdellodrilus occurs at an angle of about fortyfive degrees to the sagittal axis. The origin of the ectoderm, the entoderm and the mesoderm is approximately the same in each form.

1. THE FIRST SOMATOBLAST

The first somatoblast in each instance is derived from the large posterior macromere D (text figs. 23-26). The cell d(X) is extremely large and occupies a median posterior position. In Clepsine (Whitman) d- (X) is called the 'neuro-nephroblast.'


EMBRYOLOGY OF BDELLODRILUS 191

It divides into two, four and finally eight large cells called the teloblasts; the middle stratum of the germ bands is derived from them. These eight teloblasts are arranged into two groups of four cells each. Each group, which later is composed of four rows of cells, produces the middle stratum of the germ band on the corresponding side. The inner row of each band lies ultimately near the median ventral plane and gives rise to the corresponding half of the nervous system. The adjoining rows — 'nephroblasts' — give rise to the nephridia. The derivatives of the outer row are still in doubt, but probably take part in the formation of the ectoderm.

In Dinophilus (Nelson) d^ (X) is formed by a laeotropic division of the macromere D (text fig. 25) ; D is much smaller than X. Immediately after the formation of X, x^ is budded off to the right at a low level. Next x^ is budded off to the left at a higher level than x^; x^ is next formed by a dexiotropic division from the dorsal side, a little to the left. Next X divides equally and produces X and X, right and left. These two large cells correspond to the proteloblasts in Bdellodrilus. Finally X on either side divides equally, and produces the two teloblasts on each side of the median plane. These four cells, according to Nelson, correspond to the posterior teloblasts of Nereis. They also correspond to the neuroblasts and nephroblasts of Bdellodrilus. The division of X in Dinophilis and Nereis differs no more than do the corresponding divisions in Nereis and other annelids (Amphitrite, etc.). At the time of the closure of the blastopore in Dinophilus, the descendants of X are distributed dorsally and laterally to the posterior stem cells. In Neries the main bulk of the descendants of X lay on the vegetal side of the stem cells.

In Unio (Lillie) the 'first somatoblast' X is formed by an unequal division of D (text fig. 24) in a median posterior position; xi is budded off from X, just behind C on the vegetal pole; next x^ is budded off from X symmetrically with x^ on the right side, just posterior to d^; next x^ is formed from X towards the apical pole, posterior to d'-^; then x^ is budded off from X anteriorly, towards the vegetal pole. This division of X does

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


192


GEORGE W. TANNREUTHER




Fig. 23 Nine-cell stage of Clepsine, upper pole (after Whitman).

Fig. 24 Nine-cell stage of Unio from behind (after Lillie).

Fig. 25 Nine-cell stage of Dinophilus, left side (after Nelson).

Fig. 26 Nine-cell stage of Bdellodrilus, left side.

not occur in this manner in Dinophilus, Bdellodrilus or even in Nereis. The fourth division in the above three forms is equal and bilateral, while in Unio the fifth cleavage of X is the first bilateral division and forms X, X right and left. Next X, X on either side divides nearly equally and gives rise to the shell gland. These four cells might be regarded as the posterior teloblasts, which occur in other forms, as in Nereis and Dinophilus.

In Bdellodrilus X is formed by an equal division of the macromere D (text fig. 26), and takes a median posterior position First xi is budded off from X to the right, posterior to C. Then x2 is budded off to the left, symmetrical with x^ and posterior to d^ Next x^ is formed from the median dorsal anterior edge of X, between d^ and c^ Now the first bilateral division of X takes place and forms the proteloblasts X, X, right and left. Each of the proteloblasts bud off x^ x^ and x" respectively. At the


EMBRYOLOGY OF BDELLODRILUS 193

next division each proteloblast divides nearly equally, and gives rise to X^i\ neuroblast, and X*^^, nephroblast, on each side of the median axis of the embryo. Next each nephroblast divides nearly equally and produces X'-'^ and X'^\ Now a very interesting thing happens; either X^^) or X^^^ divides (but never both in the same egg) and produces the three nephroblasts on each side, which are designated as X^^)^ x^^' and X^^*'. In Clepsine only two of these teloblasts are concerned in the formation of the nephridia. The lateral teloblasts, as stated above, are probably ectodermal.

These four forms unquestionably show that there is a marked similarity in the cleavage of the 'first somatoblast,' not only in widely different individuals in the same group, but in individuals of widely separated groups. This comparison could be extended to other groups or forms, but the above will suffice for our purpose.

2. THE SECOND SOMATOBLAST

In Clepsine, the 'second somatoblast' has rather a unique origin. It is formed at about the twelve-cell stage. D, after the formation of X, becomes directly the 'second somatoblast' or M. (These cells are differently designated by Whitman; D is represented by x, X by x^ and the mesoblasts by x and xy.) M divides nearly equally and produces the right and left mesoblasts, from which the inner stratum of the germ bands is formed.

In Bdellodrilus, M is formed by a very unequal division of the macromere D, at the twenty-four-cell stage (fig. 85). The larger cell or M is formed in front of X. It is inclined a little to the left of the median axis. The first division of M is equal, producing the mesoblasts, one on either side. These primary mesoblasts now bud off a number of small cells, directly beneath d^ and c^ (figs. 86-87). It is very difficult to make out the exact number of these small cells, since they are not visible externally. There are at least twelve formed, six on either side from each mesoblast. After this small group of secondary mesodermal cells are formed, the mesoblasts M, M, give rise to


PLATE 7

EXPLANATION OP FIGURES

78 Two-cell stage, horizontal section; CD dividing.

79 Four-cell stage; horizontal section taken above the center of the egg.

80 Same as the preceding, with plane of section below center.

81 Nine-cell stage, parasagittal section, to right of median plane.

82 Horizontal section of a nine-cell stage, taken four sections from the top. Taken from embryo composed of 21 sections, each eight micra in thickness.

83 Same as the preceding; sixth section from top.

84 Same as figures 82 and 83; fifteenth section from top.

85 Parasagittal section of a twenty-four-cell stage; plane of section little to left of median axis. This figure shows the unequal division of the macromere D in the formation of the second somatoblast.

86 Thirty-three-cell stage. Parasagittal section to left of the median plane. Shows the formation of the secondary mesodermal cells (m cells) from the primary mesoblasts.

87 About the same stages as the preceding, to show the distribution of the yolk in different cells.

88 Horizontal section of an eighteen-cell stage, fourth section from top. Series composed of 20 sections, each eight micra in thickness.

89 Same as the preceding; seventh section from top.

90 Taken from series same as figure 88; eighth section from top.


194


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 7



88

Tannreuther, del


195


PLATE 8

EXPLANATION OF FIGURES

91 Taken from the same series as figures 88 to 90; eighth section from the top.

92 Horizontal section of an eighteen-cell stage; sixth section from the top; shows the nem-oblasts and nephroblasts.

93 Same as the preceding; tenth section from top; this figure shows the persistence of the cleavage spindles after the cell membranes are distinct.

94 Same as 92 and 93; fourteenth section from top; taken from a series of 20 section each eight micra thick. This figure shows the upper side of the macromere D wedged in between the other cells.

95 Transverse section of an embryo represented by figure 64; section taken at plane 2 — 2, or at a region corresponding to plane 2 — 2, of figure 98.

96 Transverse section of stage corresponding to figure 63; section taken at plane 2 — 2 ; figures 95 and 96 shows germ bands only partially covered by the ectoderm.

97 Transverse section of embryo represented in figure 69; section taken at level marked by line 2 — 2; here the germ bands are completely covered by the ectoderm.

98 Longitudinal section, near median line, of stage represented by figure 69.

99 Longitudinal section of an embryo represented bj' figure 71 ; plane of section near median line.


196


EMBRYOLOGY OF BDELLODRTLUS

G. W. TANNREUTHER


PLATE 8



98 P ^^WM

Tannreuther, del.


197


198 GEOKGE W. TANNREUTHER

the inner stratum of the germ bands. The plane of division in the formation of the primary mesoblast is at right angles to that of the secondary.

In Dinophilus, M is formed at the twenty-nine-cell stage, by an unequal division of D. M is much larger than D, as in Bdellodrilus, and is in front and below X, slightly to the left of the median plane. The division of M is now delayed until the seventy-two-cell stage, when two small cells are budded off anteriorly towards the vegetal pole, close to the line of junction of the two mesoblasts. At the next division two small cells are budded off, one on either side of the first pair. The following cleavages are teloblastic and produce the mesoblastic bands. The mesoblasts do not move into the cleavage cavity as in many other annelids, but remain on the surface until covered by the ectoderm.

In Unio, at the thirty-two-cell stage, M is formed by a very unequal division of the macromere D. The first division of M is equal and bilateral. Their position is immediately behind the entomeres. The next division of the two mesoblasts is very unequal, two small cells, m, m, are budded off at the posterior lip of the blastopore. Later the mesoblasts are included within the segmentation cavity, where they take up their final position behind the archenteron and give rise to the definitive mesoblastic germ bands with lateral teloblasts.

From the forms compared above it is very evident that there is a remarkable similarity with Bdellodrilus, not only in the early cleavage stages, but in the establishment of the germ bands as well. Thus cells having the same origin and lineage have the same final result in a wide series of forms (d"* the mesoblasts). Again, cells of unlike origin have a different fate (first and second somatoblasts) ; or cells of a different origin may have the same fate (d^ of annelids and the second and third generation of ectomeres in polyclades) . Then cells of the same origin may have a different fate (a-- in Unio and Bdellodrilus). These contradictions, however, are far less striking than the resemblances. The 'first somatoblast' in each of the above four forms gives rise to the ventral plate and all or nearly all of the trunk ectoderm, while


EMBRYOLOGY OF BDELLODRILUS 199

the 'second somatoblast' produces the definitive mesodermal elements of the adult animal.

3. VARIATIONS IN THE METHOD OF MESODERMAL FORMATION

All annelids and molluscs which have been carefully studied show that the ectoderm arises from the three generations of ectomeres, the mesoderm from M and the entoderm from the remaining cells. There are, however, a few minor variations in forms like Clepsine, Crepidula and Nereis. In polyclades the mesoderm is directly associated with the ectomeres. The second and third generations of ectomeres, as in Discocoehs, produce the mesoblast, and the macromeres the entoblast. In molluscs and annelids the mesoderm is more closely associated with the macromere D. There is considerable variation in the cleavage of D in the formation of the 'second somatoblast;' in some forms D is given over entirely to the mesoderm; in other forms it shares equally, or in still others it contributes but little to the mesoderm.

Forms in which the mesoblast has two sources

(a) Ecto-mesoblast and (b) coelo-mesoblast. In Thalassema, Torrey distinguished between ecto-mesoblast, from the ectoderm and coelo-mesoblast from M. He states that the coelomesoblast is present in two bands, each consisting of five sub-equal cells. These are closely applied to the body wall and lie in the usual position on each side of the neural rows, but are more widely separated than in most anneUds. The ecto-mesoblast on the other hand, derived from the first and second quartette of ectomeres, is present in great abundance and many of the cells have already undergone considerable differentiation in the formation of the muscles. He further states that the two mesoblast cells, M, M, are the last to sink in at gastrulation, instead of the first, as in the case where development is more direct (Nereis and Amphitrite). The two coelo-mesoblast bands of five cells each are shown to have the same origin and early history as the mesoblast bands in other annelids. The coelo-mesoblast, which is meagerly developed in the trochophore, is clearly correlated with


200 GEORGE W. TANNREUTHER

the long duration of its free swimming and almost stationary larval existence. In all forms where there is a tropchophore stage of long duration, as is the case in all annelids with equal cleavage, the two coelo-mesoblast cells do not, in the early stages at least, bud like teloblasts. This is tue in Hydroides, some species of Polygordius, in Thalassema and Podarke.

Many of the annelids and molluscs show that the so-called ecto-mesoblast (designated, as larval mesoblast or as mesenchyme by some authors) arises from certain ectodermal cleavage cells of the second or third quartette of ectomeres and. is entirely independent of the coelo-mesoblast. In Thalassema (Torrey) ten large ectodermal cells sink in. from the ectoderm and. give rise to all the mesenchyme. Three of these cells are from the a, c and d quadrants of the third, quartette and seven from the first quartette of ectomeres. The most important source of mesenchyme in Thalassema is from the three cells of the third quartette (3d.-"-\ 3c- •^■-•\ and Sa---). The first two sink into the cleavage cavity, just before gastrulation and at first lie close to the two coelo-mesoblast cells. They soon migrate laterally and bud off simultaneously small cells toward, the M cells. They divide like teloblasts, but in the reverse order to the ordinary direction. So close is the connection of these cells with the coelo-mesoblast that one would be certainly led to think that they formed a part of these bands, unless their cytogeny had been carefully followed. Similar conditions are described by Treadwell in Podarke obscura. The progeny of these two cells forrn almost the entire mesenchyme of the post throchal region and become differentiated for the most part into muscles of the digestive tract. The progeny of the other ectodermal cell migrates to the mid-ventral line. The ecto-mesoblast cells of the first quartette sink into the primary body cavity later than those of the third; their exact cell lineage has not been traced, but probably give rise to gut musculature. This mesoblast has commonly been considered as purely larval and transitory. In some instances it is possible to determine its exact origin, but in many others merely the general region from which it arises.


EMBRYOLOGY OF BDELLODRILUS 201

During the last thirty years embryologists have differed in their conception of the origin of the mesoderm and of its phylogenetic significance. Hatschek ('78) was among the first to distinguish between mesenchyme and mesoderm, but held, after studying the embryology of Polygordius, Echiurus and Eupomatus, that these two morphologically different mesoblasts, arise from a common foundation. This same view was later put forth by the Hertwigs ('81) in their 'Coelomtheorie,' which, according to Meyer, has formed the foundation of all later work on mesoderm. Roule ('89 and '94), Burger ('91 and '94), Fraipont ('88), Hacker ('95), and others, have described the mesoblast as having a single origin. On the other hand, those who have studied the embryology of annelids and molluscs, consider the origin of the mesenchyme distinct from that of the mesoblast or coelo-mesoblast. This later view was first described by Kleinenberg ('78 and '86), and later by Whitman ('87), by Berg ('90), by Schimkewitsch ('94), by Meyer ('01), by Torrey ('03), and others.

A larval mesoblast was first described by Lillie ('95) in Unio. It arises asymmetrically from the derivatives of a- - and later migrates into the segmentation cavity, where it divides equall}^ and. becomes symmetrically arranged on either side of the midline. The derivatives of these two cells become metamorphosed into 'myocytes' and larval adductor muscles, which are functional only during larval life.

Treadwell ('97) regards both mesenchyme and mesoderm as morphologically the same tissue, the apparent difference in their mode of origin being of no significance. And, further, Wilson regards the larval mesoblast (ecto-mesoblast, because of its origin from the ectoderm) as a distinct tissue from that of the definitive mesoblast or ento-mesoblast, and states that it is homologous with the mesenchyme of the turbellarian ancestors of the annelids, while the mesoblast from which the adult structures arise is phylogenetically younger and is represented prophetically in the ontogeny of such a form as Discocoelis (polyclade) by the peculiar lateral division of M, and states that the ecto-mesoblast and endo-mesoblast are phylogenetically of


202 GEORGE W. TANNREUTHER

different origin. This same point was previously urged by Meyer.

The condition, however, found by Wilson in Nereis and Lumbricus does not indicate a hard and fast distinction between the two kinds of mesoblast. In Nereis, cells from the anterior end of the germ bands separate early and pass forward into the segmentation cavity where they give rise to the larval musculature. This corresponds exactly in structure and function with the larval mesoblast of Unio (Lillie) and Podarke (Treadwell). In Lumbricus the origin of the mesenchyme is similar to that in Nereis. These two kinds of larval mesenchyme have also been described by Eisig ('98) as occurring in the same individual (Capitella, a polychaete annelid).

In Thalassema and Podarke the larval mesenchyme arises directly from the ectoderm, while in Nereis and Lumbricus it arises from the anterior ends of the mesoblast bands. According to Treadwell, no one has yet proven that no 'mesenchyme' arises from the germ bands in cases where a larval mesenchyme exists. If we accept Wilson's view that mesenchyme and mesoderm are different phylogenetically, we must regard the two sets of larval mesenchyme which have the same structure and function, as non-homologous, or we must regard the mesenchyme and mesoderm as morphologically the same tissue and the difference in their modes of origin as of no significance. Furthermore, Wilson has pointed out that the trochophore, as it occurs at present, is more than a mere ancestral stage, for it contains in a concentrated form the anlage of the whole future body. According to Mead, the ectoderm behind the first septum in Amphitrite arises from a group of cells which surround the proctodaeum of the young trochophore and are descended from a single cell, the 'first somatoblast.' The same is true of other trochophore forms. There is no need to assume phylogenetically a new formation of ectoderm for the body as distinct from that of the head. Neither is there any necessity to assume a distinct phylogenetic origin of the larval mesoblast from that of the mesoderm.


EMBRYOLOGY OF BDELLODRILUS 203

It is evident that in Nereis and Lumbricus, both kinds of mesoblast have the same origin, and simply shows a more complete concentration of the mesoderm than in Thalassema and Podarke, where the mesenchyme is formed direct from the ectoblast. The mesoblast cells collected at the posterior end of the trochophore, which are derived from M, represent the mesoderm of the body. It is morphologically continuous with that of the head, as in Nereis, and is concentrated at this point to provide for the elongation as new segments are formed. The difference in the concentration of the mesodermal elements, as to whether they have a single or double origin in no way interferes, as already pointed out, with the morphological unity of the tissue, and as to the source of its origin, whether from the ectoderm or from the endoderm phylogenetically, we are not able to say (Treadwell).

Meyer ('01) in his study of the phylogenetic significance of the two kinds of mesoblast, gave a view directly opposed to that expressed by Treadwell. After an exhaustive review of the whole mesodermal question, he concludes that the great mass of evidence, both embryological and anatomical, points to the conclusion that in annelids, at least, there are two entirely distinct forms of mesoblast, the ecto-mesoblast (primary mesoblast) and the coelo-mesoblast (secondary mesoblast). Of these two he considers the primary mesoblast to be phylogenetically the older, and as a rule, to be derived from the ectoderm. The coelo-mesoblast, on the other hand, is regarded as a later formation, which has originated from the gonad cells.

The formation of the ecto-mesoblast in annelids and molluscs from certain cells of the first, second, third and fourth generation of micromeres, can well be regarded as vestiges or survivals of the process which occurs in all four cells of the second and third quartettes of certain polyclads. The origin of the ecto-mesoblast from the ectoderm in annelids and molluscs, partially bridges the gap between them and the polyclads. In order to have a complete homology of the mesoderm in the polyclads, annelids and molluscs, it is necessary to find a polyclad in which there is a double origin of the mesoderm. The development of the polyclad Leptoplana (Wilson) is the nearest representative to complete the homology. In Leptoplana only a part of the four quadrants of the second quartette contributes to the entire mesoderm, the typical condition in polyclads being that all of


204


GEORGE W. TANNREUTHER


the second and third quartette is mesodermal. The behavior of d^ in the polyclad Discocoehs, is also very suggestive. Here the division of d^ is equal and gives rise to two symmetrically placed cells at the posterior end of the embryo, comparable to the primary mesoblasts found in annelids and molluscs. Some investigators have even suggested that these two posterior cells in the polyclads may give rise to the mesoblast bands in this particular group. This latter point, however, has never been verified.

Table 3 shows that the first, second, third and fourth generation of micromeres, in a series of widely separated forms, may contribute to the formation of the mesoderm.


TABLE 3



1st gen.


2d gen.


3d gen.


4th gen.


Annelids:






Thalassema


part of a, b and c quad's


none


1 cell each of

a, b and c

quadrants


d^ part mes.


Bdellodrilus


none


none


none


d^ all mes.


Molluscs:






Unio


none


a2-2 (larval)


none


d^ all mes.


Crepidula


none


aS b^, c2


none


d^ part mes.


Physa


none


none


b^, c^


d^ part mes.


Podarke


none


none


a^3-2-2-2 (,3-2-l-2. (J3-2-2-2


d^ part mes.


Polyclads :






Discocoelis


none


all mes.


all mes.

none


Leptoplana


none


part of each quadrant


none


none


In case of the ecto-mesoblast a complete series could be arranged, in which all of the cells of certain quartettes contribute to the mesoblast, to those forms in which only a small part of certain quartettes is mesoblastic. Again in case of the coelo-mesoblast we have a wide range of variation, in which all of d^ is mesodermal, to those in which only a small part of d^ is mesodermal. As far as records show, Capitella is the only annelid in which


EMBRYOLOGY OF BDELLODRILUS 205

d^ does not contribute to the coelo-mesoblast. Here then we have quite a unique series ranging from those forms where the entire mesoderm is ectodermal in origin, or where it is both ectodermal and entodermal, to those where it is entirely entodermal. From the above it is evident that the entire mesoblast of polyclads is derived from the ectomeres, and, if homologies be any significance, it would be fair to conclude that this mesoblast is represented by the ecto-mesoblast in the annelids and the molluscs.

The origin and development of the mesoblast in Bdellodrilus contributes but little to the phylogenetic significance cf the primary and secondary mesoblast. Here, beyond question, when considered from the standpoint of their origin, they are one and the same tissue. Both are formed directly from the primary mesoblasts. The secondary mesoblast cells are budded oE from the two primary mesoblasts before the germ bands begin their development. Similar conditions are found in other forms, as in Lumbricus; here, however, the secondary mesoblast is formed later directly from the anterior ends of the mesoblastic germ bands. The difference is only in the point of time in their formation. In Bdellodrilus there can be no hard and fast distinction made between the two kinds of mesoblast. Both must be considered as the same tissue phylogenetically.

4. VARIATIONS IN THE SOURCE OF THE ENTODERM

In general, as stated above, the ectoderm originates from the three generations of ectomeres, the mesoderm from d^, and the entoderm from the remaining cells. The origin of the three germ layers, however, depart somewhat from the above rule in some of the annelids and molluscs. In some species cells from the first, second and third quartettes contribute to the mesoderm; in others d^ gives rise to entoderm as well as mesoderm. In all annelids and molluscs. A, B and C, after the formation of the first three sets of ectomeres, are distinctly entodermal. The macromere D, after the formation of d^ is likewise entodermal. In some forms D is the same size as its fellows, in others


206 GEORGE W. TANNREUTHER

it is reduced until it is little more than a mere nucleus, while in others it has completely disappeared as an entomere, and is given over entirely to the formation of mesoderm.

In annehds, in a gradually decreasing series, D (Nereis) is the same size as the entoblast cells A, B and C. In Dinophilus it is about half the size of these cells. In Bdellodrilus D is little more than a mere nucleus; while in Clepsine D is given over entirely to the formation of the mesoderm. In molluscs it is a fairly common condition to find the entoblast cell D smaller than A, B and C, or even greatly reduced. In Crepidula it is very little reduced; in Unio it is more than half reduced, while in Ischnochiton, D is often little more than a mere nucleus. The second somatoblast, M, may contribute to the formation of entoderm as well as mesoderm. In forms like Crepidula M is mostly entodermal. In Fiona (Casteel) the division of M in the formation of the entoderm is very similar to that in Crepidula. In Unio two small cells are budded off from M, which lie near the entoderm, and are probably concerned in the formation of that layer.

In some of the annelids the primary mesoblasts bud off small cells directly posterior to the macromeres. This number varies; in Nereis there are six to ten, and in Aricia there are but two. In many of the other annelids and also in some of the molluscs, where their cell lineage has been traced, it is found that these small cells give rise to entoderm. There are at least sixteen to twenty species of annelids and molluscs in which similar cells have been found (small cells from the primary mesoblasts.) Diverse accounts of their behavior and fate have been given by different investigators. Table 4 shows the fate of these small cells in a few of the annelids and molluscs.

In the mollusc Alpysia, according to Carazzi, each primary mesoblast buds off four small cells. Three of these are mesoblastic and one is entoblastic. This interesting condition might be considered as a transitional form or as a connecting link between those forms in which these small cells are entirely mesodermal and those in which they are entodermal. Again we could arrange a series of annelids and molluscs in which at one extreme


EMBRYOLOGY OF BDELLODRILUS


207


TABLE 4


ENTODERM


MESODERM


NOT CERTAIN


Crepidula


Amphitrite


Dreissensia


Nereis


Arenicola


Patella


Podarke


Umbrella


Spio


Thalassema


Planorbis


Serpulorbis


Fiona


Unio?


Cyclas


Ischnochiton


Limax


Aricia


Physa fontanalis




Phj^sa hyponurum




Aplysia




the entoblast derived from M is greater in amount than the mesoderm, as found in Crepidula, and at the other extreme, where but two rudimentary cells of M are entoblastic, as in Aricia.

According to Wilson, a series of this nature may indicate a gradual elimination of the entodermal element from the macromere D of the fourth quartette, and finally its complete transformation into the mesoblast. Kovalevksy ('71) suggested that this transformation shows quite forcibly that the mesoblast pole cells are to be regarded, phylogenetically, as derivatives of the archenteron, because of their close association with the posterior entoblast cell, D.

The primary entoblasts. A, B, C and D, undergo but little change until late development in those forms which possess a larval stage, and may remain in this condition until after the trochophore is developed, or until after the blastopore is closed. In those individuals with a fetal type of development, they often remain distinct until after the germ bands are completely formed, as in Clepsine.

GENERAL ADAPTATION AND INTERPRETATION OF CLEAVAGE

The cleavage of eggs of widely separated forms exhibit unique resemblances. At certain stages of development these resemblances exceed their differences. Is the persistence of these features due to the influence of ancestral inheritance, or are they due more to the adaptive conditions of their environment, to


JOURNAL or MORPHOLOGT, VOL. 26, NO. 2


208 GEORGE W. TANNREUTHER

meet the highest need of the developing animal? It has been demonstrated, again and again, in anneUds, molluscs and even in polyclads, that homologous cells of like generations give rise to like parts in the developing embryo and the adult. The occurrence of these conditions in such widely separated forms furnishes a very interesting and important phase in the study of cell hneage. The tendency has been rather to emphasize these resemblances, than to give special stress to the exact conditions which occur in any one species in its different stages of development. It is true, however, that the general form of cleavage may be inherited from a long series of ancestors, probably from some of the Turbellarian worms. But the problem of more direct importance in any one group is, why such variation in the size, form, direction and rate of cleavage?

1. IN THE CLEAVAGE OF BDELLODRILUS

In Bdellodrilus we have a determinate type of cleavage, i.e., the fetal as well as the adult structures can be shown to have a definite or direct cell lineage, and can be traced back to the unsegmented egg. The structure of the ovum is quite homogeneous, and at the time of maturation, the egg can be definitely oriented as to the future axis of the body. Before the first cleavage is complete, the parts of the ovum which give rise to the different germ layers can be traced or ascertained with a fair degree of accuracy, i.e., definitely localized parts which • give rise to definite organs or structures.

"Adaptation in cleavage can manifest itself only in three possible ways or modes of cleavage variation, which are, as has been pointed out by Lillie, Mead, Conklin and others, the following: first differences in the rate of cleavage; second differences in the size; and third, differences in the direction of cleavage."

The general plan of cleavage in Bdellodrilus, is similar to that of other forms. The ectoderm is derived from the four basal cells, by three successive horizontally formed cleavages. The mesoderm from a fourth cleavage of the posterior macromere D and the entoderm from the remaining cells. The first cleavage


EMBRYOLOGY OF BDELLODRILUS


209


in Bdellodrilus is meridional and very unequal. In the two-cell stage the larger cell is posterior and the smaller cell anterior. The larger cell divides first and very unequally, while the smaller cell divides nearly equal (text figs. 1-3 and fig. 5). In the fourcell stage D is posterior, C right, A left and B anterior, inclined a little to the right. Thus it is very evident that the four-cell stage illustrates a difference in the rate of cleavage, a difference in the size of the cells, and a difference in the direction of the cleavage. The significance of these variations may be emphasized as follows:


a. Difference in the rate of cleavage of cells

If we compare a thirty-two-cell stage of Bdellodrilus with other forms or perhaps, better, with an ideal ovum, in which there is a uniform rate of cleavage in the formation of the cleavage cells, a uniform size and a uniform direction of cleavage, a distinct variation occurs as shown in table 5.


TABLE 5






CKEPIDULA


IDEAL OVUM


NEREIS


BDELLODRILUS


First generation of ectomeres


12 9 5 2 4

32


16 8 4


16 8 4


8


Second generation of ectomeres


11


Third generation of ectomeres


4


Mesoblast


2


Entoblast


4 i 4


7



32


32


32


It is evident that in the first generation of ectomeres Bdellodrilus departs very far from the ideal condition. The first generation contains eight cells instead of sixteen. This means that the cells have divided more slowly than in the ideal ovum. In an ideal ovum these cells form the prototroch and the entire region in front of it, with the apical plate in the center. In Bdellodrilus, this region is degenerate and no trace of the apical plate appears. This indicates an adaptive modification — eight cells instead of sixteen — due to a degenerate frontal region.

In the second generation of ectomeres, the ideal number is


210 GEORGE W. TANNREUTHER

eight, while in Bdellodrilus it is eleven. This increase above the ideal is due entirely to the rapid succeeding divisions of one cell — d^, the first somatoblast. The other cells of the quartette have not divided, while d^ has given rise to three new cells. Does the behavior of d^ suggest any significance, or is it adaptive? From d- the ectoderm of the trunk region, the nephridia and the entire nervous system is derived ; d^ is not only the largest but the most actively dividing cell of the entire embryo; hence its rate of cleavage is well adapted to its resulting formations.

The number of ectomeres in the third generation is the same as in an ideal ovum; d^ however is often formed before aor b^ of the second generation. This interesting phenomenon is due to the tendency of the basal cell, D, and its derivatives to divide more rapidly than those of A, B or C. The differences in the rate of cleavage in the first, second and third generation of ectomeres, no doubt possess prospective significance, looking forward to the definitive parts. This may fairly be called adaptation in the rate of cleavage.

In Bdellodrilus the more rapidly dividing cells do not form the first functioning parts. The cells of the first quartette are the first to function, in the production of cilia for the movement of the embryo. The variation in the rate of cleavage is not due to the varjdng conditions of the media, or the dividing ovum would be uniformly affected, as a whole. Nor is it diie to the size of the individual cells, as the largest cells divide more rapidly. At the thirty-two-cell stage the ideal ovum contains four entodermal cells, while in Bdellodrilus there are seven. Here the cleavage is carried to the end without any resting stage of the four basal cells. This is due to the fact that the larva develops very rapidly and the entodermal cells must keep pace with the rapid development in order to reach their final position, just where they are needed.

b. Variation in the size of cells

The relative sizes of the cells in the early cleavage of the eggs of Bdellodrilus are adapted to the later developing parts.


EMBRYOLOGY OF BDELLODRILUS 211

The largest cell at the four-cell stage is D. Its first division is very unequal, and the smaller cell is less than one-tenth the size of the larger. It is the first ectomere of the first generation formed. The second division, in most instances, is equal; when unequal, the largest cell passes into the upper product, and forms d^, the first somatoblast. The third division is unequal, and d^ the smaller product, is again uppermost; and finally, the fourth division is very unequal and only a small portion remains as the macromere D. The greater bulk, d^ becomes the second somatoblast. In each of the above instances the larger cells form a large part of the embryo and the adult, while the smaller cells, in every instance, form a very insignificant portion. The unequal division in each instance is evidently adaptive, for the great bulk of the material passes into the two somatoblasts and gives rise to the muscular, nervous and excretory systems.

c. Variation in the direction of cleavage

Here only some of the special cleavages will be emphasized. The first division of the second somatoblast is equal, and each part forms equal parts of the mesoderm. Next, each primary mesoblast buds off five or six small cells beneath the first quartette of ectomeres. These small cells remain quiescent for a considerable period and later give rise to the dorsal mesoderm. Immediately after these small cells are formed, the mesoblast bands are begun by a forward prohferation of cells from the anterior face of the primary mesoblasts. The plane of division iS at right angles to that of the small cells. These bands extend forward between the ectoderm and the entoderm, and at the same time the entodermal cells extend posteriorly between the mesoblast bands and the group of small cells, thus separating the primary and secondary mesoderm. Here the direction of the cleavages place the. cells where they are later used in the formation of some special part, adapted for that particular region.

The first somatoblast buds off x^ to the right, x^ to the left, and x^ median dorsal anterior. X now divides equally and


212 GEORGE W. TANNREUTHER

t

each proteloblast buds off a small cell, x"*, one to the right of x^ and the other to the left of x^. Again each proteloblast buds off a small cell, x^, on either side of x^ At the next division each buds off a small cell, x^ on the ventral anterior edge. Later, x^ is budded off from each neuroblast on the ventral side. These small cells give rise to the trunk ectoderm and the larger cells to the nephridia and nervous system. Here again the cells are formed just where they are needed; the smaller on the exterior or outer surface while the larger remain within.

2. ADAPTATION IN THE CLEAVAGE OF OTHER FORMS

In following the variation of cleavage cells in annehds and molluscs, special cells can be arranged in a complete series, from those of an almost insignificant size to an extremely large cell. In following these variations, step by step, we can not fail to be convinced that these variations are adaptive to the future needs and habits of the larva and of the adult animal.

In forms with equal cleavage, the first somatoblast gives rise to the ectoderm of the trunk region. In Polydorgus, Podarke, Hydroides, Eupomatus and others with equal cleavage, d^ is the same in size as the cells of the other quadrants. Equal cleavage has been offered by some as due to a lack of differentiation in the early stages but in such forms as Podarke with equally cleavage, very early differentiation occurs, and the prominence of these early functioning parts varies according to the size of the initial cell from which they are formed.

In forms with unequal cleavage, the first somatoblast differs in size from the remaining members of the same quartette. Beginning with Amphi trite, the relative size of d- increases successively in Chaetopterus, Arenicola, Nereis, Capitella, Aricia, Spio, Clepsine and Bdellodrilus. Those forms with equal cleavage pass through a distinct trochophore stage and are characterized by an almost equatorial prototroch, a very large exumbrella, and with a very slow trunk development. In those with unequal cleavage, especially in the second generation of ectomeres, there is a gradual decrease in the prominence of the


EMBRYOLOGY OF BDELLODRILUS 213

trochophore to its approximate or complete disappearance; on the other hand, there is a gradual acceleration in the time of the trunk development, varying according to the increase in the relative size of the first somatoblast or X. Treadwell states that the extra amount of material stored in the macromere D is in some way related to the amount of somatic and mesoblastic material needed in the future organism. This statement is true of the condition that occurs in such annehds as Bdellodrilus and Clepsine.

GENERAL SUMMARY

The undivided egg of Bdellodrilus philadelphicus is nearly oval. Its median longitudinal axis through the region of the polar bodies corresponds to the median axis of the future adult. The polar bodies occupy the region which later becomes the anterior end of the embryo.

The first cleavage plane is nearly at right angles to the median axis of the resulting individual, and divides the egg into two very unequal parts. The second cleavage occurs at an angle of about forty-five degrees to the first. It divides the smaller cell nearly equally and the larger cell very unequally; the larger cell divides first. In a four-celled embryo the large cell D is posterior, B is anterior, inclined a little to the right; A left and, C right.

The ectoderm is separated from the four macromeres by a series of three oblique cleavages. The first generation of ectomeres is formed in a dexiotropic direction. The second generation laeotropically and the third in a dexiotropic fashion.

In the fourth generation of micromeres, d^ is mesoblastic. The other cells of the fourth quartette, together with the four macromeres, form the entoderm. The cleavage of the entodermal cells is carried to the end without delay, in the formation of the digestive tract, and the interior of the embryo becomes a solid mass of entodermal cleavage cells, which later become differentiated into the epithehal portion of the alimentary canal. As the core of entodermal cells grows posteriorly,


214 GEORGE W. TANNREUTHER

it separates the primary and secondary mesoderm. The extreme ends of the digestive tract are ectodermal. Among other annehds and also in molluscs, so far as is known, the entodermal cells are not broken up into cells but enter directly into the formation of the digestive tract.

The posterior cell, d^ (X), of the second generation of ectomeres is the largest cell of the segmenting ovum. The derivatives of X are symmetrically placed with reference to the median plane of the future individual. The large cell, X, gives rise to the trunk ectoderm, the nervous and the excretory systems. The nervous system is derived from the two neuroblasts. The brain is formed from the extreme anterior end of the neural rows.

The largest cell, d* (M), of the fourth generation of micromeres, gives rise to the entire mesoderm. It is the first cell to divide in a bilaterally symmetrical manner. The primary mesoblast cells, M, M, bud off five or six small cells each, beneath the first quartette of ectomeres, which give rise to the secondary mesoderm on the dorsal side of the embryo. Immediately after these small cells are budded off, the primary mesoblasts, by a teloblastic proliferation of cells, produce the mesoblast bands.

The embryo increases but little in bulk before the germ bands are formed. The embryo as a whole, during its early stages of development, is extremely plastic and may vary considerably in its transverse and longitudinal axes. The developing embryo is completely turned on itself, and the anterior and posterior ends are in immediate contact. The outer surface is ventral and the turned in portion is dorsal. This pecuHarity of development is foreshadowed in the position taken by the early cleavage cells.

At the beginning of the germ-band formation, the embryo begins to rotate on its transverse axis. This movement is due to the action of cilia, which are produced by the ectodermal cells on the median ventro-anterior end of the embryo; the rotation alternates.

As growth continues within the cocoon, the ends of the embryo soon begin to overlap. The embryo may assume almost


EMBRYOLOGY OF BDELLODRILUS 215

any position in the cocoon during its later stages of development. The embryo is completely developed before emergence; the trochophore stage is completely suppressed; the gastrulation is of the epiboHc type.

Columbia, Mo. November 3, 1914

LITERATURE CITED

Balfour, F. M. 1893 Elements of embryology. London.

Berg, R. S. 1888 Zur Bildungsgeschichte der Excretionsorgans bei Ciido drilus. Arb. a. d. zool. Inst. VViirzburg, Bd. 7, Heft 3. BiGELOW, A. M. 1899 Notes on the first cleavage of Lepas. Zool. Bull.,

vol. 2, no. 4. BtJRGER, O. 1891 Beitrage zur Entwicklungsgeschichte der Hirudineen. Zool.

Jahrb., Bd. 4. Carazzi, D. 1900 L'embriologie dell'Aplysia limacina. Anat. Anz., Bd. 18. Casteel, B. D. 1904 The cell lineage and early development of Fiona marina,

a nudibranchiate mollusc. Proc. Ac. Nat. Sci., Philadelphia. Castle, W. E. 1896 The early embryology of Ciona intestinalis Flemming

(L). Bull. Mus. Comp. Zool. Harvard College, vol. 27, no. 7. Child, C. M. 1900 The early development of Arenicola and Sternaspis.

Arch. f. Entwick. der Organismen, Bd. 9, Heft 4. Conklin, E. G. 1897 a The embryology of Crepidula. Jour. Morph., vol. 13.

1897 b Cleavage and differentiation. Biol. Lecture, Woods Hole. EisiG, H. 1898 Zur Entwickelung der Capitelliden. Mittheil. a. d. Zool.

Stat. Neapel, Bd. 13. Fraipont, J. 1887 Le genre Polygordius. Fauna u. Flora d. Golfes von

Neapel, Monographic, tom. 24. GoTTE, A. 1882 Ueber die Entwickelung der Chaetopoden. Leipzig. Hatschek, B. 1885 Entwickelung der Trochophora von Eupomatus uncina tus. Arb. zool. Inst. Wein, Bd. 6. Heath, H. 1899 The development of Ischnochiton. Zool. Jahrb., Bd. 12,

Heft. 4. Hertig, O. und R. 1881 Die Coelomtheorie. Jenaische Zeitschr., Bd. 14. Kleinenberg, N. 1879 The development of the earthworm. Quart. Jour.

Micr. Sci., vol. 19. Kowalevsky, a. 1871 Embryologische Studien an Wiirmern und Arthropoden.

Mem. de I'Acad. Imp. de Sc. de St. Petersbourg, tom. 16, no. 12. Lang, A. 1884 Die Polycladen des Golfes von Neapel. Flora und Fauna

des Golfes von Neapel, Bd. 6. LiLLiE, F. R. 1895 The embryology of Unionidae. Jour. Morph., vol. 10.

1899 Adaptation in cleavage. Biol. Lecture Woods Hole.

1901 The organization of the egg of Unio. Jour. Morph., vol. 17. Mead, A. D. 1897 The early development of marine annelids. Jour. Morph.,

vol. 13.


216 GEORGE W. TANNREUTHER

Meyer, E. 1890 Die Abstammung der Anneliden. Biol. Centralb., Bd. 7. Moore, J. P. 1895 The anatomy of Bdellodrilus illuminatus, an American

Discodrillid. Jour. Morph., vol. 10. Nelson, A. J. 1904 The early development of Dinophilus. Proc. Ac. Nat.

Sci., Philadelphia. RouLE, L. 1899 Etudes sur le developpement des annelides et en particulier

d'un Oligochete limicole marin (Enchytraeoides marioni). Ann.

Sci. Nat., (7), tome 7. Salensky, W. 1885 Developpement de Branchiobdella. Arch, de Biol.,

tom. 6, fasc. 1, p. 1. ScHiMKEWiTSCH, W. 1895 Zur Kentniss des Baues und des Entwickelung des

Dinophilus vom weissen Meere. Zeit. wiss. Zool., Bd. 59. ToRREY, J. C. 1903 The early embryology of Thalassema mellita (Conn).

Ann. New York Acad. Sci., vol. 24, no. 3. Treadwell, a. L. 1898 Equal and unequal cleavage in annelids. 'Biol.

Lecture Woods Hole.

1901 The cytogeny of Podarke obscura. Jour. Morph., vol. 17. Whitman, C. O. 1878 The embryology of Clepsine. Quart. Jour. Micr. Sci.,

vol. 18.

1887 The germ layers in Clepsine. Jour. Morph., vol. 1. Wilson, E. B. 1887 The germ bands of Lumbricus. Jour. Morph., vol. 1.

1889 The embryology of the earthworm. Jour. Morph., vol. 3.

1891 The origin of the mesoblast bands in annelids. Jour. Morph., vol. 4.

1892 The cell lineage of Nereis. Jour. Morph., vol. 6. WiERZEJSKi, A. 1905 Embryologie von Physa fontinalis. Zeit. f. wiss. Zool.,

Bd. 62.


THE MORPHOLOGY OF NORMAL FERTILIZATION IN PLATYNEREIS MEGALOPS

E. E. JUST

THREE PLATES (THIRTY FIGURES)

1. INTRODUCTION

In a previous paper on Platynereis megalops, which described the egg-laying habits, it was stated that insemination takes place in the body cavity of the female and, further, that the eggs will not fertilize when inseminated in sea water. The present paper is a description of the normal fertilization process in Platynereis. An experimental analysis of fertilization in Platynereis appears elsewhere (Just, '15).

2. NORMAL FERTILIZATION OF PLATYNEREIS

The living egg. The egg of Platynereis is compressed and irregular in shape while in the body cavity. Those eggs which happen to be uninseminated when laid gradually round out in sea-water as almost perfect spheres equatorially, but with a rather shorter polar axis. The large, centrally placed, germinal vesicle is slightly elongated in the polar direction. The largest eggs, fully rounded out, measure 180 to 200 fx. They are almost perfectly transparent, have an equatorial ring of oil drops, and a well marked transparent exoplasm or cortical layer of protoplasm with very faint granules forming a delicate mesh. In short, the living egg closely resembles that of Nereis; it is larger (but of. Wilson, '92) not so deeply pigmented, and lacks the characteristic yolk spheres of the Nereis egg.

A. Fertilization in the living egg

We may consider the fertilization of the egg under the following heads: (1) insemination, (2) penetration of the sperm, and (3) copulation of the germ nuclei.

217


218 E. E. JUST

1. Insemination. In Platynereis insemination normally takes place in the body cavity ( Just, '14). The eggs, when laid, have the sperm attached within a thin hull of jelly, the secretion of the cortical layer. If the worms be allowed to deposit eggs in India ink ground up in sea-water it can be proved satisfactorily that a hull of jelly, as in Nereis, envelops inseminated eggs. This jelly, absent in uninseminated eggs, is formed from the exoplasm of the egg as the result of stimulation through sperm attachment. In sea-water the zone between India ink particles and the vitelline membrane gradually widens, not so much because of the slow diffusion of the jelly from the egg, as because of the swelling of the extruded jelly.

Insemination in some way brings about oviposition. The presence of the sperm in the female is a stimulus to egg laying; as in Nereis (see Lillie and Just) the presence of the sperm in the sea-water brings about the shedding of the eggs. The first result of the attachment of the sperm to the egg is jelly formation through cortical secretion, with the consequent formation of the perivitelline space; and this process must begin in the body cavity, since eggs have a thin jelly investment when laid. As in Nereis the vitelline membrane is preformed; the sperm does not cause 'membrane formation.'

For twenty to thirty minutes after oviposition, the sperm remains external to the egg. During this time profound changes take place in the egg, many of which doubtless are to be interpreted as changes incident to maturation, the mechanism of which is released with the breakdown of the' cortical substance and the consequent formation of the perivitelline space. These changes: breakdown of the germinal vesicle, formation of the spindle, polar body formation, and cytoplasmic movements, are easily followed in the living egg.

2. Penetration. In Nereis a striking phenomenon of sperm attachment is the fertilization cone (Lillie, '11, '12). In Platynereis no sharply defined cone is found. There are, however, cytoplasmic disturbances at the point of sperm entry. In polyspermic eggs the cytoplasm may form a low blunt protrusion, with as many as five spermatozoa attached to it, but this is not


FERTILIZATION IN PLATYNEREIS MEGALOPS 219

a cone. Twenty-five minutes after oviposition a slender strand of protoplasm may be discerned across the perivitelline space and beneath the point of sperm entry; even this does not seem to be constant, but appears to be formed only in the animal hemisphere. This protoplasmic strand lies, first, in a radius of the egg, but gradually bends so that it now lies almost tangential to the egg. It is found after sperm entry.

After thirty minutes the spermatozoon is engulfed. Often the formation of the sperm aster is discernible, the middlepiece and tail remaining outside. The maturation asters are always visible in the living egg. Mathews ('06) has called attention to the difTerence between the structure of the asters of living eggs and of fixed material. The difference is certainly striking in Platynereis. Instead of the short stiff astral fibres of chrom-osmic material or the long slender ones of mercuric fixation, one sees in the living egg beautiful broad rays sweeping through the cytoplasm.

3. Copulation of the germ nuclei. About fifty minutes after egg-laying, the germ nuclei copulate, the cleavage asters form, and at sixty minutes the egg divides unequally. The egg at this time exhibits a stratification of protoplasmic stuffs. During maturation the cytoplasmic currents shift the materials. The equatorially placed oil drops, about eighteen in number, gradually become massed at the vegetative pole, the coarser (yolk) granules lie above these; at the clearer animal pole are the male and female pronuclei. Beneath the polar bodies the cytoplasm is most transparent. The asters are very distinct. One cannot get an adequate picture of these structures from sections. In the living egg they are incomparably clear; large broad rays which bear little resemblance to the short stiff fibres seen in the sections.

The penetration path of the spermatozoon may often be followed, the copulation path always followed. The spermatozoon enters at any point of the egg and through this, as in Nereis (Just, '12), the first cleavage plane passes along the copulation path of the germ nuclei.


220 E. E. JUST

B. Observations on the sectioned egg

Observations of the phenomena of fertiHzation in the Hving egg were supplemented with a study of sectioned material.

Technique. Eggs were fixed in Meves' fluid for thirty minutes, one hour, or twelve hours. Aceto-osmie-bichromate mixtures (Mathews, 1 '99; Bensley) ; Bouin's fluid, modified by the addition of an equal volume of water; and Gilson's mercuric-nitric mixture were likewise used. Although very destructive to the yolk and oil, the modified Bouin proved helpful in the study of certain details in connection with sperm penetration.

The difficulties of fixation, which are great in this egg, as in Nereis, may in large measure be overcome by the subsequent treatment. The following methods were used after fixation with Meves :

(a) Clearing with double distilled anihn oil from 80 per cent alcohol.

(b) Clearing in cedar oil from 95 per cent alcohol.

(c) Clearing in cedar oil from 95 per cent alcohol after treatment with glycerine (eggs put in 70 per cent alcohol plus an equal amount of glycerine).

(d) Clearing in xylol from 95 per cent alcohol or from absolute alcohol.

In all cases xylol was used before imbedding in paraffin or in paraffin with some admixture of Johnston's rubber-asphalt mass. It was found that avoidance of absolute alcohol left the eggs less brittle and therefore less refractory in cutting. By far the most natural contours of both the Platyfiereis and the Nereis eggs are preserved through the use of aniline oil after 80 per cent — a clearing agent that I have used successfully for several years. Staining was with iron hematoxylin alone. Sections were cut four micra thick.

Spermatozoa, after fixation, were studied for the most part unstained after the methods of Koltzoff, de Meyer, etc. The iodine mixture recommended by Mayer for Volvox proved in ^ From the legend of Mathews' figures it appears that he used aceto-osmicbichromate mixtures.


FERTILIZATION IN PLATYNEREIS MEGALOPS 221

valuable. For permanent preparations Bensley's staining mixtures were used.

1. Stages previous to the penetration of the sperm. The egg of Platynereis rivals in structure the beauty of the Nereis egg. A section of an uninseminated egg (fig. 1) teased out of the. female directly into Meves' fluid gives many of the details. The cytoplasm is sharply marked off into two regions: the exoplasm made up of clear cortex and zone of oil and yolk and the deeply staining endoplasm.

The outer portion of the exoplasm is a mesh of pale blue delicate fibrils, the alveoli of the cortical jelly. The outer limits of this cortical layer — slightly more dense than the deeper portions — is studded with black granules immediately below the vitelline membrane. The inner border of the cortex arises from a zone of closely-packed, deep-staining bodies, from which apparently the walls of the cortical alveoli project. Below this inner border is the region of oil drops which lies in the equatorial zone, among spherules which prove, from their later behavior, to be yolk spheres, although even in the best preparations, the fine granules of which they are composed tend to shrink from their spherical walls (c/. LiUie, '11; figure of Nereis egg fixed in Fleming) . These yolk spheres are evenly crowded against the deeply stained basal ♦ area of the cortex. Around the germinal vesicle and closely applied to it is the endoplasmic mass, made up of fine granules which take the stain very tenaciously. Its outer limits are uneven, encroaching on the area of oil drops and yolk spheres as blunt projections.

Scattered throughout the germinal vesicle, as in Nereis, are the chromosomes — fourteen tetrads. These lie among many black granules of varying size. Although an attempt has been made to study their number, distribution, etc., and to ascertain any constant characters, nothing now can be said further of them. These granules tend to be spherical and to grade down to minute bodies.

The whole egg, therefore, exhibits a granular structure, both living and fixed, as Mathews some time since ('06) for echinoderm eggs and more recently Kite for some other eggs have shown.


222 E. E. JUST

Lillie ('06), too, in a most elaborate study on the egg of Chaetopterus, has determined the granular structure of the cytoplasm. Vacuoles found in mercuric-nitric or picro-acetic preparations are filled with yolk or oil in Meves' preparations or in the living Platynereis egg (c/. Wilson, '98, on the cytoplasmic structure of eggs, including that of Nereis).

The egg of Platynereis, as compared with that of Nereis fixed with the same methods, does not show so clearly the radial striation in the cortical layer or the homogeneous yolk spheres.

Ten minutes after laying the germinal vesicle is breaking down and maturation asters, formed outside its wall, are pushing into its substance. The deepest of the cortical alveoli are often still unemptied; the whole process of jelly extrusion can easily be followed from its beginning in inseminated eggs. On one or between two of the apices of the wavy vitelline membrane the spermatozoon is found attached by its perforatorium. Sperm head, middle-piece, and tail are readily distinguished (fig. 2).

Fifteen minutes after laying, the cortical jelly has been wholly extruded (fig. 3) and the first maturation spindle formed, with the chromosomes in late prophase. The endoplasm, with the extra-chromatin substance of the germinal vesicle, imbeds the spindle. In toto mounts of the egg at this stage, as is true of the Nereis egg, give no view of the spindle. One sees only a deeply stained core of substance which incloses the spindle. The egg is irregular in shape and the vitelline membrane is closely applied.

The spermatozoon is visible on the membrane (figs. 3 and 4) above a group of granules similar to those more thinly scattered throughout the periphery of the egg. These granules are markedly like those described by Meves and are doubtless 'mitochondria;' but in Platynereis they cannot possibly have the significance that Meves ascribes to them in the eggs of various forms. The granules appear massed beneath the point of sperm entry, but these masses assume no definite form. I have purposely figured those that give the nearest approach to cone formation (figs. 3, 4, and 5). A slender strand of cytoplasm may extend toward the membrane just below the perforatorium.


FERTILIZATION IN PLATYNEREIS MEGALOPS 223

The granules in the region may appear as a disc, but never as a retracted cone, as in Nereis. The cortical breakdown has released the close application of the yolk spheres to the inner cortical margin; they are now irregularly spaced and among them lies the granular cytoplasm.

The figures (2 to 5) also give good pictures of the spermatozoa. They are much like the living spermatozoon. The head is almost spherical, the perforatorium a large blunt cap; the middle-piece and tail are often clearly defined.

2. Penetration of the spermatozoon. The penetration of the sperm head begins at twenty to twenty-six minutes after laying (c/. Nereis, forty-five minutes after insemination). The first maturation spindle, in the metaphase, is oriented in the polar plane of the egg; the inner endoplasmic mass which incloses the spindle is, at this stage, triangular in section; the outer aster of the spindle is near the apex of the triangle. The base of the triangle is less blunt than in previous stages and reaches farther outward along a radius of the egg. The various stages of penetration are shown in figures 6 to 18. The sperm substance enters the egg as a slender black thread, which gradually increases in size at its inner end. The sperm head, in my preparations, is usually homogeneously black, but often the external bulb is not so dark; or lighter areas appear along the entering thread; (particularly figs. 10, 11, 12, and 14). Often, especially in sections stained for twelve hours only, in stages just after the attachment of the perforatorium to the cytoplasm, the head appears, not as a homogeneous chromatin mass, but as a slightly differentiated body. One gains, therefore, the impression that the spermatozoon flows into the egg (c/. Koltzoff and Lillie, who, with different methods, find Nereid spermatozoa extremely ductile) .

Cytoplasmic changes due to sperm entry are clearly marked during the later stages of penetration ; striae appear in the cytoplasm around the entering spermatozoon, the area stains more deeply, and a projection from the endoplasmic mass reaches out toward the point of sperm entry (see figs. 14 to 17) (c/. on these points. Foot, Gardiner, Vedjovsky, Jenkinson, and Lillie, '12).


224 E. E. JUST

As the head is drawn into the egg, the inner bulb turns with its growth. Finally, the portion forming the external bulb is engulfed. The middle piece and tail, as in Nereis, never enter the egg (fig. 17). They may often be found in sections outside the membrane after penetration of the sperm head (see figures).

I have never found the spermatozoon in the Nereis or in the Platynereis egg at the time or in the form figured by Wilson ('96 and '00).

Does the sperm head rotate? I could not positively determine the rotation of the sperm head in the egg of Platynereis. In the first place, a definite cone organ, like that of Nereis, is lacking, and secondly, the middle-piece does not enter the egg. The history of the sperm penetration is known practically for every minute from entrance to pronuclear copulation. Meves' fixation alone was not depended upon. The Bouin preparations gave results much like those of Bonnevie's with picro-acetic mixtures on the Nereis eggs. While absolutely worthless for cytoplasmic detail, they were helpful in determining the structure of the sperm nucleus after penetration. The evidence favors rotation; the turning of the inner sperm bulb (fig. 17) and the position of the long axis of the sperm and aster as often found at right angles to the radius of the egg (fig. 22) .

The sperm aster does not arise until the nucleus is beyond the yolk region (figs. 14 to 22). Within the endoplasm, the aster once formed, quickly divides equally, but the amphiaster does not long retain its equal poles, for one sperm centrosome and its aster gradually dwindle in size. Rays arise between one or both of the sperm centrosomes and the inner centrosome of the maturation spindle, thus forming a secondary spindle. The sperm nucleus lies nearer the larger sperm centrosome (see figs. 20 to 25).

3. Copulation of the germ nuclei. The egg chromosomes, after the formation of the second polar body, form fourteen chromosome vesicles which fuse to establish the egg nucleus (figs. 26, 27), all vestiges of the egg aster disappearing. The sperm nucleus enlarges as its asters become smaller. At the time of apposition, but one sperm aster is found (fig. 28) . I believe that one sperm


FERTILIZATION IN PLATYNEREIS MEGALOPS 225

aster begins to wane soon after the formation of the homodynamic amphiaster and finally disappears. One aster can always be found (fig. 29). The opposing nuclear membranes break down and one nucleus forms with the single sperm aster. Soon a small aster appears on the nuclear membrane (fig. 30), the nucleus breaks down, and the heterodynamic first cleavage spindle forms.

3. DISCUSSION

The case of Nereis and . Platynereis, with respect to the entrance cone offers an interesting parallel with that of Toxopneustes and Arbacia (Wilson and Mathews). In both Nereis and Platynereis, however, the middle-piece is left outside the egg. The absence of cone-organ in Platynereis makes the question of rotation obscure, whereas in Nereis the evidence is indisputable.

Bonnevie ('08), in her paper on Nereis, has mentioned certain cytological differences between the '4arge and small varieties" of Nereis eggs. As indicated above, the time of sperm entry is earlier in Platynereis. It is also true that the polar bodies are formed earlier, the first cleavage is earlier, and the subsequent rhythms are faster, so that the larval stage is reached earlier.

So far as both Nereis and Platynereis are concerned, the role of the middle-piece or its contained centrosome, as the chief actor in fertilization, is wanting. There are spermatocytes with intra-nuclear centrosomes^ (see Julin on Styleopsis). But if this hypothesis be postulated (c/. Packard in 1914) for Nereis sperm, this next step, as was pointed out by Lillie in 1912, should also be taken: the centrosome gradient must be quantitatively different from its base at the middle-piece to the tip of the sperm head: "If intra-nuclear centrosomes are the causes of the formation of the sperm aster, not only must they exist at every level, but also (that) they must decrease in size from the base to the apex of the sperm nucleus!"

2 See also Hegner and Newman for intra-nuclear centrosomes in oocytes.


226 E. E. JUST

According to Schaxel, the middle-piece does not enter the egg of echinoderms. Meves will not admit this for echinids and doubts that in Nereis the middle-piece is left outside the egg while denying the centrosome the chief part in fertilization. In Platynereis, as in Nereis, by diverse methods it can be shown that the middle-piece does not enter the egg. We are thus forced to conclude that, whatever its role, the middle piece in Platynereis can play no part, either in heredity or through a centrosome in the dynamics of fertilization.

Marine Biological Laboratory Woods Hole, Mass.

LITERATURE CITED

BoNNEviE, Kristine. 1908. Chromosmenstudien II. Heterotypische Mitose

als Reifungscharakter. Arch, fiir Zellforschung, Bd. 5. Bensley, R. R. 1911. Studies on the pancreas of the guinea pig. Am. Jour.

Anat., vol. 12. Foot, K. 1897. Origin of the cleavage centrosomes in AUolobophora. Jour.

Morph., vol. 12. Gardiner, Ed. C. 1898. The growth of the ovum, formation of the polar

bodies, and the fertilization in Polychoerus caudatus. Jour. Morph.

vol. 15. Hegner, R. W. 1908. Intra-nuclear mitotic figure in primary oocytes of a

copepod, Canthocampus. Biol. Bull., vol. 14. Jenkinson, J. W. 1904. Maturation and fertilization of the axolotl egg.

Quar. Jour. Micros. Sci., vol. 48. JuLiN, J. 1893. Structure et developpement des glandes sexuelles, ovogenese

spermatogenese et fecondation chez Styleopsis grossularia. Bull. Sc.

de France et Belgique, 24. Just, E. E. 1912. Relation of the first cleavage plane to the entrance point

of the sperm. Biol. Bull., vol. 22.

1914. Breeding habits of the heteronereis form of Platynereis megalops at Woods Hole, Mass. Biol. Bull., vol. 25.

1915. An experimental analysis of fertilization in Platynereis megalops. Biol. Bull., vol. 28.

Kite, G. L. 1913. Studies on the physical properties of protoplasm. Am.

Jour. Physiol., vol. 32. KoLTZOFF, N. K. 1909. Studien uber die Bestalt der Zelle. II. Untersuchung en Tiber das Kopfskelett des tierschen Spermiums. Arch, fur Zell forsch., Bd. 2. Lillie, F. R. 1906. Observations and experiments concerning the elementary

phenomena of embryonic development in Chaetopterus. Jour. Exp.

Zool., 3, 1906.


FERTILIZATION IN PLATYNEREIS MEGALOPS 227

LiLLiE, F. R. 1911. Studies of fertilization in Nereis. I. The cortical changes in the egg. II. Partial fertilization. Jour. Morph., vol. 22.. 1912. III. The morphology of the normal fertilization. IV. The fertilizing power of portions of the spermatozoon. Jour. Exp. ZooL, vol. 12.

LiLLiE, F. R. AND Just, E. E. 1913. Breeding habits of the heteronereis form of Nereis limbata at Woods Hole, Mass. Biol. Bull., vol. 24.

Mathews, A. P. 1899. The changes in the structure of the pancreas. Jour. Morph., vol. 11.

1906. A note on the structure of the living protoplasm of echinoderm eggs. Biol. Bull., vol. 11.

1907. A contribution to the chemistry of cell-division, maturation, -and fertilization. Am. Jour. Physiol., vol. 18, No. 1.

Newman, H. H. 1912. Maturation of the armadillo egg. Biol. Bull., vol. 23.

Packard, C. 1914. The effect of radium radiations on the fertilization of Nereis. Jour. Exp. Zool., vol. 16.

Vedjovsky, F. und Mrazek, a. 1903. Umbildung des Cytoplasma wahrend der Befruchtung und Zellteilung. Nach der Untersuchungen am Rhynchelmis-Ei. Arch, fiir mik. Anat., Bd. 62.

Wilson, E. B. 1892. The cell lineage of Nereis. Jour. Morph., vol. 6.

1897. Centrosome and middle piece in the fertilization of the seaurchin egg. Science, vol. 5, No. 114.

1899. On the protoplasmic structure in the eggs of echinoderms and some other animals.

1900. The cell in development and inheritance. The Macmillan Co. Wilson, E. B. and Mathews, A. P. 1895. Maturation fertilization, and polarity of the echinoderm egg. Jour. Morph., vol. 10.


JOURNAL OF MORPHOLOGY, VOL. 2fi, NO. 2


DESCRIPTION

All figures were drawn with the camera lucida with Leitz xV oil immersion objective and No. 5 ocular, except where otherwise stated. All figures from sections of inseminated eggs of Platynereis megalops. All sections from eggs killed in Meves' fluid and stained in iron haematoxylin.

PLATE 1.

EXPLANATION OF FIGURES

1. Section of an unfertilized ovocyte. The oil drops are a delicate brown, the granular yolk spheres very lightly stained. The cortex is intact.

2. Ten minutes after laying. The cortex is partially reduced. The head, middle-piece, and tail are clearly shown.

3 to 5. Fifteen minutes after laying. The granules are massed below the point of sperm attachment; the perforatorium is still attached to the membrane 6 to 8. Twenty minutes after laying.

6. The perforatorium is touching the cytoplasm. The granular mass has disappeared.

7. The perforatorium is in the cytoplasm.

8. A somewhat tangential section, showing the very beginning of penetration. 9 to 13. The penetration stages, twenty-five minutes after laying, mesophase

of the first maturation division. The figures show that there is no constant disposition of granules at the point of sperm entry — certainly nothing of the nature of a cone, as in Nereis.


228


FERTILIZATION IN PLATYNEREIS MEGALOPS

E. E. JUST


PLATE 1


m


. \




K


. (•




/.- . ».





^*v^f«i-.vi-< _, , ..



v^w'


A v-^.-r^.v >".?.?


/




229


PLATE 2


EXPLANATION OF FIGURES


14, 15. Penetration stages, twenty-five minutes after laying. Note the turning of the inner sperm bulb.

16 to 18. Twenty-seven minutes after laying. 1 he middle piece is shown in fig. 17. In 16 and 18 the middle-piece was found in adjacent sections.

19 and 20. Thirty minutes after laying, telophase, first maturation division. The sperm head is still within the zone of oil drops and without an aster.

21 to 22. Thirty-two minutes after laying; early prophase, second maturation division.

21. The sperm head is at right angles to a radius of the egg, the aster forms around the granule at the tip of the sperm head.

22. Formation of sperm aster within the endoplasm.

23. Thirty-five minutes after laying. The amphiaster is in contact with the egg aster. The spermatozoon is in an adjacent section.


230


FERTILIZATION IN PLATYNEREIS MEGALOPS

E. E. JDST



,**»^'<*)-/!« ; •3«**l ' & '^•'* •


©



-t^TT


PLATE 2


■iii


^



%



231


PLATE 3


EXPLANATION OF FIGURES


24 to 30; oc. 1, iV oil im. Later stages, showing marked inequality of sperm asters. Note relation of the spermatozoon to the larger aster. 25a and b. Forty minutes after laying.

26. Forty-six minutes after laying. The egg aster is degenerating.

27 to 29. Copulation stages.

27. The smaller sperm aster could not be found. Two egg and three sperm nuclear vesicles are shown.

28 to 29. Formation of the male and female nuclei. 30. Origin of the first cleavage spindle.


232


FERTILIZATION IN PLATYNEREIS MEGALOPS

E. E. JC8T


PLATE 3




266


CILIATED PITS OF STENOSTOMA

WM. A. KEPNER AND J. R. CASH

University of Virginia

FOUR FIGURES

The material for this paper was found upon glea and sediment deposited upon submerged leaves and twigs taken from both near the surface and the bottom of pools in the vicinity of the University.

The animals are about 1 to 2 mm. long and 200 mi era broad at the widest region. The body is oblong, spindle shaped, and is widest in the region of the mouth; it is grayish white and its epidermis, which contains rhabdites, is thickly covered with short cilia. The mouth is on the ventral side, about 150-200 micra posterior to the anterior end. The urinary system consists of a single nephridium

The ciliated pits are by far the most striking organs of the body which are externally visible. These are small invaginations in the epidermis which are located lateral-dorsally about 100 micra from the anterior end of the body. Their shape is, in general, similar to small sacs, but their lateral walls are highly contractile so that the pit may be made to assume the form of a deep cut or that of a shallow, concave disc. The ciliated pits have a diameter of about 50 micra and a depth of about 40 micra.

This paper is concerned with the minute anatomy of the ciliated pits and their development. In order to carry out such a study the animals were fixed in Flemming's stronger fluid, which consists of 15 parts 1 per cent chromic acid, 4 parts 2 per cent osmic acid, 1 part glacial acetic acid; time of fixing 25 minutes. Chrome-aceto-formaldehyde, hot and cold solutions of acetosubhmate, and Zenker's fluid were all tried, but without success,

235


236 WM. A, KEPNER AND J. R. CASH

either causing great distortion or disintegration of the animals. The worms were cut into sections, some three and some five micra thick and the sections stained with iron haematoxylin and counter stained with Bordeaux red. Macerations stained with such intra vitam stains as Wright's stain and methylen blue were verj^ valuable in corroborating results.

HISTOLOGY OF THE PIT

The histology of the pit involves an understanding of the epidermis. The epidermal cells of the animal have the power to secrete a protective mucus-like substance. That such is the case can readily be seen by placing the animal in an abnormal solution of not too rapid killing power. The animal will at once enshroud itself in a thick sheath of protective mucus within which it swims around. Such a phenomenon will be more fully described in a later part of this paper.

The pit is associated with a region of the central nervous system known as the ciliated pit-ganglion. The pit, as well as this ganglion, is a modified region of the general epidermis. The marginal walls of the pit are formed by cells transitional in structure between the general epidermis and the low cells at the bottom or fundus of the pit. As the invagination which forms the pit takes place this transition takes place until there is a layer of low, small epithelial cells lining the fundus of the pit. The boundaries of these fvmdus cells are less pronounced than the boundaries of the general epithelial cells and their nuclei less frequent. In certain regions of this lining of the fundus the few nuclei which are present are indefinitely placed, which fact suggests that there is no basement membrane.

On the exterior of the body, lying close upon the fundus of the pit, is a homogeneous mass of mucus-like substance. The marginal walls of the pit are thickly covered with cilia which appear to be longer than the cilia of the general body epithelium, but no cilia at all were found upon the low cells lining the fundus nor were any seen projecting above the homogeneous mass of mucus-like substance.


CILIATED PITS OF STENOSTOMA 237

The ciliated pit-ganglion is by far the most conspicuous feature of the pit. It is located just within the body and lying around the base of the ciliated pit in a cup-like manner. The cells of this structure are only indistinctly separated from those of the dorsal ganglion or 'brain' by a few muscle fibers and have the same characteristic, granular nuclei as those of the 'brain.' Some of these ciliated pit-ganglion cells are seen to send processes through the epithelium of the fundus which lies in contact with the homogeneous body of mucus-like substance. These we take to be sensory rods of highly special nature which enable the organ to detect very slight changes in its surrounding medium. These sensory rods are shown clearly, as dark blue structures, in intra vitam staining with Wright's stain and in many of the regular sections. Figure 4 shows such a section. The fact that the ganglion cells arise from the epithelial cells which line the fundus of the pit also supports the idea that these processes are left behind by the cells as they migrate inward to enter into formation of the ciliated pit-ganglion.

Rightful interpretation and appreciation of the above statements will only be obtained through a study of the origin of the ciliated pits.

THE ORIGIN OF CILIATED PITS

The origin of the ciliated pits can readily be studied in specimens which are dividing. The first appearance of the pits is seen in two sharp, abrupt depressions of the epidermis, one on each side of the animal. From the bottoms of the depressions (i.e., the region which will be the fundus of the new pit) the cilia disappear. Figure 1 shows an early pit in this stage of development. Ventral to this abruptly lowered region a crowded mass of mesenchymal cells is formed which represents the anlage of the 'brain. '^

1 We are indebted to Prof. Bohmig, through the kindness of Prof. L. von Graff, for the following quotation from page 34 of O. and R. Hertwig's Die Coelomtheorie, Jena, 1881. In regard to the Platyhelminthes they say: "In der Abteillung stammt wahrscheinlich der motorische Teil der Centralorgane des Nervensystems im Anschlusse und die Muskulatur aus dem Mesenchym, der sensorielle Teil im Anschlusse an die Sinnesorgane aus dem Ektoderm."


238


WM. A. KEPNER AND J. R. CASH



Fig. 1. An early stage in the formation of the ciliated pit and its ganglion, (end.) Endoderm or wall of enteron. (g) General epidermis lowered at (a) to form the rudiment of the fundus of pit. Note absence of. cilia in this lowei'ed region and migrating mitotic cells (mc, m'c')- (mes) Mesodermal cells crowded about the forming ciliated pit-ganglion (g). X 1500.


This sharp depression is already a rudimentary pit with its non-ciliated fundus and its ciliated marginal walls but lacks a ciliated pit-ganglion. At this time, about the fundus of the rudimentary pit mitoses arise which send into the mesenchymal space between the fundus and the anlage of the 'brain,' which has already been formed, a proliferation of cells which radiate from beneath the developing fundus of the pit. This mass of cells is the beginning of the ciliated pit-ganglion.

Thus there are established at the outset two parts of the ciliated pit. a) The epithelium of the pit; b) The rudiments of a ciliated pit-ganglion.


CILIATED PITS OF STENOSTOMA 239

The epithelium of the growing pit is extended as a region which is morphologically different from the general epidermis in that its cells are lower and are repeatedly dividing to yield additional cells to the formation of the ciliated pit-ganglion ; also in that the cells which hne the fundus of the pit have irregularly placed nuclei, and have lost their cilia, while the cilia on the marginal cells have become longer than the cilia on the general body epithelium. These characters are shown in figures 2 and 3. Up until this stage in their development the cells of the pitepithelium retain their power to elaborate rhabdites, as is illustrated in figures 2 and 3.

As the pit grows larger no rhabdites are to be found in the epithelium of its fundus. But before these rhabdites have totally disappeared the formation of a peculiar body is started, which in the mature pit is a highly refractive, homogeneous layer, which Ott ('92)2 i^as called the 'homogeneous mass.'

The nature of this 'homogeneous mass' can best be arrived at by observation of the specimens during fixing. As an animal lies in contact with the slide, if it be fixed by dropping the fixing fluid upon it, it will adhere to the slide on account of the protective discharge thrown out by the cells of the general epithelium. To avoid such trouble it was necessary to apply the fixing fluid with a dash as the animal swam around in a small drop of water on the slide. Thus any adhesions which would injure the fixed specimen were avoided. The details of this trouble can be plainly observed under the binocular microscope. If an entire specimen be treated with methylen blue, a protective blue sheath of mucus with imbedded rhabdites (stained deep blue), will be seen to be formed around the animal. If this sheath be removed from the specimen and the stain again applied the epidermal cells fail to respond and the protective sheath is not formed the second time.

Thus it is evident that in an effort to protect itself the epidermis not only discharges rhabdites but also a mucus which stains with methylen blue less deeply than the rhabdites. Now, since

2 Ott; Jour. Morph., vol. 7, 1892.


240 WM. A. KEPNER AND J. R. CASH

in the fundus of the mature pit there are no rhabdites, it is suggested that, as the cells of the developing epithelium are physiologically differentiated, they lose their power to elaborate rhabdites and develop a greater capacity to secrete a permanent, refractive, mucus-like glea which protects the greatly exposed and extremely sensitive fundus of the pit. So we draw the conclusion that the only difference between the mucus secreted by the cells of the general epithelium and the mucus which composes the 'homogeneous mass' of the ciliated pit is that the 'homogeneous mass' is permanent, perhaps more dense, and withstands the action of reagents better than does the temporary secretion of the general epithelial cells. We have made comparison of these two substances by staining with methylin blue, in which case they stain alike, both staining in living specimens a rather dark blue as contrasted with the intensely dark blue of the rhabdites. So much for the development of the epithelium of the ciliated pit and its secretion product.

DEVELOPMENT OF THE GANGLION OF THE CILIATED PIT

As has been stated previously in this paper, beneath the forming ciliated pit there is a mass of cells which we take to be mesenchymal in origin. This statement is made in abeyance since we are not concerned at present with the origin of the 'brain.' We have, however, been able to see that the 'brain' arises from these cells. But the important point which we endeavor to make is that the ciliated pit-ganglion has a distinct origin from the epidermis.

With the earliest formation of the pit-depression at the surface of the body there occur mitoses in its epithelium which send into the mesenchymal region a number of cells which locate themselves between the epithelium of the fundus and the mesenchymal cells which form the 'brain,' as shown in figure 2. There is, however, a distinction at the very outset between the cells of the 'brain' and those which are forming the ciliated pit-ganglion, as shown by the above figure. This proliferation of cells arising from the epidermis continues to grow with the development of the superficial part of the pit.


CILIATED PITS OF STENOSTOMA


241


-^ 'T? ^ -7mes



Fig. 2. Later stage of formation of ciliated pit and its ganglion. Note widened fundus with its low epithelium that yet has rhabdites (rh). Mitoses (mc) in region of fundus continue to be present. Ganglion (g) has enlarged, (mes) Mesoderm that develops into 'brain' and commissure, (end) Endoderm. X 1500.


Throughout the growth of this ganghonic mass of cells thennuclei have a constant chromatin pattern. At the earliest and intermediate stages of the development of these cells their nuclei tend to be oval while their cytoplasmic bodies are more or less elongated, pyriform, or spindle shaped. Figures 1, 2 and 3. In the final stages of their development the cell bodies become less distinct until in the mature ganglion there is only a network of fibers or cytoplasmic strands supporting many spheroidal nuclei. Figure 4.


242


WM. A. KEPNER AND J. R. CASH

br


/


hm








Fig. 3. Later stage in foi-mation of ciliated pit and ganglion. Note appearance of 'homogeneous mass' (hm), with rhabdites (rh) yet present in the fundus epithelium. Ciliated pit-ganglion (g) has now fused with the fibrous part of the 'brain' (br); (end) Constricted enteron. (c) Commissure of 'brain' forming. X 1500.

The interesting outcome of this development is a sensory epithelium from which many cells have retreated, leaving behind a low, secreting epithelium through which they leave elongated processes of themselves.' Figure 2. These processes are the sensory ends of the ganglionic cells which have been described. Thus w^e have the development of a ciliated pit whose marginal cells are covered with extremely long cilia which may protect the delicate fundus against impacts of external objects by practically closing the mouth of the pit to any particles of matter which might enter and in any way injure the sensitive

' It cannot be definitely stated that all ot these ganglionic cells have such processes, since on account of the nature of the case, only a few such processes in each animal can be sectioned parallel to their axes.


CILIATED PITS OF STENOSTOMA


243



— l>r


Fig. 4. Ciliated pit with its 'homogeneous mass' (hm) ; fundus epithelium (fe); marginal epithelium with its cilia (mgc), and pit-ganglion (g) well established; the latter receiving a bundle of fibres from 'brain' (br). (mus) Muscles. X 150C.


base. Over the base is spread the 'homogeneous mass' elaborated by the epidermis of the fundus into which the sensory rods of the ciliated pit-ganglion cells extend and test the chemical nature of the water, conveying the sensations obtained to the pit-ganglion, which merges into the 'brain.'

In these results we have been able to confirm the description of Ott ('92) so far as the general structure of the pits is concerned, but have not, however, been able to agree with his description of the fundus of the pit. He says "the cilia of the


244 WM. A. KEPNER AND J. R. CASH

epithelial cells could be seen passing through the homogeneous mass," and that — the cilia on the small cells at the base of the pit are Uke those of the epithelial cells of the integument except that thej^ are much longer. They range from 8 micra to 15 micra in length."* We have found that the cilia on the marginal walls of the pit are longer than those on the general surface of the body but have found in no case any suggestion of cilia related to the fundus of the pit or its 'homogeneous mass.' According to Ott/ Landsberg says "that the bottom of each pit is covered with a thick layer of homogeneous substance which may be regarded as mucus. Below this is a thin layer of ciliated epithelial cells whose cilia project through the homogeneous layer. Next to this is a much thicker layer which is made up of mostly pyriform cells, although there are other histological elements scattered through it. Next to this layer is the ganglion which is connected with the nerve." This description is very much in accordance with our results with the exception of the statement that there are cilia on the low cells at the fundus of the pit. Ott says "There are three possible methods by which the three layers described by Landsberg might be produced: 1) By a division of the epithelial cells, 2) by a migration of cells from the brain ganglia to the walls of the pits, 3) by a migration outward of some of the epithelial cells to form a second outer layer. If a new layer of cells was formed by the first method we ought certainly to find numerous spindles vertice to the surface in every developing pit."" Now we have assumed the first mentioned method of formation of the ciliated pit ganglion mainly on account of the fact that we have seen these numerous spindles as described by Ott (fig. 2) . We find them in nearly every section that passes through the fundus and ganglion.

In one other respect our conclusions differ from a previous description of the general structure of the pits by von Graff ('13). He says "Das Nervensystem besteht aus zwei langgestreckten Half ten, deren jede durch eine swache Einschntirung

Jour. Morph., vol. 7, p. 291, 1892. Same reference, p. 291. ^ Same reference, pp. 292-3.


CILIATED PITS OF STENOSTOMA 245

in eine hintere, durch eine breite Kommisur verbundene und eine kleinere, vordere Partie zerfallt. Die letztere hildei die beiden Griihschenganglien, in welche sich die Wimpergriihshen einsenken. (Das Tierreich, s. 20.) According to our observations, the ciliated pit-ganglion does not arise from the 'brain' but arises independently from the epidermis.

In conclusion, it is interesting to observe the striking parallelism presented by this organ in its function and mode of origin with the olfactory organ of a vertebrate so far as its function (i.e., its function in the fish) and its mode of origin is concerned.

This organ functions as a tester of the chemical nature of the water which passes through or over it as do the olfactory organs of the fish. Moreover, this organ arises as a modified region or plate in the epidermis. Some cells of this plate sink beneath the base of the plate to form the ganglion of the pit. All this is closely analagous to the following description of the origin of the olfactory ganglion as given by Minot ('92).^

The ectodermal cells of the olfactory plate multiply, the karyokinetic figm'es being found next to the outer or free surface of the layer; the cells thus produced assume the appearance of medullary neuroblasts and at four weeks are found migrating toward the mesenchymal surface, so that the base of the layer of the olfactory ectoderm becomes crowded with nuclei; the protoplasm of these neuroblasts is collected on one side of the nucleus in a pointed mass; the cells now grow forth from the ectoderm and constitute the anlage of the ganglion between the ectoderm and the brain. (P. 637.)

CONCLUSION

The ciliated pit and its ganglion in this flatworm arise from the general epithelium in a manner closely analogous to the mode of origin of the olfactory epithelium and olfactory ganglion of the vertebrates.

' Italics our own.

^ Minot; Human Embryology.


Huber GC. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day. (1915) J Morphol. 26: 247-358.

The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day

G. CARL HUBER

From the Department of Anatomy, University of Michigan, and the Division of Embryology, Wis tar Institute of Anatomy and Biology, Philadelphia

THIRTY-TWO FIGURES

CONTENTS

Introduction 247

Material and methods 249'

Ovulation, maturation, and fertilization 255

Pronuclear stage 257

Segmentation stages 265

2-cell stage 265

4-cell stage 273

8-cell stage 275

12 to 16-cell stage 279

Summary of segmentation stages, rate, and volume changes 280

Completion of segmentation and blastodermic vesicle formation 2S6

Blastodermic vesicle, blastocyst, or germ vesicle 300

Late stages of blastodermic vesicle, beginning of entypy of germ layers. . . . 307

Development and differentiation of the egg-cylinder 317

Late stages in egg-cylinder differentiation, and the anlage of the mesoderm 336

Conclusions 352

Literature cited 356

INTRODUCTION

The early developmental stages of placental mammals, embracing the stages of sex cell maturation and fertilization, of segmentation, of blastodermic vesicle and germ layer formation, though subject of numerous contributions extending over many years, have in no form been completely investigated. The literature dealing with the phenomena of maturation and fertilization as observed in placental mammals has in recent years been enriched by a number of studies to the extent that for certain of the mammals — bat, rabbit, guinea-pig, mouse, and rat — the data at hand are sufficiently complete to enable a clear and comprehensive presentation, based on observed facts, and permit of comparison with similar phenomena as observed in other vertebrate and invertebrate forms. As concerns the process of segmentation in placental mammals, there are still lacking sufficiently comprehensive observations embracing a number of forms to enable a clear and succinct presentation of the rate of blastomere formation, the cytomorphosis of the cells, and of the relative position of the several segmentation stages in the genital tract. This is no doubt owing to the difficulty of obtaining the necessary material timed so as to admit of proper staging, and the impossibility of making extended observations on living material. Our knowledge of the phenomena of blastoderm vesicle formation, though comprehended in its general phases, is lacking in detail, except for a very hmited number of forms. The process of germ layer formation is of such fundamental importance to a clear comprehension of later developmental stages, both in phylogeny and in ontogeny, that a brief account of observed facts in any one form may not be regarded as wholly without value.

Opportunity presented itself, while stationed at The Wistar Institute of Anatomy and Biology, to collect and fix an extended series of embryological stages of the albino rat. This material has proven sufficiently comprehensive to enable a presentation of the several developmental stages of this mammal, beginning with the pronuclear stage and extending to the stage of the anlage of the mesoderm. For this period, which extends to about the tenth day after insemination, only very few of the essential stages are lacking, though for certain of the stages confirmatory preparations would have been desirable. The material at hand, however, seemed sufficiently complete to present a connected account of the stages it is hoped to cover. The embryology of allied forms, especially of the mouse, has received much more extended study than has that of the rat, though the development of the rat has received especial consideration by Fraser, Christiani, Selenka, Duval, Robinson, Widakowich, and as concerns maturation and ovulation, by Sobotta and Burckhard, Kirkham and Burr. The pertinent literature will be considered in connection with the presentation of my own results.

MATERIAL AND METHODS

The material on which this investigation is based was obtained from albino rats (Mus norvegicus albinus, Donaldson)^ taken from the extensive rat colony of The Wistar Institute of Anatomy and Biology. The experience gained in the breeding, feeding, and growth experiments, extending over many years, conducted by Donaldson and his associates and resulting in numerous excellent publications, was at my disposal while collecting this material. The material used was all carefully timed, so that sequence of stages was obtained with some degree of certainty. With care and experience, it is possible to regulate and observe insemination, so that stages may be approximated quite accurately. Kirkham and Burr state that on several different occA^sions we have observed actual pairing" of the albino rat. Widakowich states that he was unable to obtain accurate data as to the age of the embryos except by observing coitus. According to this observer, a female rat permits many males to copulate in the course of several hours, receiving males 30 times or oftener, when suddenly she drives them away. Sobotta and Burckhard, on the other hand, admitted males a few hours after parturition, depending on the fact that many mammals ovulate soon after parturition. Though attempts were made, they were unable to observe pairing, and they state that the 'Dieners' charged "with the care and feeding of the rat colony were only seldom able to observe attempts at pairing. At The Wistar Institute no difficulty is experienced in pairing albino

^ Melissinos and Widakowich state having used as material the albino rat, variety Mus rattus albinus. Donaldson has conclusively shown, that by reason of physical characters — blood crystals, shape of the skull, etc. — the albino rat kept as pet or laboratory animal cannot be Mus rattus albinus, but must be Mus norvegicus albinus.

rats. Dr. J. M. Stotsenburg, to whose experience and careful records I am greatly indebted for the trustworthiness of the material collected, made use largely of females who had born one litter. Pairing was seldom attempted a few hours post partem, as was done by Sobotta and Burckhard, but usually about 30 days after the birth of a litter, which may have been nursed or otherwise disposed of. The great majority of females used in pairing were at the time free from 'domestic cares.' The females employed were kept in separate cages for some time before giving birth to young and until the time of mating. About 30 days after the birth of a litter, a male was placed in the cage with the female. If the female was in heat, copulation usually took place soon after. The male was left with the female for an hour to an hour and a half, during which time several pairings would occur, and at the end of which time the female would try to hide from the male, climb the side of the cage and defy him with her teeth. The male albino rat is not prostrated by the sexual act, the same male serving for several successive copulations. In case the female was not in heat, this soon became evident and the male removed, to be again placed into her cage 24 or 48 hours later. The time when the copulation was first observed was noted on the card attached to the cage and gave the time from which the age of the embryo or respective stage was reckoned. The time given is, therefore, that of 'insemination,' a term which Long and Mark have introduced to indicate "the introduction of the male sexual elements into the genital tracts of the female by the act of coitus or otherwise." This time could be accurately noted, while 'semination' which "applies to the access of the spermatozoa to the eggs in the oviducts, the coming into contact of the male and female reproductive cells" can not be accurately timed. The success attained in pairing albino rats as above stated, obviated the necessity of depending upon chance material or resorting to 'artificial insemination' as described for the mouse by Long and Mark. I am at loss to understand why Widakowich should regard the age determinations of Sobotta and Melissinos (mouse embryos) more accurate than his own, reckoned from the time of observed coitus. The slight though observable variation in the rate of development in a series of ova of the same animal, more marked when supposedly similar stages of several animals are investigated, precludes the accurate timing of stages.

As fixing fluids, there were used Zenker's fluid, sublimatealcohol, Flemming's fluid, Bouin's fluid, and Carnoy's fluid. After a few trials, all were discarded in favor of Carnoy's fluid, prepared by mixing 6 parts of absolute alcohol, 3 parts of chloroform, and 1 part of glacial acetic acid. This somewhat illogically compounded fluid penetrates rapidly and does not cause shrinkage. Tissues are fixed in it for several hours, then washed in several changes of absolute alcohol in which it has been my custom to store the tissues. The following procedure was practiced in all stages up to about 12 days after insemination: The animals were anaesthetized and the head severed from the body, to admit of free bleeding. The rat was then fastened to a board, and thorax and abdomen opened by a mid-sagittal incision, the abdominal walls pinned back, and the intestine elevated toward the thorax. With as little manipulation as possible, the ovaries were separated from their attachment, the mesometrium cut, the uterine horns elevated and the vagina severed. The whole genital tract was then placed on a clean slide and arranged in approximately normal position. Slight tension was maintained by tying a thread to the connective tissue removed with each ovary and bringing the threads along the reverse side of the slide and tying them to the vagina. If the slide is clean, the mesometrium of each uterine horn may be spread out evenly and caused to adhere to the slide. Ovaries, oviducts, -and uterine horns may thus be spread out in normal position and each uterine horn fixed as a straight tube. When thus arranged on the slide, the preparation was placed in a relatively large quantity of Carnoy's fluid, fixed, and then transferred through several absolute alcohols. For nearly all the material used in this study, the method of fixation was as here given. In the earlier stages of material collection, attempts were made to obtain segmentation stages in warm normal salt solution. Several were thus obtained and were used to control the observations made on sections, as will be discussed later. By cutting the oviduct at about its middle, freeing it from its mesosalpinx and cutting the uterus about 1 cm. below the insertion of the oviduct, a pipette fitted with a rubber bulb and filled with warm normal salt solution can be inserted into the uterine cavity and moderate pressure made. It is usually possible to wash into a watch crystal a certain number of the contained segmenting ova. Before reading the article by Widakowich, essentially the same method as employed by him, for isolating implanted blastodermic vesicles was developed. This may be quite readily done after fixation in Carney's fluid and teasing under a stereoscopic binocular. Vesicles sectioned in situ, however, gave on the whole more satisfactory results, so that teasing out implanted vesicles was not resorted to.

The fixed tissues were imbedded in paraffin, using xylol as a clearing fluid. For stages including those falling within the period ranging from the first to the fourth day after insemination, the ovary and oviduct to its insertion in the uterus, were embedded en masse. For stages falling within the period of fifth to sixth day after insemination, the uterine horns were divided into segments measuring about 1.5 cm., and sectioned parallel to the plane of the mesometrium. For later stages, after the enlargements in the uterine horns are distinctly evident, these were removed and cut severally in the three planes. The great majority of the sections were cut at a thickness of 10 ju ; certain ones at a thickness of 5 /x ; a few at a thickness of 7 m- The sections were fixed to the slide by the water-albumen method. The great majority of the series were stained in hemalum, counterstained in Congo red. This solution, which presents certain advantages as a counterstain for embryologic tissues, is prepared as follows: 0.5 gms. of Congo red (Griibler) is placed in 100 ccm. of distilled water and the water brought to boiling. This should give a clear solution. Before cooling, add 100 ccm. of distilled water and 10 ccm. of absolute alcohol. The Congo red solution thus prepared may be kept many weeks. After staining the series in the usual way in hemalum, they are differentiated in acid alcohol, and passed through several washes of 'tap water' into distilled water. They are then stained in the Congo red solution, which may be diluted with distilled water about five times. With the diluted solution, the counterstaining requires one to two hours. The sections are then rinsed in distilled water, differentiated in 80 per cent alcohol, dehydrated, cleared, and mounted in damar. Certain of the series were stained in Heidenhain's iron-hematoxylin and counterstained in Congo red. The drawings accompanying this contribution were nearly all drawn on coarse 'Ross board,' with the aid of the camera lucida at a magnification of 1000 diameters, using pencil and India ink. Such drawings admit of liberal reduction, and give a detail not readily obtained otherwise. Free use has been made of the Born method of reconstruction, especially for earlier stages. The majority of the models thus obtained are here reproduced.

I desire to express my sincere thanks and appreciation of the very material aid given me by Mr. Wayne J. Atwell, then Assistant in the Department of Histology and Embryology of the University of Michigan, in the making of the reconstructions of the oviducts included in this account.

OVULATION, MATURATION, AND FERTILIZATION

When this study was projected, it was the purpose to begin it with the stages of maturation and fertilization. During the time of material collection, there appeared the contribution of Sobotta and Burckhard: "Reifung und Befruchtung des Eies der Weissen Rate," covering these stages fairly completely. Duplication of their work did not seem necessary, so that my own studies begin with the pronuclear stage, to which stage the above mentioned investigators had carried their observations. Therefore, as concerns the process of ovulation, maturation, and fertilization as observed in the albino rat, I am confined for my data to the literature; from which a brief resume is here made.

The normal gestation period for non-lactating albino rats may be roughly estimated as from 21 to 23 days. As has been shown by King, the period of gestation of lactating albino rats varies from a minimum of 24 days to a maximum of 34 days. The average number in a litter is six. In lactating females suckling five or less young and carrying five or less young, the period of gestation usually does not exceed 23 days and may thus be considered as normal. In lactating females suckling five or less young, while they are carrying more than five young, the period of gestation may be prolonged from one to six days. In lactating females suckling more than five young, the period of gestation is always prolonged, and may be prolonged to a maximum of 34 days. Daniel's studies on the white mouse lead him to formulate the following law: "The period of gestation in lactating mothers varies directly with the young suckled." Such exact relation between the number of young suckled and the extent of the prolongation of the gestation period was not observed by King for the albino rat.

In the albino rat, ovulation occurs spontaneously and is not dependent on copulation, which act, however, may precede or follow ovulation. Kirkham and Burr state that ovulation usually occurs about 24 hours after parturition and that the developing ova can be traced in the ovary through the two oestrus cycles preceding their discharge. Long, in his study No. 3, by Mark and Long, finds that ovulation must occur in the albino rat on an average not less than 18 hours after parturition. Sobotta and Burckhard state that ovulation always occurs within 36 hours post partem, though at very variable periods, often only a few hours after the completion of parturition; again, much later. A second ovulation period apparently occurs some 30 days post partem, as would appear from the ■successful pairings conducted by Dr. Stotsenburg. This agrees with the observations of Melissinos, who found that pairings were more numerous when attempted 29 days after parturition, than when attempted 20 to 21 days after parturition, as practiced by Sobotta. Semination probably takes place in the ampullar portion of the oviduct. Eelatively few spermatozoa enter the oviducts and Sobotta and Burckhard estimate that the life of the spermatozoa in the genital tracts of the albino rat is only about 10 hours.


The phenomena of maturation and fertilization in the albino rat have been carefully studied by Sobotta and Burckhard, from whose account the following brief summary is taken: The behavior of the ovum of the albino rat with respect to the formation of polar bodies is very similar to that of most other mammals studied. The first polar body is given off within the ovarian follicle, the second in the oviduct and only after semination. The first maturation spindle, developed from the nucleus of the oocyte of the first order, forms usually immediately after parturition. Kirkham and Burr state "it is usually formed less than 24 hours after parturition." It is short and broad, with the chromatin scattered. The first maturation spindle lies near the center of the ovum, then passes toward the surface assuming a tangential position, and only with the beginning of metakinesis, takes a radial position. The chromosomes of the first maturation spindle, estimated as numbering 16, appear in the form of modified rings, which af e divided transversely across to form short rounded rods with a longitudinal direction in the diaster stage. The first polar body is formed in the ovarian follicle and appears to be relatively large. It is evident only in the ovarian ovum, and appears to be lost soon after its formation. Its fate is doubtful. The first polar body is nearly always missing in tubal ova. Kirkham and Burr state that "the rare occurrence of the first polar body associated with the egg in the tube is to be attributed to its rapid disintegration, which begins as soon as it is formed, and may lead to complete disappearance before ovulation occurs." The second maturation ^'division begins immediately after the completion of the first, without an intervening resting phase. The spindle formed is narrower and longer than the first, with the chromatin massed. In its monaster stage, it lies in a tangential position, with the chromatin in diads, and with the lines of division at right angles to the axis of the spindle. The appearance of the second maturation spindle in the monaster stage marks the end of the maturation phenomena in the ovary. The monaster stage of the second oocyte division was not observed in the ovary by Sobotta and Burckhard, but was seen by Kirkham and Burr. The first division Sobotta and Burckhard regard as a reduction division, a heterotypic longitudinal division ; the second as an equatorial division, a homeotypic longitudinal division. Ovulation probably occurs during the monaster stage of the second maturation division.

The tubal ova are surrounded by a relatively thin oolemma to which are adherent a variable number of discus cells. They are smaller than the ovarian ova; the latter measuring 60 /x to 65 iJL, the tubal ova 55 n to 60/x. The recently discharged tubal ova are to be found in the distended ampullar portion of the oviduct, where they are found clumped together surrounded by discus cells. Semination takes place in this region. The spermatozoa usually enter while the tubal ova are in the monaster stage of the second maturation division, after which metakinesis begins. The second maturation spindle assumes a radial position in the metakinetic phase. The second polar body is smaller than the first, and usually lies compressed between the oolemma and the ooplasm, and is evident during fertilization and segmentation. The spermatozoan head penetrates the thin oolemma and the ooplasma; the long middle piece and tail following the head into the ooplasma, as has been shown by Coe, and Kirkham and Burr. The long middle piece, soon after penetrating the ooplasma, presents an increase in stainability, and its spiral thread becomes evident. The spiral thread, as Duesberg has shown, has its origin in the mitachondria of the spermatid. It may be, therefore, that the male sexual cell introduces mitachondria to the egg cell at the time, of fertilization. Some little time after the penetration of the sperm head, this enlarges and becomes vacuolated, and diplosomes with polar rays become evident. As the sperm head begins to metamorphose, tending to the formation of the male pronucleus, the chromosome group of the dispireme of the second maturation spindle, undergoes metamorphosis to form the female pronucleus. This enlarges rapidly to form a vesicular nucleus which lies free in the ooplasm, while the metamorphosing male pronucleus, usually smaller, is accompanied by a deeply staining thread-like structure, derived from the middle piece. The centrosomes of the first segmentation spindle are by inference derived from the sperm centrosome. The data here given, as concerns the maturation and fertiUzation phenomena pertaining to the albino rat, unless otherwise credited, have been drawn from the account of Sobotta and Burckhard, whose account is accompanied by excellent figures.

Long has studied in living ova of mice and rats the phenomena of maturation and fertilization. Tubal ova were placed in Ringer's solution on an especially constructed slide and spermatozoa introduced. It was possible to seminate the ova of rats with rat spermatozoa and to observe the formation of the second polar body. The formation of the second polar body, "usually near the first polar cell, may begin within five minutes to two or more hours after the spermatozoa are introduced. The constriction may be finished three-fourths of an hour later." The first appearance is an elevation clearer than the rest of the cell. The swelling becomes higher, and at one side of the elevation there appears a depression which is the beginning of the constriction which presently encircles the whole swelling and cuts it off from the egg." Nothing could be said as to the changes which the chromatin undergoes after the spermatozoa have penetrated the egg. The eggs remained alive and apparently normal for about twelve hours, after which they began to degenerate.

pronuclearI stage

As has been stated, my own observations on the development of the albino rat (Mus norvegicus albinus) begin with the pronuclear stage. The material at hand for this stage is listed in table 1, page 258.

Thus there are present in the series 34 ova showing a pronuclear stage and 9 ova showing the second maturation spindle in the monaster phase. The latter may be dismissed with the brief statement that they represent unfertilized ova. In rat No. 108, with 7 ova in the stage of the second maturation spindle, killed 24 hours after the observed copulation, there was found no trace of spermatozoa in the oviduct. Two reasons may be offered for the non-appearance of fertilization in this case:

TABLE 1


HECORD


HOURS AFTER BEGINNING OF INSEMINATION


NUMBER OF OVA


STAGE OF DEVELOPMENT


NUMBER


Pronuclear


Second maturation spindle


106

107 108 109 110


24 hours

24 hours

24 hours

24 hours, 15 min.

24 hours, 15 min.


8 11

7 9 8


00 CO O 00


1

7 1



Total


43


34


9


Ovulation may have occurred so late that the spermatozoa may have died before the ova reached the ampullar portion of the oviduct. This explanation, it would seem, is invalidated by the fact that the position of the ova in the oviduct, as shown by graphic reconstruction, is essentially the same as in the other four rats studied, and in which fertilized ova were found, so that ovulation must have preceded the killing of the animal by some hours. The other reason, more plausible, attributes non-fertilization to a pathologic condition of the genital tract. In this rat, one ovary was distinctly pathologic, with periovarian capsule greatly distended with a sanguinous liquid, while the upper end of the uterine horn with adjacent oviduct on the other side, as seen in sections, presented evidence of inflammation and epithelial desquamation, in part occluding the lumen. It seemed evident, therefore, that the spermatozoa introduced in the genital tract were unable to penetrate to the oviduct and consummate fertilization. The other two unfertilized ova, found with ova in the pronuclear stage, were in oviducts in which no spermatozoa were found. Both in the mouse and the rat, relatively few spermatozoa reach the upper end of the oviduct ; too few, it would seem, to consummate fertilization of all the ova in certain cases. In all of the ova which contained the second maturation spindle, this was in the monaster phase and in tangential position. In size, shape, and chromatin configuration, all presented the characteristics described and figured by Sobotta and Burckhard and Kirkham and Burr, therefore, need not be considered further.

The stage of pronuclei was observed in over 100 ova of tlie white rat by Sobotta and Burckhard. According to these observers, the two pronuclei show in the earlier stages of their development, large chromatin-like nucleoli, the number of which varies. Some little time later, one or several such chromatoid nucleolar bodies with irregularly formed chromatin masses arranged on the linin network are to be observed. At a still later time, the chromatin becomes distributed over the linin network, throughout the nuclear space, giving the appearance of a fine chromatin network. One of the pronuclei is, as a rule, somewhat smaller than the other. This is regarded as the male




Fig. 1 Tubal ova, albino rat. X 200. A, rat No. 110, 24 hours, 15 min., ovum in pronuclear stage, larger nucleus female pronucleus; B, and C, rat No. 59, 2 days, 2-cell stages, thin oolemma showing in C, only partially seen in B; D, rat No. 62, 2 days, 22 hours, 3-cell stage, the nucleus of the unsegmented blastomere in the monaster phase, only one of the other two cells showing in the figure.

pronucleus, since near it the 'sperm centrum' was now and then observed. The pronuclei lie in about the center of the ovum. The pronuclear stages of my own material, observed in 34 ova, obtained 24 hours after the beginning of insemination — thus at the end of the first day of development — all present essentially the same stage of metamorphosis. As may be seen in .A. of figure 1, the nuclei are distinctly membraned, and are of relatively large size. The ovum here sketched measures in the stained preparation 70 n by 62 p., and is, therefore, of slightly oval form. Sobotta and Burckhard give 55 ix to 60 ii as the size of the tubal ova, and 60 /x to 65 /x as the size of the ovarian ova in the white rat. Kirkham and Burr give the diameter of the hving unsegmented egg of the rat as of 0.079 mm. As may be seen from A and B, of figure 2, the tubal ova, even when free in the oviduct, are not of necessity spherical in shape, but often shghtly compressed, as may be clearly seen in four models of tubal ova in the pronuclear stage, reconstructed at a magnification of 1000 diameters, in my possession. Depending on the plane of section, the diameter of a tubal ovum may thus vary to the extent of 5 /x to 8 m- The two nuclei in the preparation shown in A of figure 1, measure, the larger one, regarded as the female pronucleus, 23 m by 16 /x, the smaller 17 m by 15 ix. Essentially all of the chromatin is distributed over the linin network in fine granules, the larger nucleus presenting one large, faintly



Fig. 2 Models, made after the Born method, of two tubal ova of the albino rat in the pronuclear stage. X 200. A, rat No. 106, 24 hours; B, rat No. 110, 24 hours, 15 min. Reconstructions made at a magnification of 1000 diameters, figure reduced in reproduction.

staining chromatoid nucleolus. The ooplasm is finely granular, distributed so as to give the section a slightly mottled appearance. When compared with figures given by Sobotta and Burckhard (figs. 21 to 24, plates 9-10) showing pronuclear stages of the ova of the rat, my own seem to fall in about the middle of this series, thus some little time after their formation, but not immediately preceding the stage of segmentation spindle formation. In the albino rat, and perhaps in other mammals, the pronuclear stage, in its various phases of nuclear metamorphosis, must constitute a stage covering a relatively long period. If it is assumed that semination occurs about 10 to 12 hours after the beginning of insemination, such assumption being justified by the observations of Sobotta and Burckhard, according to whom the life of the spermatozoa in the genital tract of the white rat is only about 10 hours, and if it is recalled that in


DEVELOPMENT OF THE ALBINO RAT 261

living rat ova Long found that the constriction of the second polar body may be completed three-fourths of an hour after its inception, then it must be evident that the pronuclear stage extends through a period which exceeds 10 to 12 hours, since in none of my pronuclear stages obtained 24 hours after insemination was evidence of first segmentation spindle observed.

In order to determine accurately the relative position of the ova within the oviduct during the pronuclear stage and the stages of segmentation, oviducts containing ova were reconstructed after the Born wax plate method. In form, relations, and general structure, the oviduct of the albino rat is essentially the same as that of the mouse as described by Sobotta. The oviduct of the rat measures from fimbriated end to termination in the uterine horn from 2.5 cm. to about 3.0 cm. It presents eight to ten fairly constant major folds, the middle group of which is closely applied to the ovarian capsule. The upper or distal folds pierce the capsule, ending in the fimbriated end found within the capsule, while the lower or proximal folds, proximal with reference to the uterine horn, effect connection with the uterine horn. These relations are essentially the same as those described by Sobotta for the oviduct of the mouse. This observer recognizes four segments in the oviduct of the mouse, characterized by epithelial lining, nature and extent of folding of the mucosa, and thickness of the musculature. The first segment, which falls to the infundibulum, presents a thin musculature and high mucosal folds with epithelial lining consisting of relatively short cylindrical cells with distinct cuticular border and long cilia. As characteristic of this portion of the tube there are further described accessory nuclei compressed between the epithelial cells. Only this portion of the oviduct is ciliated. In the second segment, the lumen is large and the folds of the mucosa prominent. They are covered by a non-ciliated epithelium, without distinct cuticular border. The musculature is relatively thin. In the third segment the musculature is well developed with circularly and longitudinally disposed cells. The lumen is narrow and the folds are nearly absent, while the epithelium is of a simple columnar variety. The fourth segment, not so well characterized, consists of the loops which make con


262 G. CARL HUBER

neetion with the uterine horns, with folds and epithelium much as in the third segment, and a prominent musculature. In all essentials, this description applies to the oviduct of the albino rat, except that in the first segment the accessory nuclei described by Sobotta as found between the epithelial cells were not evident in the rat. In figure 3, is reproduced a model of a wax reconstruction of the right oviduct of rat No. 106, killed 24 hours after the beginning of insemination, and containing eight ova in the pronuclear stage. This oviduct measured from fimbria to termination in the uterine horn 3.2 cm. It presents 10 major folds, which folds may be recognized with more


Fig. 3 Model of right oviduct of rat No. 106, 24 hours. X 10. Fimbriated end and infundibulum removed in the drawing so as to expose underlying loops; their relative position given in dotted outline. The position of the ova, which are outlined in circles, is shown as if seen through a transparent wall. The relative position of three of the eight ova found within this tube cannot be revealed in this view of the model.

or less clearness in all the models made and here reproduced. The slight difference in the relative position of these folds as seen in the several figures may be accounted for by the varying degrees of tension to which the tissues were subjected prior to fixation. In rat No. 106, the ovaries with oviduct and upper end of the uterine horn, were excised and placed in the fixing fluid without applying any tension. Of these 10 major folds, the four distal ones, those beginning with the fimbriated end, fall to segments one and two of Sobotta' s designation, having a wide lumen and folded mucosa. In the figure, the position of the ova is indicated by small black circles. By reason of the relation of the folds, only five of the eight ova can be brought


DEVELOPMENT OF THE ALBINO RAT


263


to view in the aspect of the model sketched. The position of the first and the last of the series is correctly given. The ova are situated in a loop of the oviduct which is about 8 mm. from the fimbriated end. By the end of the fiirst day after the beginning of insemination, the ova have thus travelled about onefourth the length of the oviduct. In figure 4 is reproduced a model of a detailed reconstruction of that portion of the oviduct



Fig. 4 Model of the segment of the right oviduct of rat No. 106, 24 hours, containing the ova the general position of which is shown in Figure 3. X 50. The wall is in part removed, so as to expose the lumen. Note the character of the folds of the mucosa. The relative position of the eight contained ova, all in the pronuclear stage, is clearly shown.

containing the ova, representing a loop of the tube with one side cut away, this to show the extent and character of the mucosal folds, the width of the lumen and the relative position of the several ova. The figure presents these facts so clearly that lengthy description is deemed unnecessary. The several ova are distributed through a tube segment measuring about 2.5 mm. in length. They lie free in the lumen, apparently bathed in a fluid from which there is only a small amount of precipitation at the time of fixation. Their position in the oviduct at


JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


264


G. CARL HUBER


this stage, free in the lumen, is well shown in figure 5, which is from a longitudinal section of a loop from the left oviduct of rat No. 109, showing three ova, with but few remaining discus cells and a thread of coagulum linking the ova together, an appearance quite characteristic at this stage. The figure was drawn by aid of camera lucida from a single section. All of the ova, of which there are seven, distributed through this loop, contain two pronuclei ; in none of the ova figured do the two pronuclei




- . %


Fig. 5 Camera lucida drawing of a portion of a section of the left oviduct of rat No. 104, 24 hours, 15 min. X 100. Three ova with a few discus cells, are shown as lying free within the lumen. The ova are in the jironuclear stage, not shown in this section, but readily ascertained by tracing through the series. The loop of the oviduct here shown in section is cut longitudinally, thus the folds of the mucosa are not prominent.


fall in the same section. My series contains seven oviducts with pronuclear stages, with accompanying ovary, cut serially. Only one of the oviducts, rat No. 106, was reconstructed in wax. In the other six, graphic reconstructions were made. This permits analysing the loops, determines their sequence, but does not readily admit of measuring their length. In the six oviducts graphically reconstructed, the position of the ova, the number of which varies from one to seven in the several tubes, is essentially as in the wax reconstruction figured. It would appear,


DEVELOPMENT OF THE ALBINO RAT


265


therefore, that in the albino rat, 24 hours after the beginning of insemination, the ova are to be found in the pronuclear stage, with the ova distributed in the end of the third to the beginning of the fourth major loop of the oviduct, a portion of the oviduct having a relatively wide lumen and lined by a much folded mucosa and possessing a relatively thin muscular wall, having thus migrated about one-fourth of the length of the oviduct.


SEGMENTATION STAGES


2-cell stage. The material on which my own observations of this stage are based is listed in table 2.


TABLE 2


RECORD NUMBER


HOURS ATTER BEGINNING OF INSEMINATION


NUMBER OP OVA


STAGE OP DEVELOPMENT


60

59 58 61 62


1 day, 18 hours

2 days

2 days, 17 hours 2 days, 18 hours 2 days, 22 hours


7 8

8

8

11


2-cell stage

2-cell stage / 7, 2-cell stages; \ 1, 3-cell stage

2- cell stage / 10, 2-cell stages; \ 1, 3-cell stage


Thus in all 40 ova after the completion of the first segmentation division and 2 ova in the 3-cell stage, in each of which the undivided blastomere presents a nucleus in mitosis.

My own material lacks stages showing the formation of the first segmentation spindle, the conjugation of the two pronuclei, and the first segmentation division. I am forced to proceed from the pronuclear stage to that showing the first two blastomeres. It was not possible to supplement my material after this was sectioned and the stages determined, .since it was only after leaving The Wistar Institute that this gap in my series was recognized. This is the more to be regretted since neither Melissinos, Sobotta and Burckhard, nor Kirkham and Burr, all of whom have considered maturation and fertilization as observed in the albino rat, discuss these stages in their account. In the albino rat, the fusion of the two pronuclei on the first segmentation


266 G. CARL HUBER

spindle, and the first segmentation division would appear to fall to a period ranging from the beginning to near the middle of the second day after the beginning of insemination, probably about 30 to 32 hours after insemination. In the mouse, in which these stages have been very completely and carefully investigated by Sobotta, the conjugation of the pronuclei and the first segmentation spindle formation falls to the end of the first day after copulation. These phenomena appear to be passed through rather quickly in the mouse ovum, covering a period of only about one and a half to two hours.

The 2-cell stage with resting nuclei extends through a relatively long period. In the mouse it extends through nearly an entire day, as shown by Sobotta, who found 2-cell stages present through a period ranging from 25 hours to 48 hours after copulation. Melissinos often observed the 2-cell stage with resting nuclei in both mice and rats in material gathered 24 hours after copulation and to 44 hours thereafter. It is to be regretted that this observer does not differentiate riiore specifically between ova of mice and rats in his description. As a rule it is impossible to determine except by inference to which of the two varieties of ova his account refers. It may be assumed that the statements made apply equally well to the ova of either the mouse or the rat.

In my own material, the 2-cell stage was observed during a period extending from 1 day, 18 hours to 2 days, 22 hours after the beginning of insemination, thus for a period extending over more than 24 hours. In the albino rat, the first two blastomeres are equivalent cells of essentially the same size and structure, as may be seen from B and C, of figure 1, drawn respectively of ova found in the right and left oviducts of rat No. 59, killed two days after the beginning of insemination, and regarded as representative ova. The two cells of each ovum are not spherical, but of slightly oval form, with relatively large, distinctly membranated nuclei, with fine chromatin granules scattered on the linin network and a number of relatively large chromatoid nucleoli. The cj^toplasm presents a granular appearance, the granules being evenly distributed throughout the cell. In my own material, I seldom find the two cells lying in the same plane,


DEVELOPMENT OF THE ALBINO EAT 267

but one cell, as a rule, rises slightly higher than the other. This is more clearly seen in reconstructions than in sections. In figure 6 are shown reconstructions of the 2-cell stages, figured in B and C of figure 1. In B, of the figures, the plane of section is at right angles to the vertical axis of the reconstruction as shown in B of figure 6, while in C of figure 1, the plane of section is parallel to the vertical axis of the reconstruction shown in A of figure 6. The equivalence or non-equivalence of the first two blastomeres of the segmenting mammalian ovum has been the subject of discussion since the time of Van Beneden's fundamental observations on the segmentation of the ovum of the rabbit. This discussion has been summarized a number of times in recent years, and need not be entered into here. Suffice to say that the consensus of opinion of the more recent contributors



Fig. 6 Models, obtained by reconstruction after the Born method, of the 2-cell stages of the albino rat. Rat No. 59, 2 days. X 200.

is, that the first two blastomeres of the mammalian ovum are equivalent in size and structure if the stage is observed soon after its formation. As above stated, the 2-cell stage of the mammalian ovum extends through a relatively long period, probably about 24 hours. The two cells do not as a rule divide synchronously, the division of one preceding the other by some little time, resulting in a 3-cell stage. The cell to divide first increases slightly in size and presents a clearer protoplasm prior to its division. In a 2-cell stage, viewed in this phase of cytomorphosis, one of the cells appears slightly larger with clearer protoplasm than does the other cell, explaining the difference in size and structure observed by Van Beneden and by other observers who concur in his views. I am convinced that a difference in the size of the two cells may be accounted for by the plane of section in which they are cut, even though the' nuclei of both cells are included in the section. In the figures of sections of the 2-cell


268 G. CAEL HUBER

stage of the mouse, given by Sobotta and Melissinos, the nuclei of the two cells lie in about their center and essentially in the same plane. In my own material of the 2-cell stage of the albino rat it is not unusual to find the nuclei of the respective cells nearer the opposite poles of the two cells than at their centers, as shown in C, of figure 1. In B of this figure, where the two nuclei appear as lying much nearer the center of the cells, they are in reality placed much as in C, as is shown by the reconstruction.



Fig. 7 Model of the right oviduct of rat No. 59, 2 days. X 10. Not quite the entire oviduct was available for reconstruction, the upper end of the uterine horn thus not shown in the figure. The position of the four 2-cell stages, each of which is outlined in a circle, found within the tube, is shown as if seen through a transparent wall.

To determine the position of the segmented ovum in the 2-cell stage in the oviduct, reconstructions were made of two oviducts. In figure 7 is shown a reconstruction of the right oviduct of rat No. 59, killed two days after the beginning of insemination. In preparing the material for embedding, this oviduct was cut not quite at its insertion into the uterine horn. The portion of the oviduct reconstructed measures 2.29 cm. Nine major loops are shown. The four ova in the 2-cell stage found in this tube are situated in the sixth to the seventh loop at a distance of about 1.4 cm. from the fimbriated end. This portion of the oviduct falls to segment three of Sobotta' s designation. It is lined by non-ciliated epithelium resting on a mucosa with inconspicuous secondary folds, but presenting four or five characteristic major folds. This portion of the oviduct is closely


DEVELOPMENT OF THE ALBINO RAT 269

applied to the outside of the ovarian capsule, and conspicuous in all of the figures of models of the oviducts here presented. The detail of the distribution of the ova in the tube is given in figure 8, a reconstruction under a higher magnification of the segment of the oviduct containing the ova. The lumen is exposed so that the character of the mucosal folds may be seen. The ova are spaced in a segment of the tube measuring 3 mm., and are



Fig. 8 ]Model of the segment of the right oviduct of rat No. 59, 2 days, containing the four 2-cell stages as shown in figure 7. X 50. Note the absence of prominent folds in the mucosa. The segment presented in the reconstruction measures 3 mm. The four 2-cell stages contained in this tube are relatively widely spaced.

in this case more widely separated than is usual for this stage. In figure 9, there is reproduced a reconstruction of the left oviduct of rat No. 62, killed 2 days, 22 hours after the beginning of insemination. This tube was also cut a little before its insertion into the uterine horn. The portion reconstructed measures 2.45 cm. In it there are found five ova in the 2-cell stage, situated about 2 cm. from the fimbriated end, and in the last loop of the third segment of the oviduct. The five ova are closely


270


G. CARL HUBER


grouped between two opposing folds of the mucosa. Their general relations are shown in figure 10, a reconstruction under higher magnification of the segment of the oviduct containing



Fig. 9 Model of the left oviduct of rat No. 62, 2 days, 22 hours. X 10. Not quite the entire oviduct was available for reconstruction, thus the relative position of the upper end of the uterine horn is not shown in this figure. Fimbriated end and infundibulum removed in the drawing, so as to expose the underlying loops; their relative position is given in dotted outline. The position of five 2cell stages, found within this tube, is given as if seen through a transparent wall.



Fig. 10 Model of the segment of the left oviduct of rat No. 62, 2 days, 22 hours, containing the five 2-cell stages, the general position of which is shown in figure 9. X 50. Note the compact grouping of the ova.


the ova, cut so as to expose the lumen. At the magnification used it was not possible to reproduce in the model the exact shape of the several ova, their relative position is, however, correctly given. In all, ten oviducts, containing 40 ova in the 2-cell stage, are included in my series. Of these, two, as above given, were reconstructed by the Born method. The other eight were reconstructed graphically, beginning with the uterine


DEVELOPMENT OF THE ALBINO RAT 271

« 

end of the tubes. In six of these, the ova are quite closely grouped as given in the reconstructions shown in figures 9 and 10. In the remaining two they were more widely spaced, about as shown in figures 7 and 8. In the oviducts taken from rats Nos. 58, 61, 62, killed respectively 2 days, 17 hours, 2 days, 18 hours, and 2 days, 22 hours, after insemination, the ova are found in a portion of the tube which corresponds very closely to that shown in the reconstruction presented in figure 9. In rat No. 60, killed 1 day, 18 hours after insemination, the ova are more widely spaced and are situated in a segment of the oviduct approximately one loop nearer the fimbriated end than that given in figure 7, a model of the oviduct of rat No. 59, killed two days after insemination.

In one of the segmented ova of rat No. 60, the two blastomeres resulting from the first segmentation division are distinctly separated by a space equal to about one-half of the diameter of each of the cells. No oolemma is discernible. The two separated cells appear normal in size, shape, and structure, as do also their nuclei. They lie free in a slightly distended portion of the lumen, and appear not to have been separated as a consequence of manipulation. The possibility of each developing separately is suggested, and may be offered as a possible explanation of the occurrence of very small embryos now and then found among others showing normal development. King states that "On dissecting pregnant females (rats) one frequently finds one or more embryos that are much smaller than the rest. While in some instances such small embryos appear normal and are presumably either runts or embryos that have resulted from superfecundation, in the majority of cases they are pathological, probably because of faulty implantation of the ovum." My own material contains pathologic ova and embryos in different stages of development. This portion of the material will be considered in Part II, where the possibility of the occurrence of half embryos will be discussed.

As may have been seen, the 2-cell stage of the albino rat covers a period of somewhat more than 24 hours, extending from about the middle of the second day until toward the end of the third


272 G. CARL HFBER

day after the beginning of insemination. During this period the segmented ova migrate in the oviduct for a distance equahng nearly half its length. The trustworthiness of the material, it would seem to me, is shown by the fact that in the shorter time stages the segmented ova are situated nearer the fimbriated end, while in the longer time stages they approach the region of the insertion of the oviduct into the uterine horn. This is clearly shown in the reconstructions shown in figures 7 and 8. A 3-cell stage was observed only twice: in one of eight ova contained in the oviducts of rat No. 58 (2 days, 17 hours) and in one of eleven ova found in the oviducts of rat No. 62 (2 days.



Fig. 11 Two views of each of three models of 4-ceIl stages of the albino rat. Rat No. 50, 3 days, 1 hour. X 200. A, B, and C, gives a side view, A', B', and C a vertical view, of each of the three models.

4.

22 hours). All the other ova found in these two animals were in the 2 -cell stage. In the two 3-cell stages noted, the undivided blastomeres of each ovum presented a nucleus in mitosis; in one, in the monaster phase, in one, in the diaster phase. The division of the first two blastomeres, resulting in the 4-cell stage, it would appear, occurs in the albino rat toward the end of the third day. The material gathered at the beginning of the fourth day after insemination presents throughout a 4-cell stage. In D of figure 1 is shown reproduced one of the sections of a series of six sections including one of the ova in the 3-cell stage. Only one of the two cells resulting from the division of one of the first two blastomeres is included in the section; the cell in mitosis represents the undivided blastomere.


DEVELOPMENT OF THE ALBINO RAT


273


4-cell stage. The material includes the oviducts of two rats, Nos. 50 and 63, killed 3 days and 1 hour after the beginning of insemination, with twelve ova in the 4-cell stage. In figure 11, there are shown two views of each of the models obtained by reconstruction after the Born method, at a magnification of 1000, of the three 4-cell stages found in the oviducts of rat No. 50. The drawing of the reconstructions do not present the conventional figures of the 4-cell stage of the mammalian egg. In none of the twelve ova of this stage was the plane of section such as to include all of the four cells in one section. ^ Nearlv all



Fig. 12 Cross-section of right oviduct of rat No. 50, 3 days, 1 hour. X 100. This section contains two cells of a 4-cell stage of the albino rat, slightly compressed between the folds of the tubal mucosa.


lie in a portion of the tube which presents a relatively narrow lumen, and appear as if slightl}^ compressed between the folds of the mucosa. I am not disposed to regard this as a resultant of fixation, due to contraction at the tim.e of fixation. In figure 12 is reproduced a cross section of the right oviduct of rat No. 50, passing through a 4-cell stage. It is evident that in shape the two cells included in the section, conform in the main to the form of the lumen, the mucosa appearing as slightly retracted to one side of the egg mass. This conformity in shape of cell mass to the form of the lumen I find quite general in my material showing segmentation stages of the albino rat, to some extent


274


G. CARL HUBER


even in the 2-cell stage, more clearly shown in the 4-cell and later segmentation stages, as will appear from further reconstructions presented. It would seem to me reasonable to assume that these cell masses are of such plasticity that they are molded by the tubal mucosa rather than thej^ would compress the mucosa and maintain an inherent form. A number of segmented ova in presumably the 6- and 8 -cell stages were removed from oviducts by injection and studied in warm normal salt solution, in a living state. In the warm normal salt solution the morula masses



Fig. 13 Model of right oviduct of rat No. 50, 3 days, 1 hour. X 10. A short segment of the upper end of the uterine horn, lower part of the figure, is included. The fimbriated end and a part of the infundibulum removed in the drawing so as to expose the underlying loops; their relative position is indicated in dotted outline. The position of the four ova in the 4-cell stage, at the beginning of the last loop of the oviduct, is shown as it seen through a transparent wall.


presented a nearly spherical form, conforming to the conventional illustrations of the same. In none of the sections of fixed material of my series was this the case. The form of the cell mass, assumed by the segmenting mammalian ovum in early stages of segmentation, therefore, seems to me a question more for academic discussion than one of fundamental importance. The right oviduct of rat No. 50 (3 daj^s, 1 hour) was reconstructed after the Born method. This model is reproduced in figure 13, and includes the uppermost end of the uterine horn. The oviduct


DEVELOPMENT OF THE ALBINO EAT 275

measures 2,8 cm. and contains four ova in the 4-cell stage, situated at the beginning of the last loop leading to the uterine horn, 2.25 cm. from the fimbriated end, thus in the fourth segment of the oviduct as of Sobotta's designation. In figure 14 is reproduced a detailed reconstruction of the segment of the oviduct containing the ova, with the convex portion of the wall of this loop, as shown in figure 13, removed. The section reproduced in figure 12, passes through the lower of the three upper ova, shown in reconstruction in figure 14. In the figure of the reconstruction as also in that of the section, is shown the groove in which these three ova lie. The other oviducts con


Fig. 14 Model of the segment of the right oviduct, rat No. 50, 3 days, 1 hour, containing the four ova in the 4-cell stage, the general position of which is shown in figure 13. The convex portion of the wall of the loop containing the ova is removed, so as to expose the lumen.

taining 4-cell stages were reconstructed graphically, beginning with the uterine end. The position of the ova in each is essentially as given in the model reproduced in figure 13.

8-cell stage. In rat No. 57, killed 3 days, 17 hours after the beginning of insemination, there are found in the left oviduct, six segmented ova in the 8-cell stage and one segmented ovum in the 11-cell stage. The right ovary and oviduct was injured in the process of embedding and could not be used for sectioning. The ova are spaced in the loop of the oviduct which terminates in the uterine horn. Six of the segmented ova were reconstructed, the seventh was not detected at the time the reconstructions were made. The six models obtained are reproduced


276


G. CARL HUBER


in figure 15, two views of each model being shown. Five of the models, A to E, show 8-cell stages. In F, there is figured an 11-cell stage, three of the cells having completed the next following division. As may be seen from the figures, the form of these morula masses is not spherical but in the main slightly oval, with further irregularities better shown in the models than in the illustrations, due to the fact that the egg masses conform to the shape of the lumen of the oviduct in the region in which they are found. The mucosa lining the segment of this



Y) 1 ;




-j^


Fig. lo Models, obtained by reconstruction after the Born method, of 8cell and 11-cell stages of the albino rat. Rat No. 57, 3 days, 17 hours. X 200. Two views of each model is presented. A-A', to E, E' are of models of 8-cell stages; F and F' of a model of a 11-cell stage.


oviduct containing the ova presents four quite regular longitudinal folds. In figure 16, there is presented a model of a detailed reconstruction of the segment of the oviduct containing the ova, their relative position in the tube and their relation to the major folds is clearly shown. One of these folds it was necessary to in part remove so as to bring to view in the drawing certain of the ova. In figure 17, there is reproduced a portion of one of the sections of the series from which the model shown in figure 16 was made. The fold of the mucosa occupying the center


DEVELOPMENT OF THE ALBINO RAT


277



Fig. IG Model of the segment of the oviduct, rat No. 57, 3 days, 17 hours, containing the ova shown in fig. 15. X 50. A portion of the wall of the oviduct and a part of the major folds of the mucosa are removed in the drawing so as to expose the contained ova. The relative position of the seven ova found in the tube is shown, as also the extent and character of the folds of the mucosa. The exact form of each of the several ova could not be reproduced in the model at the magnification used; their position is given correctly.







%s^§^4'j



Lcfi^


Fig. 17 Camera lucida drawing of a portion ot a section of the left oviduct of rat No. 57, 3 days, 17 hours. X 100. This section is of the series of sections from which the models shown in figures 15 and 16 were made. Sections of four 8-cell stages, as seen in a single section, are included. The close proximity of three of these ova, their relation to the tubal wall and mucosal folds is to be noted.


278


G. CARL HUBER


of the drawing, and greatly occluding the lumen, is the fold removed in the model. In this very fortunate section four of the morula masses are included; all are of the 8-cell stage and represent in section the four ova which are placed closely together as seen in the model figured in figure 16. In figure 19, A, there is reproduced at higher magnification another of the sections of the series,, including the right one of the three ova in close apposi


Fig. 18 Model of the left oviduct of rat No. 51, 4 days. X 10. A short segment of the upper end of the uterine horn was included in the reconstruction, lower end of the figure. The position of three of the morula masses, 12-cell to 16-cell stages, in the terminal part of the oviduct is to be noted, a further one is located in the upper part of the uterine horn. These are shown as if seen through a transparent wall. A fifth morula, situated in the uteriiie horn about 1.5 cm. from the entrance of the oviduct, is not included in the figure.


tion as seen in figure 17, showing six of the eight cells, each cut in the plane of its nucleus. In both of these figures (figs. 17 and 19) the morula masses, as seen in the sections drawn, present a quite regular oval outline. In succeeding sections, in which the mucosal fold and the wall of the oviduct approximate, the cross diameter of each of the four morula masses becomes greatly reduced, they appearing in the final sections of the series in which they are included, as narrow, non-nucleated bands of protoplasm.


DEVELOPMENT OF THE ALBINO RAT 279

This series, it seems to me, corroborates the statement previously made, that the detail of form of the living segmenting ova of certain mammals, while in transit through the oviduct, is in a great measure dependent on the configuration presented by the lumen of the oviduct in the particular region in which they are found.

12-cell to 16-cell stages. Rat No. 51, killed 4 days after the beginning of insemination, presents the end of the segmentation stages in the oviduct. In the genital tract of this animal there were found eight morula masses, five on the left side and three on




Fig. 19 Sections of morula stages of the albino rat. X 200. A, 8-cell stage, rat No. 57, 3 days, 17 hours; six of the eight cells, each cut in the plane of its nucleus, are included in the section figured. B, C, and D, 12-cell to 16-cell stages, from right oviduct, rat No. 51, 4 days.

the right side. It is somewhat difficult to determine definitely the number of cells constituting each of the morula. The number appears to vary between 12 and 18, though nearly all of the morula masses show certain nuclei in mitosis. The left oviduct with a short adjoining segment of the uterine horn was reconstructed. Slight tension was applied to the tissue prior to fixation, which accounts for the elongation of the proximal loop of the oviduct. The model is reproduced in figure 18. As is evident on study of this figure, three of the morula masses are situated in a portion of the oviduct just prior to its insertion in the uterine tube. These are closely grouped between folds of the mucosa. A fourth morula is found in the uppermost part of the uterine

JOURN.^L OF .MORPHOLOGY, VOL. 26. NO. 2


280 G. CARL HUBER

cavity, just distal to the opening of the oviduct, lying free in a slightly distended portion of the lumen. This morula is of irregular discoidal form, presenting an appearance which suggests that it was fixed soon after it escaped from the oviduct. A fifth morula, of regular oval form, comprising very probably 18 cells, all of which present resting nuclei, is lodged in a shallow pit of the uterine mucosa a little over 1 cm. from the tubal opening. This portion of the uterine horn was not included in the reconstruction, the position of this morula is not, therefore, indicated in the figure. It is evident that this tube was fixed while the several morula masses were in transit from the oviduct to the uterine horn, which occurs, to judge from the material at my disposal, at the end of the fourth day after the beginning of insemination. The morula masses of the right tube are situated in the oviduct just before its point of insertion into the uterine horn, in about the same relative position as are the three upper morula masses of the left side, as shown in the reconstruction. They are of discoidal form, in close relation and appear to comprise, the one 12, the other two 14 to 16 cells. In B, C and D of figure 19 are reproduced sections of each of these three morula stages. The figures, however, are delusive in that the section for each passes through the greatest diameters of the respective morula.

The material at hand permits the conclusion that in the albino rat the segmenting ova pass from the oviduct to the uterine horn at the end of the fourth day after the beginning of insemination, probably in the 12-cell to 16-cell stages. With the beginning of the fifth day, as will appear from further discussion, all of the ova are to be found in the uterine horn.

SUMMARY OF SEGMENTATION STAGES, RATE, AND VOLUME

CHANGES

The following summary of the data (table 3) gained by a study of the models of oviducts containing ova in stages from the pronuclear to 12-cell to 16-cell stages in which latter stage transit to the uterine horn occurs, is presented to indicate rate of transit within the oviduct. The regularity of the rate of transit


DEVELOPMENT OF THE ALBINO RAT


281


as revealed in the summary may perhaps speak for the trustworthiness of the age data as concerns my material.

It will be observed that the ova approach the uterine end of the oviduct while in the 2-cell stage; transit through the last portion of the oviduct, where the greater part of the segmentation occurs, being relatively slow. It is hoped that these data, for the accuracy of which I am dependent on reconstructions, may be of service to others who may desire to collect segmentation stages of the albino rat.


TABLE 3








DISTANCE


RELATIVE


RECORD


SIDE RECON


NUMBER


STAGE


LENGTH OF


OF OVA


LENGTH OF


NUMBER


STRUCTED



OF OVA


OVIDUCT


FROM FIM

TUBE








BRIA


TRAVERSED







cm.


cm.



106


R


1 day


8


pronuclear


3.2


0.8


0.25


59


R


2 days


4


2-cell


2.29*


1.4


0.61


62


L


2 days, 22 hrs.


5


2-cell


2.45*


2.0


0.82


50


R


3 days, 1 hr.


4


4-cell


2.8


2.5


0.90


51


L


4 days


5


12- to 16cell


2.86


2.86


1.00


Not the entire length of oviduct was available for reconstruction.


In order to obtain the volume changes of the ova during transit through the oviduct, beginning with the pronuclear to 8-cell to 11-cell stages, the following procedure was adopted. As has been shown by my figures, reconstructions were made at a magnification of 1000 diameters of ova presenting the stage in question. The sections of my series measure 10 n in thickness. In order, therefore, to obtain the correct third dimension, it was necessary to use wax plates 10 mm. thick, in actual practice, five superimposed 2 mm. plates. For the majority of the sections of my series this procedure was relatively simple. However, there was usually a question as to the thickness to be ascribed to the first and last section of any given series, since it v/as evident, both from the appearance of the section, as seen under the magnification used, and the appearance of the model, that the end sections did not measure 10 ^ in thickness, and it


282


G. CARL HUBER


was necessary to reduce proportionately the thickness of the wax plate representing them. As a rule, these were made about one-half the thickness of the other plates. The irregularities revealed by the rough model after superimposing the respective plates, not so marked as might be supposed considering the thickness of the plates used, were adjusted, not by trimming the model and cutting away wax, but by smoothing with warm irons. The possibility of error is admitted, but since all of the models were made in the same way, errors if committed were probably essentially the same for all of the models. The volumes of the models w^ere obtained by weighing the water displaced by each, and after making the necessary temperature corrections, reducing weight of water displaced to volume. The average of several determinations is given in table 4.


TABLE 4






ACTUAL VOL.


AVERAGE VOL.


RECORD NUMBER


AGE


STAGE


OF EGG MASS


PER STAGE GIVEN






IN C. MM.


IN C. MM.


106


1 day



pronuclear


0.00015058



106


1 day



pronuclear


0.00014317



106


1 day



pronuclear


0.00015775



106


1 day



pronuclear


0.00017127


0.000155693


59


2 days



2-cell stage


0.00016240



59


2 days



2-cell stage


0.00018273


0.000172565


50


3 days,


1 hr.


4-cell stage


0.00018338



50


3 days,


1 hr.


4-cell stage


0.00015520


0.000162443


57


3 days,


17 hrs.


8-cell stage


0.00018893



57


3 days,


17 hrs.


8-cell stage


0.00016040



57


3 days.


17 hrs.


8-cell stage


0.00018653



57


3 days.


17 hrs.


8-cell stage


0.00018193



57


3 days,


17 hrs.


8-cell stage


0.00019979


0.000183516


57


3 days,


17 hrs.


U-cell stage


0.00021025


0.00021025


The uniformity of the figures giving the actual volume of the egg mass, as determined by the weight of the water displaced by the models of the respective ova reconstructed, leads me to feel that the errors committed in reconstruction were not serious. The last column of the table, giving averages, is of interest since it shows a very slight increase in the volume of the egg mass during segmentation and transit through the oviduct. Following the pronuclear stage, which, as has been seen, ex


DEVELOPMENT OF THE ALBINO RAT 283

tends through a relatively long period and into the beginning of the second day, by which time the ova have migrated about onefourth of the length of the oviduct, there occur only three successive mitotic divisions, including the first segmentation division, namely mitoses resulting in 2-cell, 4-cell and 8-cell stages while the ova are in transit in the oviduct. In making this statement it is assumed that in the successive segmentations, the several cells divide synchronously, which is not in conformity with the fact. These three mitotic divisions are spaced at intervals of about 18 hours. In the next following division, the fourth, the ovum passes from the oviduct to the uterine horn. Since the normal gestation period of the non-lactating albino rat is only 21 to 23 days, this slow rate of increase in volume and multiplication of cells during the first four days of development is of especial interest and is very probably to be accounted for by the inadequacy of the food supply of the ovum during its transit through the oviduct.

The presence or absence of the oolemma has not been considered in discussing the segmentation stages of the albino rat. In my own material, the oolemma was clearly observed in certain of the 2-cell stages, but not in the 4-cell nor 8-cell stages. Widakowich reports that he has observed in the albino rat, loss of the oolemma even in the 2-cell stage. Since all of the material covering these stages was fixed in Carnoy's fluid, a fluid with a relatively large glacial acetic acid content, it may be questioned as to whether the fixative used may not be in part responsible for the early disappearance of the oolemma, though neither Hubrecht nor Sobotta considers the presence or absence of an acid in the conserving fluid of special moment in the fixation of the oolemma. Sobotta finds that the oolemma disappears in the ova of mice during the 8-cell stage. The early disappearance of the oolemma in the albino rat may be offered as an explanation of the fact that the egg mass during segmentation and transit through the oviduct does not, as a rule, present a spherical form but appears compressed and molded to fit the form of the lumen. A similar explanation is offered by Sobotta to account for the irregularity of form assumed by the ovum of the mouse after loss of the oolemma. In the forms in which the oolemma


284 G. CARL HUBER

persists through the later stages of segmentation, as for instance in the rabbit, the morula mass presents a spherical form. The transit of the ova through the oviducts is effected, very probably, through peristaltic action of the muscular coat, since only a relatively short portion is lined by ciliated epithelium. Whether or not there exists a rhythmic periodicity in the peristaltic action, it is impossible to state. The fairly regular rate of transit argues for the presence of some regulatory mechanism. The compact grouping often presented by a series of ova in transit through the oviduct, especially after reaching the portion with narrower lumen, suggests peristaltic action.

The literature dealing with the segmentation stages of the albino rat is very meagre. Grosser figures what is presumably an 8-cell stage. His figure 27 is referred to only incidentally in the text, but in the accompanying legend it is stated that the figure shows three ova of the white rat in process of segmentation, with zona pellucida, in transit through oviduct, three and one-half days after insemination." If I am right in interpreting these ova as in the 8-cell stage, this corresponds very closely to my own observation on rat No. 57, 3 days, 17 hours (figs. 15-17). It is impossible to draw definite conclusions as to the segmentation of the ova of rats from the account of Melissinos. This observer while he states that his material includes the ova of mice and rats, and while considering segmentation mentions the ova of both forms, discusses them without differentiating between the two. His figures all refer to ova of the mouse. Selenka, Robinson, and Widakowich, who have contributed to our knowledge of the embryology of the albino rat, do not include the segmentation stages, to be found in the oviduct, in their account.

The rate of segmentation and the time of transit through the oviduct, as given in the literature for certain other mammals is as follows: Sobotta has shown for the mouse that the 2-cell stage is reached about 24 hours after copulation, the ovum remaining in this stage to about the 48th horn-. The 4-cell stage was observed at about 50 hours, the 8-cell stage at 60 hours, and the 16-cell stage at 72 hours 'post coitum.' The ova of the mouse pass into the uterine horn about 80 hours post coitum.


DEVELOPMENT OF THE ALBINO RAT 285

thus the beginning of the fourth day, in a stage in whicli 16 cells up to 32 cells may be enumerated; the oolemma having been lost in the 8-cell stage. The data furnished by MeUssinos as concerns the mouse, are as follows: The 2-cell stage is obtained at the end of 24 hours after copulation, the 6-cell stage during the first 12 hours of the second day, and the 28-cell stage during the second 12 hours of the second day. The ovum is said to pass into the uterine horn at the end of the third day after copulation, retaining its oolemma. The account of Sobotta seems the more reliable. Hensen describes a 2-cell stage in the guineapig 22 to 24 hours after copulation, and Bischoff records that the ovum of the guinea-pig passes into the uterine horn while in the 8-cell to 16-cell stage, toward the end of the third day. Heape, who has described very fully the segmentation stages of the mole (Talpa europea) gives no data as to the rate of segmentation. In the explanation of the figures presented it may be noted that the ova figured, showing 2-cell to 15-cell stages, were taken from the oviduct. His figure 20, showing an ovum 'fully segmented' was obtained from the anterior end of the uterus. Assheton gives for the rabbit the following data: The 2-cell stage is obtained about 24 hours and the 4-cell stage about 26 hours after coitus. The third series of divisions begins about 28 hours after coitus, so that by the end of the second day a typical morula of 16 cells to 20 cells is to be found. Between 73 hours and 96 hours the beginning of the blastodermic vesicle formation is to be noted. Ova obtained 80 hours after coitus, still surrounded by the oolemma, were removed from the uterine horn. Data as to the relative position of the ova in the oviduct in the several stages of development discussed, are given. As concerns the sheep, Assheton states that the ova pass into the uterine horn early on the third day after mating. The pronuclear stage is to be observed the second day, and the first segmentation at the end of the second day. By the fourth day, with the ova in the 8-cell stage, they are found in the upper end of the uterine horn. The blastodermic vesicle formation begins with the 16-cell stage. Again, according to Assheton, the ova of the pig pass to the uterus about the third day after fertilization, if I read him rightly, reaching the uterus in the 4-cell stage, although ova in the 2-cell


286


G. CARL HUBER


and 3-cell stages were obtained from the upper end of the uterine horn. The presence of 2-cell stages in the uterine horn lias also been noted by Keibel, in Erinaceus europaeus, by Van Beneden in the bat, and by Hubrecht in the insectivor Tupaya javanica. Finally, it may be noted that according to the observations of Bischoff, the segmenting ovum of the dog occupies 8 to 10 days after insemination in transit through the oviduct.


COMPLETION OF SEGMENTATION AND BLASTODERMIC VESICLE FORMATION

The material covering the end stages of segmentation and the early stages of blastodermic vesicle formation is listed in table 5.





TABLE 5


RECORD XUMBER


AGE


NUMBER OF OVA


STAGE


64


4 days, 14 hrs.


5


Early stage of blastodermic vesicle formation


52


4 days, 15 hrs.


8


Morula, beginning of segrhentation cavity, early stage of blastodermic vesicle


55


4 days, 16 hrs.


1


Early stage of blastodermic vesicle


68


4 days, 16 hrs.


4


Early stage of blastodermic vesicle


53


5 days


7


Early stage of blastodermic vesicle


56


5 days


5


Early stage of blastodermic vesicle


Thus there are at hand 30 ova, showing late morula stages, the beginning of segmentation cavity formation and early stages of the blastodermic vesicle, falling in the latter half of the fifth day after the beginning of insemination. In all of the uteri from which this material was taken, the ova are spaced in the uterine horns about as in later stages of development ; they lie free in the uterine lumen, are in the main ovoid in form, their long axis presenting no definite relation to the long axis of the uterine horn. In preparing this material for sectioning, it was the custom to cut an entire uterine horn into segments measuring about 1.0 cm. to 1.5 cm. in length. These segments were then embedded so as to admit cutting longitudinally and in a plane parallel to the plane of the mesometrium. Cut in this way, the majority of the ova were cut longitudinally or nearly so, others in an oblique plane, others again, crosswise. Since it


DEVELOPMENT OF THE ALBINO EAT 287

is impossible to orient the ova prior to sectioning, the securing of desirable sections is a matter of chance. The difficulty is further enhanced by reason of the fact that owing to shrinkage as a result of the action of the fixing fluid, the ova in the vesicle stage are apt to be more or less folded, so that even though the plane of section may be that desired, the resultant sections lose in value by reason of this folding.

It has been shown that in the albino rat, the ova pass from the oviduct to the uterine horn toward the end of the fourth day. During the first half of the fifth day, the migration of the ova from the oviduct to the uterine horn appears to be completed, so that by the second half of the fifth day the ova are spaced in the uterine horn about as after fixation to the uterine mucosa. As to the factor or factors which play a role in the descent of the ova through the uterine horn and their fairly regular spacing, my own material gives no data; these changes occurring, apparently, during the first half of the fifth day, covering which my material is lacking. Widakowich, who has given especial study to these questions, presents the following considerations: In the downward migration of the ova in the uterine horn, it cannot be assumed that the ova are capable of active movement nor can their motion be ascribed to the action of gravity. While peristaltic action may play a part, it is difficult to see how peristalsis could be so regulated as to space the ova fairly regularly within the uterine cavity. The presence of a cilia^ted epithelium in the human uterine cavity during the intermenstrual period suggested the presence of a ciliated epithelium in the uterine horn of the rat. After many preparations had been searched in vain for its presence, Widakowich found short cilia, not more than 2 /x long in the epithelium lining the uterine cavity of a rat killed four days after copulation, and containing ova in the blastodermic vesicle stage. It would appear, therefore, that the uterine epithelium of the rat presents a ciliary border for only a relatively short time, and that the transportation of the ova within the uterus is effected by the cilia. Mandl also found, his material however not including the rat, that cilia are present in many anihials on the epithelium lining the uterus only at certain periods, and perhaps only relatively


288 G. CARL HUBER

short periods. While the presence of cilia may explain the migration of the ova in the uterine tube, Widakowich can offer no conclusions as concerns the regulatory mechanism by means of which the ova are spaced at fairly regular intervals in the lumen of the uterus. In none of my sections of the uteri of albino rats, obtained during the fifth day after insemination, have I been able to note the presence of cilia on the uterine epithelium, even when sections were studied under the oil immersion. After reading the account of Widakowich, their presence was looked for in all pertinent stages, but without success. Especially in rat No. 50, in which the ova were passing from the oviduct to the uterine horn was careful search made, but nothing like a distinct ciliary border, composed even of short cilia, was ascertained. In the left genital tract of this rat, as has been stated, three ova were found in the terminal part of the uterine end of the oviduct, one in the uterine lumen just distal to the mouth of the oviduct, and one a little over a centimeter from this opening. The latter was lodged in a shallow depression of the uterine mucosa, as is characteristic for stages lying free in the lumen. The question as to whether this ovum was permanently lodged is difficult to answer. If this is assumed, it is further necessary to assume that the other ova would need to pass it to reach the more distal parts of the uterine lumen.

The literature contains no definite statements as concerns the reactions of the epithelium and mucosa of the uterus to the ova soon after their appearance in the uterine cavity. Widakowich summarizes the views by stating that 'Tt is generally stated, that so long as the ova lie free, the uterus shows no changes." He himself notes that at this time the mucosa presents evidence of marked new formation of capillaries. Burckhard, who had at his disposal a large number of stages showing implantation of the ovum of the mouse, discusses at length the appearance presented by the uterus soon after the ova enter the same and the lodgment of the ova therein. This observe!' notes that in the non-gravid uterus of the mouse, the lumen lies more or less eccentric, and towards the mesometrial border.


DEVELOPMENT OF THE ALBINO RAT 289

The lumen is not suiooth, but presents numerous radially arranged folds, certain of which are relatively deep. Essentially the same characteristics pertain to the mucosa of the uterus, soon after the beginning of gravidity. As the ova pass from the oviduct to the lumen of the uterus they become lodged in certain of the mucosal folds, and generally in certain of the deeper ones to be found along the anti-mesometrial border. I find Burckhard's account of the form of the lumen of the uterine horn, of the structure of the mucosa in early stages of gravidity, and the lodgment of the ova, pertaining to the mouse, applies equally well to the albino rat. No reason can as yet be given as to why the ova are lodged in the mucosal folds in which they are found, and not in others. So far as may be ascertained from the sections, the particular mucosal folds in which the ova are found, do not differ in form and structure from neighboring folds. It is possible that by reconstruction of the epithelial lining of the entire uterine horn in pertinent stages, certain characteristics of form and position might be revealed as possessed by certain mucosal folds which make them especially favorable for the lodgment of the descending ova. Such reconstructions, however, have not been made. Burckhard states that in the mouse, about the middle of the fifth day, after the ova have been in the uterine cavity for a number of hours, there may be observed the first changes in the uterine wall. The changes consist primarily in a flattening of the uterine epithelium. In the immediate region where implantation is to occur, the lining epithelial cells present instead of a cylindric form, a cubic form. The area is sharply demarked from the surrounding epithelium, the transition of cubic to cylindric epithelium being marked by a sharp-lipped epithelial ledge. In my pwn material of the rat covering these stages, the uterine mucosa likewise presents shallow pits, in the immediate regions where the ova are lodged, lined by slightly flattened, cubic epithelium, very much as described by Burckhard for the mouse. Widakowich presents an excellent figure (fig. 2 of his contribution, rat four days after fertilization — 'Befruchtung') showing clearly the relations of the ova to the uterine wall. In this rat, the


290 G. CARL HUBER

uterine epithelium presented a ciliaiy border, present even in the shallow pit lodging the ovum sketched. He argues from this that the shallow depression and the flattening of the epithelium are not a result of pressure exerted by the vesicle, as thought by Sobotta and Melissinos, but must be due to an active change in the epithelium itself. The mucosa underlying the shallow pits presents at this stage no change of structure. I am thus in accord with Widakowich when he states that he was not able to observe in the mucosa of the rat in the early stages of gravidity, the giant cells described by Disse as found in the uterine mucosa of Arvicola arvilis, in similar stages.

The form presented by the ova of the albino rat, in the late morula stages and the early stages of blastodermic vesicle, is ovoid, as may be seen from the figures to be presented. Widakowich is inclined to believe that the form of the blastodermic vesicle of the rat is in a measure dependent on the general form of the space in which it is lodged. He figures two vesicles (figs. 1-2) one of which is nearly spherical, the other of distinctly oval form. Duval (figs. 73-83) presents vesicles having ovoid, triangular, and spherical forms. Christiani's figures covering these stages, are too schematic to be of any value in drawing conclusions. I fear Robinson's account is based on imperfectly fixed material. He states that "toward the end of the fifth day, or the commencement of the sixth day, the longitudinal axis of the blastodermic vesicle is 125 /x long. During the sixth day, that axis is diminished, first to 95 /x, and then to 64 ix, after which it again increases, and at the commencement of the seventh day, it is 121 /x." Neither Fraser nor Selenka describes nor figures the stages here considered. In the mouse, according to the accounts of Melissinos, Burckhard, and Sobotta, the form of the blastodermic vesicle in early stages is spherical.

The more specific consideration of my own material I shall introduce with a discussion of three stages taken from the uterus of rat No. 52, killed 4 days, 15 hours after the beginning of insemination. In A, of figure 20, there is reproduced the middle one of seven sections of a late morula stage. This morula is of ovoid form, measuring 85 ai in its long diameter, 54 /x in its


DEVELOPMENT OF THE ALBINO RAT 291

broad diameter — that is, in plane of sections, and since it passes through seven sections of 10 thickness, measures approximate!}^ 70 ij. in its third dimension. It consists of 24 cells, as estimated by counting the nuclei contained in its several sections. The cells vary in size as well as in shape. The nuclei are for the main of spherical form, presenting one or several large nucleoli and fine chromatin granules. This morula is found within a fold of the mucosa, each side of which presents a slight depression, lined by slightly flattened epithelium. This morula mass lies




Fig. 20 Sections of morula mass and early stages of blastodermic vesicle of the albino rat. X 200. A, B, C, rat No. 52, 4 days and 15 hours. D and E, rat No. 68, 4 days and 16 hours. A, late stage of morula; B, shows the very beginning of the formation of the segmentation cavity; C, D, E, early stages of blastodermic vesicle, in E, a distinct covering layer in the thicker portion or floor of the vesicle is evident.

free in the lumen of the mucosal fold, and not in contact with the uterine epithelium. The outline and extent of the shallow depressions found in the opposing walls of the mucosal folds conform to shape and size of the morula mass contained, which appear as if slightly retracted as a result of fixation.

In B, figure 20, is figured one of the sections of a series passing through a morula stage, comprising as estimated 30 cells and measuring 90 /x, by 60 n, by approximately 50 /x, in which the very beginning of the formation of the segmentation cavity is shown. Near one pole the outermost cells have separated slightly from the more deeply placed cells, so that an irregularly shaped


292 G. CARL HUBER

cavity, eccentrically placed and passing clearly through two of a series of five sections of 10 n thickness, is evident. The small cleftlike cavity is bounded by the surrounding cells, the outline of which is distinct. So far as may be judged from the appearance noted as presented in the two sections in which this cavity is found, this arose as a single space and as a result of the separation of the enclosing cells.

In C, of figure 20, there is presented a slightly older stage showing the blastodermic vesicle formation and measuring 80 /x, by 50 ij., by approximately 50 ju, comprising as is estimated, 34 to 36 cells. Unfortunately, the lower part of this vesicle is slightly folded as is shown in the lower left of the figure. The appearances presented in the sections are reproduced as faithfully as could be. Owing to the folding, a portion of the thin wall is cut tangentially. The more darkly colored curved line represents in reality the outer boundary of this portion of the vesicle. The segmentation cavity in this vesicle is distinctly larger than that shown in B of this figure. In the section reproduced the segmentation cavity is bounded for the greater part by four somewhat flattened cells, the increase in the size of the cavity being accompanied, it would seem, by a flattening of the enclosing cells.

In these three closely approximated stages, which, since they are taken from the same uterus are probably separated in time of development by only short intervals, the cells though varying in size and shape, show no essential or fundamental difference in structure, neither in cytoplasm nor nuclei; nor do they show any regularity in arrangement. Only few mitotic figures are to be observed; none in the morula mass shown in A, and but two in each of the other two stages, shown in B and C. Judging from these preparations, one would be led to conclude that segmentation cavity formation in the albino rat is not associated nor accompanied by active cell proliferation. This point will be referred to again after the presentation of further material at hand. In slightly older stages of the blastodermic vesicle than here considered, the thicker portion of the vesicle is designated by Sobotta and others as its floor, which is directed


DEVELOPMENT OF THE ALBINO RAT 293

toward the mesometrial border of the uterine horn, while its thinner portion is designated as its roof, directed toward the antimesometrial border. Therefore, in shghtly older stages than thus far figured, the vesicle lies with its long axis approximately at right angle to the long axis of the uterine horn. In further description of the blastodermic vesicle, I shall use the term 'floor' and 'roof as here specified. In D and E of figure 20, there are reproduced typical sections of the two blastodermic vesicles taken from the uterus of rat No. 68, killed 4 days, 16 hours after insemination. Vesicle D measures 90 n by 30 m by approximately 60 n, and is of distinctly ovoid form and slightly compressed. This vesicle is found lying free in a long but narrow fold of the mucosa, both sides of which are slightly molded in conformity with the form of the vesicle. The long axis of the vesicle is still parallel to the long axis of the uterine horn. The roof of the vesicle appears as if slightly contracted, though when traced through the series of six sections it does not appear folded. The roof is composed of only a few cells, perhaps seven in all. The segmentation cavity presents a regular outline. This vesicle supports the contention of Widakowich, that the form of the blastodermic vesicle of the rat is dependent in a measure on the form of the space in which it is found. Vesicle E, of figure 20, measuring 85 fx by 45 n by approximately 40 fx, presents a roof that is slightly folded and shows an early stage in segmentation cavity formation. A figure of the vesicle is included since it represents more clearly than any other blastodermic vesicle of the albino rat in my possession, a diiTerentiation of a layer of surface cells in the mass constituting its floor. This is a question to be more fully considered in further discussion. In all the measurements of blastodermic vesicles thus far given, even in those given for the morula mass shown in A, figure 20, it is evident that one of the short diameters is appreciably shorter than the other. The vesicles are not only of ovoid form, but slightly flattened, so that even when not folded, the form of the vesicle as seen in section, even when cut parallel to the long axis of the respective vesicles, is dependent in a measure on the plane of the section, whether parallel to the longer or the shorter


294 G. CARL HUBEE

of the two cross diameters. This may be seen from the series of drawings made of a blastodermic vesicle cut cross-wise, taken from the uterus of rat No. 68, from which were also taken the two vesicles shown in D and E of figure 20. This series of figures is shown in figure 21, in which are reproduced in serial order the seven successive cross sections into which the vesicle was cut. It measures 65 /x by 38 m by approximately 70 id, and is found at the bottom of a mucosal fold, found at the mesometrial border, and is resting with one side on the epithelial lining of a shallow pit, the other wall of this mucosal fold, also showing a shallow pit, is slightly retracted. From a study of this series



Fig. 21 A complete series of cross-sections of an early stage of blastodermic vesicle of the albino rat. X 200. Rat No. 68, 4 days and 16 hours. A to C, sections through roof of vesicle, showing segmentation cavity; D to G, sections throvigh floor of vesicle.

of sections, I feel certain that the plane of section is cross and not oblique to the long axis of the vesicle. The roof of this vesicle passes through three sections. A, B and C. The segmentation cavity has thus a depth of less than 30 n. The overlapping of the cells surrounding the segmentation cavity is to be noted, especially as seen in B of this figure. This arrangement of the cells may explain how the cavity may be enlarged without a material increase in the number of the enclosing cells — in part, by a flattening out of the cells, in part by a rearrangement of the relations of the cells. In the figures of the sections passing through the floor of this vesicle, D to G, attention is drawn to the size, form and relations of the cells and to the fact that there is no distinct covering layer. In this series of sections, there are


DEVELOPMENT OF THE ALBINO RAT 295

shown in all 35 nuclei, two of which are in a late diaster phase. By excluding the nuclei that appear to be cut in two, appearing thus in two successive sections, I estimate that the blastodermic vesicle is made up of only about 30 to 32 cells.

In figure 22, there are reproduced typical sections of four blastodermic vesicles taken from the uterus of rat No. 53, killed five days after the beginning of insemination. This uterus contained in all, eight vesicles, one of which was distinctly pathologic. The four vesicles selected for reproduction and discussion present each certain characteristics worthy of consideration. Vesicle A, which presents an early stage of segmentation cavity forma



Fig. 22 Sections of early stages of blastodermic vesicle of the albino rat, X 2C0. Rat No. 53, 5 days.

tion is of interest owing to the number of mitotic divisions it contains. As a rule, I have noticed only a few mitoses at this stage. In this vesicle, which measures 90 ^ by 55 /x by approximately 40 /x, there are no less than five mitoses to be noted, three of which are in cells forming the roof of the small segmentation cavity, and are included in the section figured. This is the only vesicle in my possession in which an increase in the size of the segmentation cavity is accompanied by active mitoses in the cells bounding it. The vesicle lies free in the uterine lumen, one wall of which is only slightly pitted. In B of figure 22 is reproduced a section of a blastodermic vesicle, measuring 90 m by 55 IX by approximately 45 m- It is evident that this vesicle

JOURNAL OP MORPHOLOGY, VOL. 26, NO. 2


296 G. CARL HUBER

passes through five sections of 10 m thickness, though one of the end sections, the fifth section of the series, seems to have fallen out during manipulations of staining and mounting, since the preceding, or fourth section does not quite complete the series. This vesicle lies free in the lumen of the uterus, and there is evident only a shallow pit in the mucosa juxtaposed. In this vesicle the cells forming the roof of the segmentation cavity are relatively numerous, and are not markedly flattened, and in one an early mitotic phase is recognized. Here again cell proliferation appears to have accompanied increase in size of segmentation cavity.

The vesicle shown in C of figure 22, measuring 130 m by 30 m by approximately 40 n, lies free in a long, narrow fold of the uterine mucosa, in close proximity to a shallow mucosal pit, lined by cubic epithelium; the pit conforming in shape and extent to the form of the side of the vesicle presented to it. Therefore, it would seem that the form of the vesicle as seen in sections of the fixed material is essentially the same as that obtained in vivo. The two vesicles, typical sections of which are shown in B and C of this figure, are almost in identically the same phase of development, although their form as seen in sections differs markedly. The plasticity of the living blastodermic vesicles is no doubt such that their form is in a great measure dependent on the shape of the mucosal fold in which they are lodged. In D of figure 22, there is reproduced a section of a blastodermic vesicle which points to a stage of development which is slightly more advanced than that shown in previous figures. The vesicle measures 100 /i by 70 ^^ by approximately 50 ^x. The roof enclosing the segmentation cavity is slightly folded; a portion of its wall is thus cut tangentially, as shown in the lower left of the figure. The segmentation cavity is distinctly larger than that shown in the preceding figures, and is bounded by a relatively large number of cells, fourteen in that portion of the roof sketched in this figure, one of which is in a mitotic phase. The mass of cells constituting the floor appears as slightly compressed, in consequence of a slight intravesicular pressure which aided in the enlarging of the segmentation cavity.


DEVELOPMENT OF THE ALBINO RAT 297

The cells forming the roof of the segmentation cavity do not appear so distinctly flattened as is the case in certain of the vesicles figured in figures 20 and 21. It would appear, therefore, that at least two factors are operative in the increase of size of the segmentation cavity after its anlage — a flattening out and consequent increase of the exposed surfaces of the enclosing cells, and secondly, a cell proliferation ; and it would appear that both of these factors may be operative from the time of the beginning of segmentation cavity formation.

Early stages in the blastodermic vesicle formation in the albino rat have been previously described by Robinson, Christiani, Duval, and Widakowich; Selenka's youngest stage is slightly older than any discussed by me. My own observations are wholly in accord with those of Widakowich in so far as his account covers early stages of blastodermic vesicle formation. He discusses and figures, however, only two vesicles, obtained four days after fertilization — 'Befruchtung,' in each of which the segmentation cavity presents a smooth and regular outline and is of appreciable size. The observations of the other observers who have considered these stages will be discussed in connection with a very brief presentation of much more comprehensive observations on the mouse in similar stages of development. Of these latter, those of Sobotta ('03) are based on abundant and apparently well fixed material. Sobotta begins his discussion with the consideration of three ova taken from the same mouse, the second half of the fourth day after fertilization, each of which shows beginning of segmentation cavity formation, one of which was cut in longitudinal axis and is figured in his figure 1. This ovum is interpreted as showing that the segmentation cavity arises not as a single space, but as a number of disconnected spaces, which later become confluent and form a single space. A similar observation was made by Van Beneden on the bat, a fact which Sobotta uses to support his contention that the mouse ova studied by him were of normal structure. Melissinos gives a number of figures showing early stages in the formation of the segmentation cavity in the mouse. His figures 21 and 22 (66 hours) are not unlike my own figures shown in B of


298 G. CARL HUBER

figure 20 and A of figure 22. According to this observer, the segmentation cavity arises as a single space, due to an accumulation of fluid secreted by the cells of the morula. This secretion is evidenced by globule-like droplets which are shown in his drawings as adhering to certain of the cells bounding the segmentation cavity. In my own preparations of the albino rat, I find no evidence which would lead to the supposition that the segmentation cavity does not begin as a single space nor do I find any evidence of secretory globules as described bj^ Melissinos. Selenka has described quite fully two blastodermic vesicles of the mouse, lying free in the uterine lumen. His account of their structure, supported by two somewhat diagrammatic figures, is as follows: The wall of the vesicle is formed by a layer of covering cells — 'Deckzellen' — constituting a covering layer — "Deckschicht or Rauber's layer." The space enclosed by this layer, for about one-third to one-half of its extent, contains the 'formative cells,' for the remainder it contains fluid. The covering cells and formative cells are said to be separated by a sharp line. The formative cells are in all parts separable into two fundamental germ layers. An inner layer, bordering the cavity and constituting the entoderm, is said to be composed of cells possessing more deeply staining nuclei, irregular outline, with tongue-like processes which extend into the cavity, and a granular protoplasm; further, of cells which are more clear, more peripherally placed, and which constitute the ectoderm. Each of these fundamental germ layers consists of a single layer of irregularly formed cells. According to the observations of Selenka, therefore, the floor of the vesicle consists of three layers of cells; an outer covering layer — ^'Deckschicht' or 'Rauber's layer' — an inner layer of entodermal cells, and an intermediate layer of ectodermal cells. Jenkinson's account reads as follows: "There are present (1) an outer layer, one cell deep, of trophoblast, which is continuous over (2) an inner mass which becomes differentiated into the embryonic epiblast and the hypoblast, and which is quite distinct from the overlying trophoblast, as my specimens invariably show." In Jenkinson's figures 1 and 2, giving early stages of the blastodermic vesicle, there is not shown a differentiation of the inner


DEVELOPMENT OF THE ALBINO RAT 299

mass into ectodermal and entodermal cells; the outer layer, covering la^^er, Rauber's cells, or trophoblast, is clearly differentiated from the inner mass by a distinct space. Duval has recognized in early stages of blastodermic vesicle formation of the mouse and rat, in the thicker part of the vesicle, entodermal and ectodermal cells. The former are of irregular form, possess granular protoplasm and are said to possess the property of ameboid movement. The remaining cells are recognized as ectoderm; a distinct covering layer is not recognized. In Christiani's figures (rat), which are, however, so diagrammatic as to be of little value and are evidently drawn from poorly fixed material, entodermal cells, ectodermal cells, and covering cells may be recognized as per legends. Melissinos (mouse), while not describing definitely a peripheral or covering layer, states that outer cells of the thicker pole, like the cells enclosing the segmentation cavity, stain less deeply than do the more centrally placed cells. In earlier stages of vesicle formation, neither in figures nor in text as given by this observer, do I find reference to a differentiation into ectodermal and entodermal cells. The observations of Selenka, Duval, Robinson, Jenkinson, and others, bearing on the structure of the blastodermic vesicle of the mouse and the rat in early stages of development have been so thoroughly and critically reviewed by Sobotta that an extended discussion has here been deemed unnecessary. It may here suflEice to say that while Sobotta' s observations were made and his discussions based on ova obtained from the mouse, my own observations made on the albino rat are in agreement with his and support the contention that in early stages of blastodermic vesicle formation a differentiation of the thicker part or the floor of the vesicle into a covering, Rauber's cell, or trophoblast layer, and a further differentiation into ectodermal and entodermal cells, is not to be made: we differ in our accounts of the beginning of the formation of the segmentation cavity. An outer or covering layer is suggested in certain of my own preparations, most clearly in that sketched in E of figure 20. However, a uniform difference in structure and reaction to staining reagents of the outer layer of cells is not present in my own preparations. None of my own preparations gives evidence of


300


G. CARL HUBER


such an early differentiation of ectoderm and entoderm as given by Selenka, Duval, Christiani, and others. Cells of irregular outline with tongue-like projections, such as figured by Selenka and Duval I have not observed. The cells constituting the floor or the thick part of the vesicle all present essentially the same structure, while the segmentation cavity, as soon as it presents appreciable size, shows a smooth and regular outline. In figures 1 and 2, of Widakowich's communication, excellent figures of early stages of blastodermic vesicles of the albino rat, there is presented no evidence of a trophoblast layer nor a differentiation of ectodermal and entodermal cells. My own figures, 20 to 22, were drawn with the aid of camera lucida at a magnification of 1000 diameters and with the use of an intense Welsbach light. They are reduced five times in reproduction. With the exceptions of cell outlines, which as sketched do not in the preparations fall in the same optical plane, and are sketched more sharply than is perhaps warranted, the figures portray quite accurately the structural appearances presented, so far as may be with the use of a single color.


BLASTODERMIC VESICLE, BLASTOCYST, OR GERMINAL VESICLE The material on hand is listed in table 6.



TABLE 6



RECORD NUMBER


AGE


NUMBER OF VESICLES


75


5 days, 15 hours


6


91


5 days, 16 hours


2


88


5 days, 21 hours


■ 7


89


5 days, 21 hours


6


73


6 days


10


74


6 days


5


99


6 days


6


lOG


6 days


10


104


6 days


6 Total 58


During the sixth day, the blastodermic vesicle of the albino rat increases in size relatively rapidly. The greater portion of its wall is, at this stage, composed of a single layer of flattened cells. The vesicles are not as yet attached to the uterine wall, though the uter


DEVELOPMENT OF THE ALBINO RAT 301

ine mucosa shows a distinct reaction to their presence. Localized thickenings of the uterine mucosa, sufficient to cause localized swellings of the uterine tube, indicating the position of the ova, are evident. I have experienced more difficulty in successfully fixing the vesicles during this stage than any of the earlier or later stages studied. Although my material contains 58 vesicles of the stage under consideration, none of them may be regarded as being well fixed, and the majority of them are so folded as a result of contraction during fixation that they are of little value as objects for especial study. That the vesicles are still unattached to the uterine wall is readily determined by the fact that the shrivelled vesicles are found lying free in the depressions of the uterine mucosa, lined by a low cubic epithelium, intact throughout, and retaining its normal relation to the mucosa. The molding in these mucosal depressions no doubt gives the size of the respective vesicles as in vivo.

It is not my purpose at this time to consider more than superficially the changes affecting the uterine mucosa during ovum implantation in the albino rat. It is hoped that this may be the subject of a future communication. It is the purpose in the present communication to confine consideration to the development of the ovum itself. Many of the observations recorded by Burckhard on the implantation of the ovum of the mouse and the formation of the decidua, I find equally adapted to similar phenomena in the albino rat. Differences are to be observed in certain details which it is not the purpose to enter into here. Grosser gives a number of excellent figures (67 to 70, and 112 to 116) showing implantation and decidua formation in the albino rat; to these the interested reader is referred for the present. The thickening of the mucosa affects primarily its antimesometrial portion. During this process of thickening, the mucosal fold in which the ovum primarily finds lodgment, becomes deepened and converted into a funnel shaped crypt communicating with the uterine lumen, and surrounded by the 'Eibuckel,' or oval fold. Burckhard's schematic figures (text figures 2 to 4) may be consulted to make the phenomenon intelligible.


302


G. CARL HUBER


In figure 23, there are reproduced representative sections of five blastodermic vesicles falling to the end of the sixth day after insemination. None of these five vesicles can be regarded as well fixed. All show a certain amount of distortion, much more evident were the entire series of each of the respective vesicles shown. The form of the blastodermic vesicle of the albino rat at this stage of development, as indicated by the molding of the uterine mucosa, is ellipsoid. Their size as in vivo, when distended and of regular outline, again as indicated by the molding of the uterine mucosa, is slightly larger than would be



_p. ecL p ecL.



Fig. 23 Sections of blastodermic vesicles or blastocysts of the albino rat.

X 200. A and C, rat No. 99, 6 days; B, D, E, rat No. 100, 6 days, y.ent., yolk

entoderm; p.eni., parietal layer of entoderm; p.ect., parietal or transitory ectoderm.


supposed from the drawings presented. By reason of this distortion, exact measurements of size cannot be given.

In A of figure 23, there is reproduced that portion of one of the sections of a blastocyst (rat No. 99, 6 days) which passes through its floor; the thin roof of this vesicle was so folded that its inclusion in the drawing was deemed undesirable. However, its floor or the germinal disc, seems to have retained its normal form and structure, presenting when traced through the series a regular concavo-convex, discoidal form. It consists in the main of three layers of cells of polyhedral type; toward the border of the disc, of two layers of somewhat flattened cells, the peripheral layer being continuous with the single layer of cells


DEVELOPMENT OF THE ALBINO RAT 303

forming the roof of the vesicle, not shown in the figure, and known as the parietal or transitory ectoderm. In the floor or germ disc, there is evident a single layer of cells bordering the segmentation cavity or blastocele and possessing a more granular protoplasm, which stains a little more intensely in Congo red. Their differentiation and characteristic reaction to staining agents is at this stage of development not quite so distinct as in slightly older stages. This layer of cells, similar to that described by Sobotta for the blastodermic vesicle of the mouse in essentially the same stage of development, he has termed the yolk entoderm, 'Dotter entoderm,' a designation which is here followed. In the more superficial layer or layers of cells no characteristic differentiation is observed. In no portion of the floor of this vesicle was a distinct covering or trophoblast layer recognized.

In the vesicle, a section of which is reproduced in B of this figure (rat No. 100, 6 days), the floor or germ disc presents essentially the same structure as that shown in A. The vesicle shown under B, was also folded, especially its roof, which was drawn to one side and was thus not cut through its entire length in the section figured. Furthermore, the section chosen for drawing does not pass quite through the center of the germ disc, but a little nearer to one of its edges, which probably accounts for the fact that there is recognized for the greater part only a single layer of cells, superimposed over the yolk endoderm, which laj^er is continuous with the parietal or transitory ectoderm forming the roof of the vesicle. The cells forming the yolk entoderm constitute a single layer and are quite distinctly differentiated; one of the cells shows a mitotic phase. The roof of the vesicle formed by the parietal or transitory ectoderm, is composed of a single layer of flattened cells with flattened nuclei, the form and structure of which is more correctly shown in the right half of the roof wall, which in the section is cut transversely, while the left half, owing to the folding, is shown as cut obliquely.

In C of figure 23 (rat No. 99, 6 days), there is shown a greatly compressed blastodermic vesicle, taken from a series of cross sections of the uterine horn. In this figure there is reproduced the fifth of a series of 10 sections of 10 /x thickness; therefore.


304 G. CARL HUBER

the third dhnension of the vesicle is approximately 100 fx. It is evident that had this vesicle been cut in a favorable plane at right angles to the present series, or parallel to the mesometrial plane, its form would have approached that of a circle. I have in my possession one vesicle of this stage of devlopment, similarly compressed, cut parallel to the plane of compression, in which almost the entire roof falls within a single section of 10 /x thickness. The structure of the vesicle shown in C is very similar to that shown in A and B of this figure. The normal form of this vesicle is quite readily reconstructed from a study of the series of sections into which it has been cut. The cells of the yolk entoderm are evident. The parietal or transitory ectoderm constituting the roof consists of a single layer of much flattened cells, with relatively few nuclei, having, as seen in cross section, a long ovoid form, which, when seen in surface view present a regular, nearly circular outline (see lowermost nucleus in the figure). In similarly compressed vesicles cut parallel to the plane of compression, the germ disc may appear as consisting of three to four layers of cells. In an imaginary section passing in a plane at right angles to that figured in C, and having perhaps a slight obliquity, the germ disc would appear as if much thicker than that shown in A and B of this figure. Such sections may readily lead to false conclusions.

It seems evident from a study of the material at my disposal that during the sixth day after the beginning of insemination in the albino rat, the blastodermic vesicle or blastocyst, which has its anlage in the latter part of the fifth day, enlarges relatively rapidly; this largely owing to a distension of the segmentation cavity or blastocele. This enlargement is accompanied by a flattening and extension of the enclosing roof cells and by a rearrangement of the cells of the floor, which is reduced in thickness to a discoidal area, the germinal disc or germ area, forming about one-fifth to one-sixth of the wall of the vesicle and consisting of two or three layers of cells. During the rearrangement of the cells which constitute the floor of the vesicle, those adjacent to the segmentation cavity or blastocele differentiate to form the anlage of the yolk entoderm. The remaining cells of the ger


DEVELOPMENT OF THE ALBINO RAT 305

minal disc, having all essentially the same structure, are of irregular polyhedral form and are mutually compressed. To designate them as a distinct germ layer at this stage seems inappropriate. A differentiation into a layer of covering cells and a layer of formative ectoderm (Selenka) is not to be made. Active cell proliferation as evidenced by mitotic figures does not appear to accompany this enlargement of the vesicle. This phenomenon seems rather to be accomplished by a rearrangement of the cells constituting its floor, however, primarily by an extension and consequent flattening of the cells forming the roof of the vesicle. A similar stage is shown for the mouse by Sobotta ('03) in his figures 3, 4, and perhaps 5, of mouse vesicles from the fifth dsiy after fertilization — 'Befruchtung'. Sobotta had at his disposal much more perfectly fixed vesicles than my material contains. The structure of these vesicles as given by this observer, both as depicted in figures and text, is very similar to the presentation given by me. He also recognizes in this stage the anlage of the yolk entoderm. Figure 30, accompanying the account of Melissinos (mouse, 84 hours) presents a similar stage, although he figures fairly distinctly a layer of covering cells, which if I read him correctly, however, is of onh^ transitory existence. None of the figures given by Robinson and Jenkinson is comparable with figures A, B, C, of figure 23 of this account.

In D, of figure 23 (rat No. 100, 6 days) there is reproduced a section of a blastodermic vesicle which on superficial study presents a somewhat later stage of development than those shown in A to C, of this figure. It is, however, only A^ery slightly older than the three vesicles discussed. Vesicle D, cut in good longitudinal direction, is in reality much more folded than appears from the section figured. Its floor or germ disc is compressed in a plane parallel to that of the plane of section, so that the germinal disc is cut obliquely and not transversely, and thus appears thicker in the section than it in reality is. A distinct layer of covering cells, continuous with the cells of the parietal ectoderm, is evident. Such a layer of covering cells is figured by Selenka, Jenkinson, and Duval. The yolk entoderm has differentiated and extends by perhaps three cells, in the


306 G. CARL HUBER

section figured, onto the layer of parietal ectoderm. Selenka and Duval, who regard the cells of the primary entoderm as having ameboid properties, are disposed to regard the entodermal cells found lining the parietal ectoderm as having wandered from their seat of origin to the side wall of the vesicle. Sobotta sees no evidence of such wandering of the primary or yolk entodermal cells, but suggests that they are drawn to their position on the wall of the vesicle during its increase in size ; their wandering, therefore, is more relative than absolute. Certain cells nearer the edge of the yolk entoderm, having attachment to the parietal ectoderm, which attachment they retain as the vesicle enlarges, are thought to be drawn from their close relation to the yolk entoderm and to appear as scattered cells lining the parietal ectoderm. Now and then, such cells may divide, resulting in further distribution. Sobotta's suggestion seems to me to be more in accord with the observed facts. In vesicle D, the roof, consisting of a single layer of flattened, parietal ectodermal cells, presents several major folds as well as minor folds. The latter particularly account for the variation in thickness of the wall of the vesicle as seen in sections. At the lower left of the figure is seen a portion of the wall as seen cut on the flat, the shape of the two nuclei here shown as seen in surface view may be compared with the long ovoid form of similar nuclei when seen in cross section.

Vesicle E of figure 23 (rat No. 100, 6 days) presents a stage that is slightly older than the other four vesicles shown in this figure. The floor of this vesicle, the germinal disc, as seen in cross section, presents the form of a triangle with its base resting on the cavity, the blastocele. When compared with the slightly younger stages this portion of the vesicle presents an increase in the number of constituent cells, arranged in irregular layers to the number of five in its thickest portion. The thickening is no doubt in part due to the slight lateral compression of the vesicle, but this does not wholly account for it. The cells constituting this thickened germinal disc are for the main of irregular polyhedral form with relatively large nuclei rich in chromatin. A distinct covering layer is not evident. On its under surface there is found a single layer of cells of yolk entoderm. The


DEVELOPMENT OF THE ALBINO RAT


307


thin-walled roof of this vesicle, the parietal or transitory ectoderm, deserves no special consideration, except to state that its variation in thickness, as seen in the section figured, is due to the plane of section — cross or oblique — of different portions of the wall, owing to slight folding. This vesicle I believe to be in stage of development and structure very similar to that shown by Sobotta ('03) in his figure 6, mouse vesicle of the first half of the sixth day, and perhaps also figure 31, of the account of Melissinos, mouse vesicle, end of fourth da}', also figure 7 of Jenkinson's article who, however, describes and figures a distinct covering or trophoblast layer.

The cell rearrangement and proliferation resulting in the thickening of the floor or the germinal disc as noted in E, of figure 23, marks the beginning of a much more distinct thickening of this portion of the vesicle, partly due to cell proliferation, in part also due to the rearrangement and enlargement of the constituent cells, during which thickening process this portion of the vesicle grows outward as well as into the cavity of the vesicle, initiating the phenomenon known as the 'inversion of the germ layers' or as 'entypy' of the germ layers, to be discussed as to its anlage in the following section.

LATE STAGES OF BLASTODERMIC VESICLE, BEGINNING OF ENTYPY OF GERM LAYERS

The material at hand is listed in table 7.


TABLE 7


RECORD NUMBER


AGE


NUMBER OF VESICLES


46


6 days, 14 hours


10


54


6 days, 16 hours


9


67


6 days, 16 hours


7


24


6 days, 17 hours


3


90


6 days, 17 hours


6


72


7 days


9


80


7 days


9


92


7 days


6 Total 59


The fixation of the blastocysts of the albino rat obtained during the seventh day after insemination was much more


308 G. CARL HUBER

readily accomplished than in those obtained during the preceding day. Of the 59 vesicles of this stage obtained, many show excellent fixation. The thin wall of the vesicle is no longer so prone to fold as in the preceding stage, and does not readily retract from the uterine epithelium or mucosa, no doubt owing to a distinct adhesion of vesicle wall to the maternal tissue. It is difficult, however, so to orient the vesicles as to obtain sections of a desired plane. The general position of a given vesicle is readily determined, since the enlargement of the uterus marking its location is very evident. The vesicles are located in approximately cylindrical cavities, known as decidual crypts, which are directed toward the antimesometrial border.

These decidual crypts communicate with the lumen of the uterus, which lies eccentric and nearer the mesometrial border, by means of funnel-shaped openings. The decidual crypts or cavities are still lined with uterine epithelium, though this is now much flattened in the immediate vicinity of the vesicle and may be found in part separated from the mucosa of this region. The vesicles are now so placed that in all of them, the thicker portion, the floor of the blastodermic vesicles of younger stages or region of the germinal disc, is directed toward the mesometrial border, thus toward the still patent lumen of the uterus, while the roof of the vesicles is directed toward the antimesometrial border, thus toward the bottoms of the decidual crypts. The general direction of the decidual crypts is in the main at right angle to the long axis of the uterine horn, and directed from the mesometrial to the antimesometrial border. They may deviate, however, from the general direction at various angles and in almost any direction. The decidual crypts as seen in cross section do not as a rule present a circular outline, but appear as slightly compressed from side to side, having thus an oval outline as seen in cross section, with the long axis of this oval space as seen in cross section approximately parallel to the long axis of the uterine horn. Since the direction of the decidual crypts can in uncut material be only approximated, the obtaining of sections cut in a desired plane becomes largely a matter of chance. In a large number of my preparations the contained


DEVELOPMENT OF THE ALBINO RAT


309


vesicles are cut in an oblique plane, which may deviate only a little from the longitudinal or may approach a cross axis, while only a relatively small number of vesicles were cut favorably in the longitudinal plane, and the majority of these are in series cut parallel to the plane of the mesometrium. The vesicles on which the special consideration of this stage is based are reproduced as seen in sections, in figure 24.

Vesicle A, of figure 24 (rat No. 46, 6 days, 14 hours), is drawn from two successive sections. The upper portion of the figure


■^w-/,.:



Fig. 24 Sections of blastodermic vesicles or blastocysts of the albino rat showing the early stagefe of entypy of the germ disc. X 200. A and B, rat No. 46, 6 days, 14 hours; C, rat No. 54, 6 days, 16 hours; ect.pl., ectoplacental cone or Trager; ect.n., ectodermal node; p.ect., parietal or transitory ectoderm; v. ent., visceral layer of entoderm; p.ent., parietal entoderm.

was drawn under camera lucida from one section, then by superimposing certain of the cells so as to give proper orientation, the lower half of the figure was added from the succeeding section. The slightly oblique plane in which this vesicle was cut made this procedure desirable. This relatively small vesicle seems in excellent state of fixation, as is evident from the symmetrical outline shown by the successive sections of the series. When compared with vesicle E of figure 23, though the two are separated in time of development by only a few hours, it is evident


310 G. CARL HUBER

that a distinct advance in development has taken place. The so-called floor of vesicle A, the region of the germinal disc of former stages, directed toward the mesometrial border, is markedly thickened, resulting in an outgrowth toward the mesometrial border and an ingrowth into the cavity of the vesicle. The outgrowth forms the anlage of the 'Trager' (Selenka) or the 'ectoplacental cone' (Duval), and appears to have developed largely as a result of an increase in size of the more superficially placed cells, since cell proliferation is not marked in this region. It is admitted that the critical stages are here lacking in my material. These stages appear to fall to the early hours of the seventh day, the material for which is lacking.

As may be seen from the figure, the cells constituting the anlage of the ectoplacental cone are of relatively large size with large vesicular nuclei, and are continuous at the base with the parietal ectodermal cells which form the roof of the vesicle or its antimesometrial portion. In the cell mass which extends into the cavity of the blastodermic vesicle or blastocyst in which there is recognized the anlage of the 'egg-plug' — 'Eizapfen,' or 'egg cylinder' — 'Eicylinder' (Sobotta) there is evident a fairly clearly circumscribed compact mass of cells, which stain somewhat more deeply than the surrounding cells and which may be designated as the ectodermal node. It represents the anlage of the true ectoderm of the embryo, as may here be stated in anticipation of further description. In all of the vesicles of this stage of development, even when cut obliquely or in cross section, this small nodule of compactly arranged cells is evident. It is circumscribed both from the cells of the ectoplacental cone as also from the cells lining the blastocele. The metamorphosis leading to the formation of the ectodermal node will receive consideration in a brief general discussion of this stage. The cells covering the egg-plug, and surrounding the ectodermal node, so far as it extends into the blastocele, are arranged in a single layer, forming a dome-shaped membrane, which appears as forced into the cavity of the vesicle consequent on development of the ectodermal node. This layer of cells constitutes the yolk entoderm, the anlage and differentiation of which has been previously


DEVELOPMENT OF THE ALBINO RAT 311

considered. The antimesometrial portion of this vesicle, its roof, consists of a single layer of somewhat flattened cells, the parietal or transitory ectoderm. The parietal ectoderm presents on its inner surface a few — four in the section figured — entodermal cells of irregular outline. These may be designated, after Sobotta, as cells of the parietal entoderm.

Vesicle B, of figure 24, taken from the same rat as was vesicle A (rat No. 46, 6 days, 14 hours) presents a very favorably cut vesicle, which, however, is slightly compressed from side to side, so that its form appears more nearly circular in the sections cut in the plane of the figure, than were they cut at right angles to this plane. This is especially true of the ectoplacental cone, which for the greater part appears in only two sections of 10 yu thickness, while in the plane of the figure it measures nearly 90 /u. Cognizance of this is to be taken in considering the relative size of the ectoplacental cone as shown in this figure. This vesicle is only very slightly older than that shown in A of this figure. Its ectoplacental cone is made up of a core of relatively large cells, bordered by more flattened cells, which in this preparation stain somewhat more deeply than do the more centrally placed cells. These covering cells are continuous with the cells of the parietal ectoderm. The cell mass projecting into the blastocele is more definitely circumscribed than in the slightly younger stage shown in A of this figure. The ectodermal node appears as an oval mass composed of compactly arranged cells, and is separable on all sides from the surrounding cells. The yolk entoderm, which may now be known as the visceral layer of the entoderm (Sobotta) passes as a single layer of cells of quite regularly cubic or short columnar form, nearly about the ectodermal node to reach the base of the ectoplacental cone, extending over on the parietal ectoderm at one side (see right side of figure). A few of the cells of the parietal entoderm, three in the figure, are evident. The parietal ectoderm forming the roof or antimesometrial portion of this vesicle consists of a single layer of flattened cells, which rest on, and are adherent to the decidual tissue; the uterine epithelium lining the decidual crypt in which the vesicle is lodged having in part disappeared in the immediate region of the vesicle.

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


312 G. CARL HUBER

Vesicle C of figure 24 (rat No. 54, 6 days, 16 hours) presents a stage which is almost identical in development with that shown in B of this figure, though in shape these two vesicles, as seen in sections, appear quite different. The vesicle shown in C is less compressed than the one shown in B, and probably presents more correctly the form of the blastodermic vesicle or blastocyst of the albino rat at this stage of development. The ectoplacental cone presents a cylindrical outline and contains two cells showing mitotic phases, both included in the section figured. Its cells, more particularly the ones bordering the periphery, present a vacuolated protoplasm, the vacuoles containing lightly colored globules which from reaction to the stain are to be regarded as blood cells or fragments of such, which blood cells are regarded as of maternal origin. In this preparation, the decidual crypt contains a small amount of extravasated maternal blood, found in part surrounding the ectoplacental cone; also in the antimesometrial portion of the crypt in relation with the roof of this vesicle. These findings will receive further consideration in the succeeding pages. The cell mass projecting into the cavity of the vesicle, consisting of the ectodermal node and the layer of visceral entoderm is slightly larger than in the preceding stage but presents no special features deserving discussion. The vesicle in the section sketched presents very few cells of the parietal entoderm. The parietal ectoderm forming the roof of this vesicle consists of a single layer of flattened cells in the protoplasm of certain of which vacuolization is evident. Certain of the cells show inclusions of lightly staining globules of a color similar to those found in the cells of the ectoplacenta, particularly evident in the lower right of the figure in which they are represented as uncolored circumscribed areas. The color reaction of these globules is like that of the maternal blood cells and fragments of blood cells found in the decidual crypt in the immediate vicinity of the vesicle, and they are regarded as blood cells or fragments of such, taken up by the cells of the parietal ectoderm at this stage in the development of the vesicle.

The blastodermic vesicles or blastocysts figured in figure 24, represent an important stage in the development of the albino


DEVELOPMENT OF THE ALBINO RAT 313

rat, as also in a number of other rodents, in that they show the anlage of the phenomenon known as the inversion of the germ layers or entypy of the germ layers. Inversion of the germ layers — Blatterumkehrung" — in the ova of rodents was probably first recognized by Reichert in the guinea-pig, mouse, and rat, though it was much more fully and correctly described by BischofT as observed in the guinea-pig and a little later by Hensen, also in the guinea-pig. Further observations on this phenomenon were recorded by KupfTer in his study of the development of the field mouse, Arvicola arvalis, and by Fraser on the gray and white rat and the mouse. Selenka gave this question special study, and in a number of monographic communications deals with the phenomenon of Blatterumkehrung as observed in three varieties of the mouse, the white rat, and the guinea-pig. Selenka's observations have formed the basis for future work on this problem. They have been widely accepted and extensively quoted. It was he who introduced the term 'Trager' to denote the cell mass which results from proliferation of the covering cells. His own words concerning this point read as follows:

Wahrend bei dem Kaninchenei, nach erfolgter Sonderung der formativen Furchungszellen in ausseres Ektodenn und inneres Entoderm, die gesammste Lage der ausseren Deckzellen zu einer dlinnen resistenten Membran zusammenschrumpft, verdickt sich bei den Nagern mit invertirten Keimblattern der mit den formativen Zellen in Contact befindliche Abschnitt der Deckschicht unter lebhafter Zellvermehrung zu einem sphari.schen oder konischen Gebilde, welches ich als 'Trager' bezeichne; * * * * j^jg Einwucherung dieses Tragers ins Innere der Keimblase hat zur Folge, dass die scheibenformigen Grundblatter (Ektodenn und Entoderm) sich nicht wie beim Kaninchen zu zwei concentrischer Hohlkugeln erweitern, sondern, ehe sie noch zu dieser Gestalt gelangten, ins Centrum der Keimblase vorgeschofen, vorgesttilpt und damit invertirt werden.

In a later publication, this observer also suggested the name 'Entypie des Keimfeldes' as a more comprehensive term than 'Blatterumkehrung' under which may be included tj^pes with inversion of the germ field without actual inversion of the germ layers. In later years Duval, Christiani, Robinson, Jenkinson, Sobotta, Kolster, D'Erchia, Spee, Burckhard, Melissinos, Widakowich, Lee and others have studied the earlier developmental


314 G, CARL HUBER

stages of rodents presenting the so-called inversions of the germ layers. O. Hertwig in his chapter "Die Lehre der Keimblatter" gives a brief resume of our knowledge of the inversion of the germ layers as observed in certain rodents, noting that three main modifications are to be observed. The first and simplest, as found in the field mouse; the second or intermediate as found in the rat and mouse; the third and most complex as observed in the guinea-pig. Hertwig's account is based largely on the observations of Selenka, the accuracy of which is now questioned from many sides.

My own conclusions concerning the early stages of the entypy of the germ layers in the albino rat are made on stages which do not portray the very beginning of this process. The vesicles shown in figure 24, in which this process is well initiated, however, present appearances, on the basis of which certain conclusions may be drawn. It is the contention of Selenka that the Triiger or ectoplacental cone is developed as a result of proliferation of covering or Rauber's cells, superimposed on the formative cells of the germ disc. He is followed in this view by Jenkinson, who states that "At a certain stage this proximal trophoblast (the so-called Rauber's cells of the rabbit) certainly becomes very thin, but it never wholly disappears, and soon thickens again to form the Trager, or, to use a modern expression, trophoblastic syncytium, which is destined to play an all-important part in the formation of the placenta." The account of Melissinos is difficult to follow, owing to his application of the term 'Raubersche Schicht.' The outer layer of the blastocyst in the region of the germinal disc is said to have a transitory existence and to disappear almost completely in the earlier stages of blastocyst formation. In a later paragraph he states, "dass nur die Raubersche Schicht existiert und sogar in den folgenden Stadien mit zahlreicheren Kernteilungsfiguren, und dass sie den Placentarconus liefert." Attention has previously and on a number of occasions been called to the fact that in the albino rat I have not been able to differentiate a distinct covering layer — Deckschicht or Rauber's Schicht (Selenka); trophoblast layer (Jenkinson) — and have expressed myself as wholly in accord with Sobotta's


DEVELOPMENT OF THE ALBINO RAT 315

observations on the mouse egg as concerns this point. He has critically reviewed Selenka's and Jenkinson's contentions as to the participation of the covering layer in the formation of the Trager or ectoplacental cone, reaching the conclusion that there is no evidence in support of this. In accord with Duval — and in this I concur — he states: Die mesometrale Spitze des 'Tragers Selenkas' ist, wie auch Duval richtig bemerkt, sogar ganz auffallig arm an Mitosen." The anlage of the ectoplacental cone or Trager, it would appear to me, is primarily the result of enlargement of its constituent cells, this enlargement of cells involving the more peripherally placed cells of the somewhat thickened germinal disc. In none of my preparations showing early stages in the formation of this structure are mitotic figures evident. Grosser in his figures 67 and 113, shows a germinal vesicle of the albino rat of 6^ days in its normal position in the decidual crypt. The vesicle there figured is about identical in time and stage of development to those figured by me in figure 24. In his figures, the Trager {Tr.) is represented as consisting of relatively few cells in which no mitoses are evident. In slightly older stages after the means of nutrition of the vesicles is improved through ingestion of maternal blood cells (Sobotta) mitotic figures may be observed in the ectoplacental cone, as shown in C of figure 24. In the rat as in Mus sylvaticus and the guinea-pig (Selenka) the ectoplacental cone arises as a solid mass of cells; in Arvicola arvalis (KupfTer) it is at first a hollow structure and is in part formed by invagination; in the white mouse (Sobotta) the form of this cell mass may vary greatly and may be solid or penetrated by a mere slit or again by a more extensive cavity.

The earlier stages in the formation of the egg-plug or eggcylinder I have not been able to follow. In the youngest stage showing this, at my disposal, A of figure 24, it consists of a central node of compactly grouped cells, of polyhedral form, quite definitely demarked from the surrounding cells, and very generally of oval form. This mass of cells I have designated the ectodermal node. In Grosser's figures (67 and 113, e, Ec) an identical structure may be observed, designated as 'Ectoderm der Em


316 G. CARL HUBEE,

bryonanlage.' The same may perhaps be observed in figure 26, plate 14, of Selenka's account. In figures 26, 28, 31, and 33 of Christiani's contribution this may be postulated, though his figures are useless for a close comparison. Duval does not figure this stage. Sobotta's ('03) figure 7, and figure 33 of the contribution of Melissinos, appear to give a corresponding stage for the mouse, but in neither of these figures is the 'ectodermal node' so clearly depicted as in Grosser's and my own figures, at least not until a somewhat older stage. Figure 6 of Sobotta ('03) may very probably be regarded as representing an intermediate stage between that shown in E of figure 23 and in A of figure 24. By a proliferation of the cells of the germinal area as shown in the former figure a stage resembling that shown in Sobotta's figure 6, is readily postulated. That the formation of the ectodermal cells is in part due to rearrangement of the cells of the germinal area I believe to be the case, since cell proliferation is not marked in this stage. The enlargement of the more peripheral cells of the germinal area, leading to the anlage of the ectoplacental cone, would of necessity cause the forming ectodermal node to force the yolk entoderm into the cavity of the vesicle, and thus form the anlage of the egg-plug and initiate the phenomenon of entypy of the germ layers. O. Hertwig, in describing the inversion as observed in the mouse and rat, after considering the formation of the Trager through proliferation of the cells of the Deckschicht, following here Selenka's account, states, referring to the Trager, Durch ihn wird der formative Teil des Ektoblasts nach dem Centrum der Blase vorgetrieben, wobei er sich in eine allseits abgegrenzte Epithelkugel umwandelt." And again, in referring to the development of the guineapig, he states: "Wie bei Maus und Ratte zieht sich das formative Ektoderm zu einer Epithelkugel zusammen." Hertwig thus appears to regard the formation of the 'Epithelkugel,' the ectodermal node, as in part at least developed owing to a rearrangement of the cells of the germinal disc. After the formation of the egg-plug or egg-cylinder that portion of the yolk entoderm which covers it is designated by Sobotta as the visceral layer of the entoderm. The scattered entodermal cells, attached


DEVELOPMENT OF THE ALBINO RAT


317


here and there to the inner surface of the parietal ectoderm, in the albino rat at no time forming a continuous layer, he has designated as the parietal entoderm. He is followed in this by Widakowich. This nomenclature has been used by me in the sense employed by Sobotta. The parietal or transitory ectoderm (Kolster's 'feinfasserige Haut') forming the roof or antimesometrial portion of the vesicles, is constituted of a single layer of flattened cells, which in the rat show no regional differentiation. The resorption of maternal blood, incidentally noted with reference to cells of the ectoplacental cone and certain of the cells of the parietal ectoderm in connection with vesicle C of figure 24, to which phenomenon attention has been drawn by Sobotta and Kolster for the mouse, will receive further consideration in the discussion of older stages.


DEVELOPMENT AND DIFFERENTIATION OF THE EGG-CYLINDER

The material at hand is hsted in table 8.



TABLE 8



RECORD NUMBER


AGE


NUMBER OF OVA


17


8 days, 17 hours (?)


2 (not all cut)


35


8 days, 18 hours (?)


6


21


7 days, 16 hours


10


66


7 days, 16 hours


7


27


7 days, 17 hours


7


89


7 days, 20 hours


5


81


7 days, 22 hours


7


94


8 days


7


95


8 days


9


96


8 days


5


For the stages showing the development and differentiation of the egg-cylinder in the albino rat I am able to present a series of stages which follow one another in close succession. The figures presented are in themselves so elucidative that an extended description is obviated. The stages under consideration fall within the eighth day after the beginning of insemination, judging from the great majority of the specimens at my disposal, although two rats (Nos. 17 and 35) killed in the latter


318 G. CARL HUBER

half of the ninth day, contained stages which are younger than nearly all of those obtained the latter half of the eighth day. I am unable to state whether this is owing to a retardation in the rate of development of the ova in rats Nos. 17 and 35, or due to an error of record. The record gives date and hour of insemination and of killing, and I have no reason to doubt its accuracy. However, the two rats in question give the only instances of marked deviation from what appears as a normal rate of development as presented by the bulk of my material. Sobotta ('11) has called attention to the difficulty of obtaining successively staged material in the mouse, and cites Kolster as contending: Man konne auf die Altersbestimmung gar nichts geben." During this stage of development the decidual crypts lodging the ova are deeper than in the preceding stage, their mesometrial portion being narrower, though they are not as yet separated from the uterine lumen. The orientation of the decidual crypts and the contained egg-cylinders is perhaps more readily made than in slightly younger stages, though not definitely enough to insure the cutting of sections in a given plane. Sections of the egg-cylinder cut in the longitudinal plane may be obtained by cutting parallel to the plane of the mesometrium or at right angles to the same. However, it is still largely a matter of chance as to whether the sections obtained pass through the midplane or at an angle thereto.

In figure 25, there are reproduced representative sections of three germinal vesicles taken from the same uterus (rat No. 35, 8 days, 18 hours) which show three closely approximated early stages in the development of the egg-cylinder. None of these three vesicles is cut in exactly the mid-longitudinal plane ; especially is this true of the ends of the vesicles. Furthermore, the antimesometrial portion of each, lower part of the figure, composed of the thin-walled parietal ectoderm, shows a certain amount of folding, so that a portion of each wall is cut en face instead of en profile. The appearances here presented by the antimesometrial portion of these vesicles is not to be confused with a 'giant cell' formation of this portion of the roof of the vesicle, described by Sobotta in his earlier publications, but corrected and retracted


DEVELOPMENT OF THE ALBINO RAT


319


in his later communications. Vesicle A, figure 25, when compared with vesicle C of figure 24, shows only a slight difference in degree of development. Vesicle A is of more elongated and of more distinctly cylindrical form. Its thin-walled portion (an


--< --«!•*> .,-\ V« 


^^/'



Fig. 25 Longitudinal sections of blastodermic vesicles of the albino rat, showing entypy or inversion of germ layers with early stages in egg-cylinder formation. The ectoplacental cone of each is not cut through its entire length and the lower portion ot each vesicle is slightly folded. X 200. A, B, and C, rat No. 35, 8 days, 18 hours, after insemination. To fit properly into the entire series these three vesicles should be from the early hours of the seventh day after insemination. ect.pl., ectoplacental cone or Trager; ect.n., ectodermal node; ex. ect., extraembryonic ectoderm, early stage of its ingrowth shown in vesicle A; p. ect., parietal or transitory ectoderm; v.ent., visceral layer of entoderm; p.ent., cells of parietal entoderm.


timesometrial portion) is longer, its cavity more extensive; this is owing to a further flattening of the cells of the parietal or transitory ectoderm. In vesicle A in the section preceding the one figured, the ectoplacental cone is thicker by about two


320 G. CARL HUBER

rows of cells than in the one figured ; the section figured not passing through the center of this structure. In vesicle A, the ectodermal node, which is distinctly demarked, no longer rests against the base of the ectoplacental cone, as in C of figure 24, but has been forced farther into the cavity of the vesicle by reason of proliferation of the cells at the base of the ectoplacental cone, resulting in the formation of a nearly cylindrically formed column of compactly arranged, polyhedral-shaped cells interposed between the ectodermal node and the base of the ectoplacental cone, but merging into the latter without sharp demarcation. To this mass of cells the name of extraembryonic ectoderm has been given by Widakowich. However, under this term this author includes also the cells of the ectoplacental cone. The ectodermal node is of larger size than in the slightly younger stage, C of figure 24, the result of cell proliferation. In the section sketched, three mitotic figures are evident in this structure. Its cells are of polyhedral shape, and show no definite arrangement. The ectodermal node and the extraembryonic ectoderm, to the base of the ectoplacental cone, together form a cylindric structure enclosed within a layer of visceral entoderm, which in the section figured is in part cut tangentially, and thus simulates an epithelium consisting of two layers of cells, but consisting in reality of a single layer of cells. Ectodermal node, extraembryonic ectoderm, and the layer of visceral entoderm together form a structure of cylindric shape which extends into the cavity of the vesicle for a distance about one-half its extent, forming the anlage of the egg-cylinder (Sobotta). Very few parietal entodermal cells are to be found on the inner surface of the parietal ectoderm. Vesicles B and C of figure 25 differ from that discussed under A, only to the extent to which the ectodermal node has been forced into the cavity of the vesicle owing to further growth of the extraembryonic ectoderm, to the extent that in C, the elongated egg-cylinder approaches the antimesometrial end of the cavity of the respective vesicle. Ectodermal node and extraembryonic ectoderm are at this stage distinctly demarked, though in close apposition. An indenture from the surface at the region of the union of these structures


DEVELOPMENT OF THE ALBINO RAT 321

with a consequent infolding of the layer of visceral entoderm is not as a rule evident, if so, only very slightly, as to the left in B ; such infolding of the visceral entoderm is not regarded as having special significance. These structures, ectodermal node and extraembryonic ectoderm, are appropriately referred to as ectodermal cylinder by Widakowich, and with the visceral entoderm, as constituting the egg-cylinder of Sobotta.

Under A of figure 26 (rat No. 17, 8 days, 17 hours), there is shown a representative section of a vesicle which is only very slightly older than that shown under C, figure 25. This vesicle was exposed, by teasing away, after fixation, the decidual tistue forming one side of the decidual crypt; this being done before embedding, so as to admit of orientation of its long axis. This accounts for the collapsed state of the thin wall of the vesicle and its slight folding, also for the fact that the ectoplacental cone is reflected upon itself. The egg-cylinder is cut in a very favorable longitudinal plane. In its antimesometrial portion, lower part of the figure, the cells of the ectodermal node now show definite arrangement in practically a single layer, with alternating nuclei. The beginning of a central cavity is evident with reference to which the cells are arranged. This cavity is the anlage of the 'Markamnionhohle' of Selenkc, more appropriately known as the antimesometrial portion of the proamniotic cavity. The cells forming the wall of the ectodermal vesicle (Ektodermblase, Selenka), derived from the ectodermal node, may now be known as the primary embryonic ectoderm (Widakowich). The extraembryonic ectoderm in the mesometrial portion of the egg cylinder has differentiated to form a relatively long irregularly cylindric structure, continuous with the base of the ectoplacental cone, composed of irregular polyhedral cells, compactly arranged and showing as yet no definite orientation. In these cells active proliferation is evidenced by numerous mitoses. The egg-cylinder is covered by a single layer of cells of the visceral entoderm. Over the antimesometrial end of the eggcylinder, the entodermal cells now present a cubic or thick pavement form, while along the sides of the egg-cylinder they are of cohmmar form, especially long in the region where the primary




■3)



V


Fig. 26 Longitudinal sections of egg-cylinders of the albino rat, showing the anlage of the antimesometrial and mesometrial portions of the proamniotic cavity. X 200. A, rat No. 17, 8 days, 17 hours; B and C, rat No. 81, 7 days, 22 hours, after insemination. A, shows the very beginning of the development of the antimesometrial portion of the proamniotic cavity developing within the ectodermal node; C shows the beginning of the proamniotic cavity developing in the extraembryonic ectoderm; ect.pl., ectoplacental cone or Trager; p.ect., parietal or transitory ectoderm; ex.ect., extraembryonic ectoderm; v.ent., visceral entoderm in B and C, the cells of this layer showing the anlage of the three zones showing absorption of maternal hemoglobin; a.met.pr., antimesometrial portion of proamniotic cavity, developing in the ectodermal node; pr.emb.ect., primary embryonic ectoderm; eci.ves., ectodermal vesicle; met.pr., mesometrial portion of the proamniotic cavity, developing in the extramebryonic ectoderm.

322


DEVELOPMENT OF THE ALBINO RAT 323

embryonic ectoderm and the extraembryonic ectoderm meet. The special cytomorphosis undergone by the columnar cells of the sides of the egg-cylinder, in contradistinction to those of the antimesometrial end, will be considered in later pages. The visceral layer of the entoderm extends to the base of the ectoplacental cone, in part passing over onto the layer of parietal ectoderm. In the section figured, cells of the parietal layer of the entoderm are not evident. The ectoplacental cone has grown in length in the direction of the lumen of the uterus or the mesometrial border. In the great majority of my preparations this structure is slightly compressed from side to side, so as to be broader in a plane parallel to the long axis of the uterus. In vesicle A, it is cut at right angles to the long axis of the uterus, thus appears as much narrower than in the other two vesicles of figure 26, which were cut in a plane parallel to the plane of the mesometrium. The increase in size of the ectoplacental cone is the result of active cell proliferation. Mitotic figures to the number of one, two or three, may now be observed in nearly every section of this structure. The parietal or transitory ectoderm, continuous with the base of the ectoplacental cone, has been reduced by this stage to a thin, practically homogeneous membrane, presenting scattered, flattened nucleated cells on its inner surface. This thin membrane is now quite firmly adherent to the wall of the decidual crypt, throughout nearly its whole extent.

Under B of figure 26 (rat No. 81, 7 days, 22 hours) there is shown a representative section of a vesicle which is slightly more advanced in development than that shown in A of this figure. The antimesometrial portion of the proamniotic cavity, the anlage of which was shown in the preceding stage, is well established. Its wall, consisting of primary embryonic ectoderm is composed of a single layer of cells with nuclei in essentially the same plane. The primary embryonic ectoderm forms a closed vesicle (Ectodermblase, Selenka) distinctly demarked from the extraembryonic ectoderm. In this as in the preceding stage the extraembryonic ectoderm forms a long cylindrical structure continuous at its mesometrial end with the base of the


324 G. CARL HUBER

ectoplacental cone. The cells are of irregular polyhedral form, compactly grouped, showing as yet no definite arrangement. Cell proliferation as evidenced by mitoses is active, amply accounting for the increase in length of this structure. The visceral entoderm encloses the long egg-cylinder as a single layer of cells and is continuous at its base with the parietal entoderm, well shown at the left of the figure. The ectoplacental cone of this vesicle is very favorably cut in a plane parallel to the long axis of the uterus. This vesicle was unusually well fixed and may be regarded as showing normal relations of the thin membranous wall, derived from the parietal ectoderm, and of the egg-cylinder, which reaches quite to the antimesometrial end of the vesicle.

Vesicle C of figure 26, obtained from the same uterus as was vesicle B (rat No. 81, 7 days, 22 hours), differs from that shown under B, in that it presents the anlage of a mesometrial portion of the proamniotic cavity. In the extraembryonic ectoderm, near its junction with the base of the ectoplacental cone, two irregular spaces may be observed. These are distinctly evident, passing through the entire section, only in the section figured. The antimesometrial portion of the egg-cylinder is not cut quite through its center, so that the primary embryonic ectoderm of the ectodermal vesicle appears as a stratified epithelium, and the antimesometrial portion of the proamniotic cavity appears as relatively small, this owing to a slight curvature shown by this egg-cylinder. The other features presented by this vesicle are sufficiently well portrayed in the figure to obviate the necessity of further description.

In figure 27, there are shown three further stages of egg-cylinder differentiation, showing progressively older stages than shown in the preceding figure. Under A of this figure, there is reproduced a representative section of a vesicle taken from the same uterus as were vesicles B and C of figure 26 (rat No. 81, 7 days, 22 hours). The figure is not of a single section, but is combined from two sections, superimposed so as to give correct dimensions and relations. The egg-cylinder of A of this figure differs from that shown in C of figure 26, in that the mesometrial



"^;\



Fig. 27 Longitudinal sections ot egg-cylinders of the albino rat showing fusion of the antimesometrial and the mesometrial portions of the proamniotic cavities. X 200. A, rat No. 81, 7 days, 22 hours; B. rat No. 96, 8 days; C,'rat No. 94, 8 days, after insemination; ect.pl., ectoplaceiital cone or Trager; p.ect., parietal or transitory ectoderm; ex.ect., extraembryonic ectoderm; ect.ves., ectodermal vesicle, with wall composed of primary embryonic ectoderm, at + junction with the extraembryonic ectoderm; a.met.pr., antimesometrial portion of proamniotic cavity; met.pr., mesometrial portion of proamniotic cavity; pr.c, proamniotic cavity; v.ent., visceral entoderm; pr.emb.ent., primary embryonic entoderm.

325


326 G. CARL HUBER

portion of the proamniotic cavity, developing in the extraembryonic ectoderm, is of greater dimension. Two relatively large spaces, bordered by a single layer of cells of the extraembryonic ectoderm, are to be observed. At the junction of the extraembryonic ectoderm and the ectodermal vesicle of primary embryonic ectoderm a further space of triangular outline may be seen. The primary embryonic ectoderm is arranged in the form of an oval-shaped vesicle, forming the antimesometrial end of the egg-cylinder. Its wall is relatively thin at the region of its apposition to the extraembryonic ectoderm, just below the triangular space above mentioned. This ectodermal vesicle is peculiar in that its cavity contains the remains of four cells. A study of the series of sections shows that these cells do not represent the crest of a fold of the wall of this vesicle, since they are not nearly so distinct in preceding and succeeding sections. It may only be conjectured that during the rearrangement of the cells of the ectodermal node, resulting in the formation of the ectodermal vesicle, certain of the cells became separated from the wall and remained free in the cavity. The primary embryonic ectoderm, forming the wall of the ectodermal vesicle is readily differentiated from the extraembryonic ectoderm, both by the fairly sharp definition of the ectodermal vesicle and by reason of the fact that its cells stain somewhat more deeply than do the cells of the extraembryonic ectoderm, as also the cells of the visceral entoderm. In the egg-cylinder shown under B of figure 27 (rat No. 96, 8 days) the antimesometrial portion of the proamniotic cavity, developing in the ectodermal node, and the mesometrial portion of the proamniotic cavity, developing as several discrete spaces in the extraembryonic ectoderm, have in part joined to form a single proamniotic cavity. The mesometrial portion of this cavity is still bridged by a septum of extraembryonic ectodermal cells, closing off a relatively large space found in its mesometrial portion. With the junction of the antimesometrial and the mesometrial portions of the proamniotic cavity, the primary embryonic ectoderm and the extraembryonic ectoderm become a continuous layer, the line of union of the two portions, however, remains evident and is readily recognized in all the egg-cylinders


DEVELOPMENT OF THE ALBINO RAT 327

of this and older stages, a question which will receive further consideration in following pages.

In C of figure 27 (rat No. 94, 8 days) the proamniotic cavity forms a continuous, single space. The figure presented is drawn from two sections; its greater portion, to the base of the ectoplacental cone from one section, the ectoplacental cone from another section. The junction of the membranous wall of the vesicle to the base of the ectoplacental cone, in the two sections used for the figure, was superimposed under camera lucida in joining the portions drawn from the two sections. It is believed that the drawing as presented gives correctly dimensions and relations of the different parts of this vesicle. The wall of the antimesometrial portion of the single proamniotic cavity is formed by the primary embryonic ectoderm, the calls of which are for the main of irregular columnar shape, with alternately placed nuclei. These cells are in active proliferation, as is evidenced by numerous mitoses. The wall of the mesometrial end of the proamniotic cavity is formed of a single layer of cells of the extraembryonic ectoderm; these cells are of quite regular shape with nuclei placed in about the same plane. They stain less deeply than do the cells of the primary embryonic ectoderm. In this egg-cylinder (C, fig. 27) the proamniotic cavity does not extend so near the base of the ectoplacental cone as in a number of other preparations in my possession, showing about the same stage of development ; in certain of these, the proamniotic cavity extends to near the mesometrial end of the egg-cylinder.

A more definite characterization of the different parts of the egg vesicle of the albino rat at the stage of development shown in C, figure 27, end of the 8th day, seems desirable, and in doing so I shall use the terminology used by Sobotta and Widakowich. The vesicle under consideration has reached a length of 0.65 mm., and a width of 0.12 mm. Somewhat more than onefourth of its length consists of ectoplacental cone or Trager. The cavity enclosed is derived from the cavity of the blastodermic vesicle with germ disc, the blastocele, and is termed by Sobotta and Widakowich the 'Dottersackhohle' or yolk-sac cavity. This cavity is bounded by a thin structureless mem JOURNAL OV MORPHOI.OCiY. VOL. 2fl, NO. 2


328 G. CARL HUBER

brane derived from the parietal or transitory ectoderm and the scattered cells forming the parietal layer of entoderm. This membrane is continuous with the base of the ectoplacental cone and presents scattered flattened cells on its inner surface. I have designated this thin membrane with cells on the inner surface as the parietal or transitory ectoderm (Kolster's feinfaserige Haut). The egg-cylinder which extends to the antimesometrial end of the yolk-sac cavity, encloses the proamniotic cavity, the antimesometrial portion of which is walled by primary embryonic ectoderm, its mesometrial portion by extraembryonic ectoderm, the two forming a continuous layer, with line of union of the two types of ectoderm evident. The uncleaved extraembryonic ectoderm is continuous with the base of the ectoplacental cone. The egg-cylinder is surrounded by a single layer of cells of the visceral entoderm, dilTerentiated so as to consist of a portion which surrounds the antimesometrial end of the egg-cylinder in relation with the primary embryonic ectoderm ; the cells of this portion being of a rather thick pavement type, constituting the primary embryonic entoderm, and further a portion which covers the sides of the egg-cylinder, with cells of a columnar type, showing special cytomorphosis. The egg-vesicles and egg-cylinders of the stage of development under consideration and for somewhat older stages show no bilateral symmetry so far as can be discerned by study under the microscope. In longitudinal sections of egg-cylinders, cut respectively in two different planes, at right angles to each other, no difference in form, relation and structure of different parts can be observed. Selenka, Kupffer, Duval, and Sobotta have previously called attention to this fact and shown that longitudinal sections of egg-cylinders may be obtained no matter whether the sections are cut parallel to the plane of the mesometrium, thus parallel to the long axis of the uterus, or at right angles to this plane. The want of bilateral symmetry is also evident in cross sections of the egg-cylinder, as may be seen from the series of sections presented in figure 28 (rat No. 27, 7 days, 17 hours). The cross-cut egg-cylinder, from several sections of which these figures were drawn, represents a stage of develop


DEVELOPMENT OF THE ALBINO EAT 329

ment very similar to that of the egg-cyhnders shown in longitudinal section in figure 26.

Widakowich, after discussing very briefly the mode of development of the egg-cylinder, discusses and figures an egg-cylinder of the albino rat, obtained 6f days after the last coitus. His figure 3 corresponds in stage of development very closely to that shown by me in A of figure 27. In his figures, there is presented an egg-cylinder showing the anlage of the mesometrial


' , ex. ect '



Fig. 28 A series of cross sections at different levels of an egg-cylinder of the albino rat after the anlage of the antimesometrial portion of the proamniotic cavity. X 200. Rat No. 27, 7 days, 17 hours, after insemination. The sections selected for the several levels drawn, A to D, are as follows: A, middle of ectoplacental cone; B and C, through extraembryonic ectodermal portion of egg-cylinder, just below junction with ectoplacental cone (B), and just above ectodermal vesicle (C); D, through middle of ectodermal vesicle. Compare with B, figure 26, a longitudinal section of an egg-cylinder of the same stage of development; p. ect., parietal or transitory ectoderm; ex.ect., extraembryonic ectoderm; pr.evib.ect., primary embryonic ectoderm of the ectodermal vesicle; v.ent., visceral entoderm; ipr.emb.ent., primaiy embryonic entoderm; a.met.'pr., antimesometrial portion of proamniotic cavity.

portion of the proamniotic cavity. Emphasis is given to the fact that in the antimesometrial portion of the egg-cylinder, there may be recognized the primary embryonic ectoderm. His own words with reference to this point read as follows:

Der Schnitt zeigt nun sehr deutlich, dass sich die Zellen, die die antimesometrale Hohle so begrenzen, dass die alte Kugel-oder Eiform dieses Teiles noch zu erkennen ist — das priniare embryonale Ectoderm — intensiver farben wie die Zellen des mesometralen Abschnittes oder die des Ectoplacentarconus — das extraembryonale Ectoderm. Die Kerne zeigen keinerlei Unterschied in der Fitrbung, wohl aber das Plasma, dass im antimesometralen Teile von dichterer Structur zu sein scheint.

This description corresponds very closely to that given by me for a similar stage. The differentiation of these two kinds of ectoderm was also recognized by Robinson, who states:


330 G. CARL HUBER

The epiblastic cylinder is closed at its distal end, the trophoblastic at its proximal, and the open ends of the two cylinders are in close apposition, but not indistinguishably fused, for the character of each portion of the ectoderm, after treatment with carmine, is still quite distinctive; the protoplasm of the trophoblast being tinged much more faintly than that of the epiblast.

Selenka, on the other hand, who has recognized in his 'Ektodermblase' with 'Markamnionhohle' a distinctive structure, believes this to blend completely with the Triiger. Since his account with reference to this point has influenced later w^orkers, I may be permitted to quote him in the original. Referring to the 'Ektodermblase' with 'Markamnionhohle/ he states:

Dieser Ektodermkeim, welcher von dem vorriickenden Tragerzapfen anfanglich sehr wohl abgegrenzt ist, indem beiderlei Gebilde sich in Folge der convexen Kriimmung ihrer einander zugekehrten Fliichen sozusagen nur in einem Punkte beriihren, fliesst endlich mit dem Trager vollstandig zusammen, und zwar bei der Waldmaus bevor, bei der Ratte und Hausmaus aber nachdem die Markamnionhohle enstanden war.

That the proamniotic cavity of the egg-cylinder of the albino rat has its anlage in two distinct cavities, the one developing in the ectodermal node in the antimesometrial portion of the eggcylinder, which is the first to develop; the other in the mesometrial portion in the extraembryonic ectoderm, was recognized by Selenka (fig. 30, plate 14, E, Markamnionhohle, E', falsche Amnionhohle), Duval (fig. 100,) Robinson, and Widakowich (fig. 3). Corresponding stages of egg-cylinder development as presented by me in figures 26 and 27, for the albino rat, are shown by Sobotta ('02), for the mouse in his figures 12 to 14 and text figures a to f. On comparison of my figures with Sobotta 's, it becomes evident that the egg-cylinder of the rat is much longer and more slender than that of the mouse. According to the account of Sobotta, the egg-cylinder of the mouse, soon after its anlage, shows by reason of a distinct transverse furrow a division into two parts, an antimesometrial portion of globular form, surrounded by a visceral layer of entoderm, corresponding to what I have designated as the ectodermal node; and a mesometrial portion which early shows the anlage of a proamniotic


DEVELOPMENT OF THE ALBINO RAT 331

cavity. A lumen is obtained in the antimesometrial portion later than in the mesometrial portion. As development proceeds, this sharp demarkation of antimesometrial and mesometrial poition is gradually lost. This, as stated in his own words, reads:

Sehen wir von dem die (der Keimhohle zugekehrte) Oberflache des Cylinders liberziehenden Dotterentoderm zunachst ab, so sieht man, dass die Furche, welche die oben erwahnten mesometralen und antimesometralen Abschnitte in Stadium der Fig. 11 u. 12 trennte, jetzt wieder wenig deutlich ist. Es bahnt sich eine Verschmelzung beider Abschnitte wiederum an, was man am leichtesten daraus ersieht, dass bald (Fig. 14) beide Abschnitte ein gemeinsames Lumen erhalten.

With the formation of a continuous proamniotic cavity, this is bordered by a single layer of 'ectodermal cells,' with alternately placed nuclei. The cells are described as being the same throughout; neither in text nor figure does Sobotta differentiate between ectodermal cells derived from the antimesometrial portion of the egg-cylinder and those derived from the mesometrial portion. Melissinos also recognizes antimesometrial and mesometrial portions in the development of the eggcylinder of the mouse, in his figure 34. According to this observer, the antimesometrial portion of the proamniotic cavity is the first to appear; later it appears in the mesometrial portion, the two cavities joining as development proceeds. The parts of the ectoderm derived from these two portions may be recognized, however, after a single proamniotic cavity has developed. This Melissinos states in the following words: "Trotz aller Vereinigung der beiden Hohlungen bleibt die Unterscheidung des normals abgesonderten antimesomtralen Abschnittes von dem mesometralen immer leicht zu machen, sei es durch eine klare Grenzlinie oder durch eine an der Peripherie des visceralen Dotterblattes befindiiche Furche." The account of Melissinos is more in agreement with the presentations as observed in the albino rat than is that of Sobotta.

Selenka, Sobotta, and Melissinos recognize three different regions of constriction to which significance is given, in the egg-cylinder of the mouse. As stated by Sobotta, the first con


332 G. CARL HUBER

striction is in the region of the original furrow which demarks the antimesometrial and the mesometrial portions of the eggcyhnder, the region of the primary amniotic fold; the second where the mesometrial cavity ends; and the third where the original blastodermic cavity reaches its mesometrial end. The three folds recognized by Melissinos, are characterized by the specificity of the ectoderm. Since his statement concerning this point is somewhat involved, I find it necessary to use his own words; they read as follows, referring to these folds he states:

Der eine derselben a liegt antimesometral und ist tier bekannte erste kugelformige Buckel (Ektoderm) mit den langlichen, cylinderpyramidalen oder polj^gonal-pyramidalen Zellen; der zweite b liegt in der Mitte und besteht aus kubisch-polygonalen Zellen, und der dritte Buckel c, aus polygonalen Zellen bestehend, liegt mesometral und ist von dem mittleren durch Einschniirung, von der Basis des Ectoplacentarconus aber durch die bekannte Urfurche des Eicylinders getrennt, in der sich das viscerale Dotterblatt zuni parietalen Dotterblatt umbiegt.

So far as I am able to determine, the account of Melissinos agrees with that given by Sobotta, as concerns the folds of the egg-cylinder of the mouse. Selenka's account need not receive special consideration.

In well-fixed egg-cylinders of the albino rat no such folds are recognized. At the line of junction of the primary embryonic ectoderm and the extraembryonic ectoderm, a slight infolding of the layers, variable in degree, is recognized. Other foldings of the wall of the egg-cylinder I have regarded as accidental and not of special significance. Therefore, I am wholly in accord with Widakowich, who has also discussed this question with reference to the albino rat and has described the low fold in the region of the junction of the primary embryonic ectoderm and extraembryonic ectoderm. Referring to that fold, he states: Dass war die einzige konstante, bald starker, bald schwacher ausgepragte Einschniirung der Proamnionhohle."

Sobotta deserves credit for having described fully the differentiation and cytomorphosis of the cells of the visceral entoderm of the egg-cylinder, and since his observations on this point apply in the main to the albino rat, they may at this time be given


DEVELOPMENT OF THE ALBINO RAT 333

consideration. During the early stages of egg-cylinder differentiation and anlage of the proamniotic cavity, the layer of visceral entoderm differentiates into a portion which is in relation with the primary embryonic ectoderm of the antimesometrial portion of the egg-cylinder, in which region the cells of the entoderm are first of short cubic shape, later of the pavement type; this portion may be regarded as forming the primary embryonic entoderm, since it forms the greater part of the entoderm of the embryo. The greater part of the visceral entoderm, that which surrounds the sides of the mesometrial portions of the egg-cylinder, consisting of extraembryonic ectoderm, differentiates into cells of the columnar type. In this latter portion, with the formation of a continuous proamniotic cavity, the entodermal cells undergo characteristic cytomorphosis. In them, as stated by Sobotta, there may be recognized three main zones: (1) a basal zone with denser protoplasm containing the nucleus; (2) a middle zone with markedly vacuolated protoplasm; (3) an outer zone in which hemoglobin granules are recognized, the latter zone staining deeply in eosin. These three zones in the cells of the visceral entoderm in the region of the extraembryonic ectoderm of the egg-cylinder may be recognized in figures 26 and 27, not so clearly as in Sobotta's colored figures, particularly his figure 17 ('03) and figure 8 ('11). However, I am able to follow closely his description in my own preparations of a somewhat older stage than thus far figured. It is Sobotta's contention that in the extravasated blood surrounding the egg vesicle, in close apposition to its thin outer wall, there may be observed many red blood cells which, though presenting normal form, show a distinctly granular content. These granules stain deeply in eosin and are in shape, size, and reaction to stain very similar to granules found in the peripheral part of the cells of the visceral entoderm. On the outer surface of the thin wall of the vesicle; on its inner surface ; in the cells lining this ; in the yolk sac cavity ; and on the outer surface of the cells of the visceral entoderm, similar granules are found. These appearances are interpreted as showing an absorption of maternal hemoglobin by the entodermal cells of the mesometrial portion of the egg-cylinder.


334 G. CARL HURER

Sobotta's statement concerning this point, which, owing to its importance, I quote in full, reads as follows:

Man wird diese mikroskopisch erkennbaren Verhiiltnisse nieht anders deuten konnen als in folgender Weise: Die Hamoglobinschollen, die durch die aussere Wand des Dottersackes in die Dottersackhohle gelangt sind, werden von der Oberflache des zylindrischen, die ganze Seitenflache des Eizylinders iiberziehenden visceralen Dottersackepithels aus resorbiert und zwar geschieht das in der Weise, dass die Hamoglobinschollen ziinachst als solche in der Zelle selbst eintreten, dann aber ini vacuolisierten Teil der Zelle gleichsam verdaut werden, wobei die einzelnen kleinen Schollen vorher zu grosseren Tropfen zusammen-fliessen scheinen.

My own observations on the albino rat as concerns this phenomenon, more particularly as concerns the structure of the cells of the visceral entoderm in the region of the extraembryonic ectoderm, corroborate Sobotta in many particulars. This question will be again and more fully considered in a contemplated later publication dealing with the implantation and decidua formation in the albino rat. It could not be considered now without a discussion of the changes involved in the development of the decidua, a question which I am not prepared to consider fully now. It may be stated, however, that judging from my own preparations and the figures of Grosser, the extravasation of blood into the egg chamber is not nearly so extensive in the albino rat as is shown in the figures of Sobotta for the mouse.

The thin membrane which surrounds the yolk-sac cavity, which I have designated as the parietal or transitory ectoderm, is derived in development from the parietal or transitory ectoderm, and the relatively few parietal entodermal cells, as described and figured for younger stages. At the stage of egg-cylinder development under consideration — with continuous proamniotic cavity — this structure appears as a thin, practically homogeneous membrane with scattered, flattened nucleated cells on its inner surface. Sobotta regards these cells as derived from the parietal entoderm, the cells of the parietal ectoderm having disappeared. As concerns this, I am unable to speak with certainty, since the Congo red solution used as a double stain is not particularly favorable in differentially coloring these


DEVELOPMENT OF THE ALBINO RAT 335

cells. However, I am disposed to regard these flattened cells as derived from the parietal ectoderm. The parietal entodermal cells are never numerous in the rat, and mitotic figures are seldom observed in them. With the extension of the vesicle with the enlargement of the blastocele, the cells of the parietal or transitory ectoderm become attenuated until they appear for the greater part as a thin cuticular membrane, and I am disposed to regard the flattened nucleated masses of protoplasm lining the inner surface of this membrane as derived from the cells of the parietal ectoderm.

Much attention has been given to certain large cells which are found in close relation with the outer surface of this thin membrane. These cells, generally referred to as giant cells (Riesenzellen) were, by Duval, Sobotta (earlier publications) and Grosser thought to be of embryonic origin and derived from the cells of the parietal ectoderm. Selenka, Disse, Kolster, Melissinos, Pujiula, Widakowich, and later Sobotta ('11) regard them as derived from the maternal tissue and as representing differentiated decidual cells. It is not my purpose to consider more fully these cells in the present communication, since the}^ are by me not regarded as of embryonic origin. My own observations as concerns them agree in the main with those of Widakowich, who, in the albino rat has followed their origin from decidual cells. Since not of embryonic origin, they have been disregarded in making the figures.

I have previously, in connection with a discussion of the structure of vesicle C, figure 24, alluded to the fact that the cells of the ectoplacental cone as also the cells of the parietal or transitory ectoderm have a phagocytic action for maternal blood cells. This Sobotta has also observed for the mouse, in which he is confirmed by Kolster who has further shown that the cells of the ectoplacental cone also take up fat particles. With the ingestion of maternal blood cells by the cells of the ectoplacental cone, more particularly, with the absorption of hemoglobin by the entodermal cells of the mesometrial portion of the eggcylinder, a period of rapid growth of the egg vesicle is initiated. To this Sobotta has called attention for the mouse; the same


336 G. CARL HUBER

is evident in the albino rat. Indeed, Sobotta presents the far-reaching conclusion that the explanation of the phenomenon of germ layer inversion or entypy of the germ layers is to be found in the dearth of food supply of the ovum in the stages preceding the formation of more definite relations between the ova or germ vesicles with the decidua. It is thought by this observer that the inversion of the germ disc has for its purpose the increase of the absorptive surface of the visceral or yolk sac entodermal epithelium, which as a differentiated layer comes to surround nearly the whole of the egg-cylinder on completion of the inversion, and is thus increased in extent and brought in relatively close relation with the maternal blood lacunae surrounding the egg vesicle.

LATE STAGES IN EGG-CYLINDER DIFFERENTIATION AND THE ANLAGE OF THE MESODERM

In the rat series there are found 24 egg cylinders showing the stages of development considered in this section; certain of them are cut longitudinally and others cross-wise.

For the special consideration of egg-cylinder formation just prior to the anlage of the mesoderm, I present two egg-cylinders obtained during the latter half of the ninth day after insemination ; one of these was cut longitudinally, the other in favorable crosssection. The egg-cylinder shown in figure 29, rat No. 40, 8 days, 17 hours after insemination, seems unusually well fixed, as evidenced by its symmetrical outline, and is cut in a very favorable plane. The sections are from a series cut at right angles to the long axis of the uterine horn. The decidual crypts lodging the egg-cylinders of this stage are by this time nearly completely separated from the lumen of the uterus, and are surrounded by a well-developed decidua. Extravasated maternal blood nearly surrounds such egg-cylinders.

Fig. 29 Longitudinal, sagittal section of egg-cylinder of the albino rat showing the final mesoderm-free stage. X 200. Rat No. 40, 8 days, 17 hours, after insemination; ect.pl., ectoplacental cone or Triiger; -p.ect., parietal or transitory ectoderm; pr.emb.ect., primary embryonic ectoderm; ex.ect., extraembryonic ectoderm; pr.c, proamniotic cavity; v.cnt., visceral entoderm, absorptive for maternal hemoglobin, cells showing the three zones described by Sobotta; pr.emh.ent., primary embryonic entoderm.


/.':^.



338 G. CARL HUBER

The egg-cylinder shown in figure 29 presents a total length of 1.15 mm., a width of approximately 0.18 mm. The ectoplacental cone presents a length of 0.4 mm. and of the proamniotic cavity, 0.5 mm., of which 0.2 mm. falls to the antimesometrial portion lined by primary embryonic ectoderm. This egg-cylinder differs only in shape and size from that shown in C of figure 27, obtained 8 days after insemination. The primary embryonic and extraembryonic ectoderm lining or enclosing the proamniotic cavity are readily differentiated. The primary embryonic ectoderm, derived from the ectodermal node, constitutes a pseudostratified epithelium, composed of relatively long columnar cells, with nuclei radially placed with reference to the lumen of the proamniotic cavity, and shows active cell division, no less than 12 mitotic figures occurring in the section figured. The protoplasm of its cells stains distinctly deeper than does that of the cells of the extraembryonic ectoderm. The cells of the latter are of cubic, short columnar, or polyhedral shape, arranged in a single or double layer, with no definite arrangement of the long axes of its nuclei. It is, therefore, possible readily to distinguish — by reason of shape and size of cells, relative position of nuclei, reaction to stain of protoplasm — between the cells of the primary embryonic and extraembryonic ectoderm, and to determine the sharp line of junction at which the two types of cells form a continuous layer, a fact which will receive further consideration in deahng with the anlage of the mesoderm as observed in slightly more advanced stages. At the mesometrial end of the proamniotic cavity, the cells of the extraembryonic ectoderm become continuous with the cells at the base of the ectoplacental cone; in the region of this junction, active mitosis are often to be observed. In this egg-cylinder the visceral entoderm m.ay readily be differentiated into two portions. The portion which surrounds the primary embryonic ectoderm to nearly the region of its junction with the extraembryonic ectoderm, consists of a single layer of broad, flattened cells which assume a cubic or short columnar shape as the mesometrial border of the primary embryonic ectoderm is approached. This portion of the visceral entoderm we have designated as


DEVELOPMENT OF THE ALBINO RAT 339

the primary embryonic entoderm. The portion of the visceral entoderm surrounding the sides of the egg-cyhnder in the region of the extraembryonic ectoderm, to near the base of the ectoplacental cone, consists of a single layer of columnar cells, regularly arranged and presenting the three zones described by Sobotta. In this stage of egg-cylinder development of the albino rat, the absorption of hemoglobin granules derived from maternal blood cells, first shown for the mouse by Sobotta and Kolster, may be readily made out. In preparations stained in hematoxylin and Congo red, in and on the outer zone of the visceral entodermal cells there may be observed granules staining deeply in the Congo red, presenting the color reaction of hemoglobin. In the middle zone of these cells the protoplasm is distinctly vacuolated, while the inner zone, containing the nuclei, presents a denser protoplasm. The transitory or parietal ectoderm consists of a homogeneous membrane, closely adherent to the maternal decidua, especially along the sides of the egg-cylinder. This layer presents scattered nucleated protoplasmic masses of spindle or dome shape on its inner surface, the relations and distribution of which may be clearly seen in the figure. Attention needs yet be drawn to the ectoplacental cone of the egg-cylinder. Its relation to the maternal decidua is very intimate, so that in places, owing to blood extravasations, it is difficult to differentiate between embryonic and maternal tissue. Many of the cells of the ectoplacental cone present a vacuolated protoplasm, the vacuoles enclosing maternal blood cells. Therefore, they are distinctly phagocytic. Sobotta has also observed and described this for the mouse. Referring to a slightly older stage after the anlage of the mesoderm, his own words read as follows:

Weiterhin sehen wir im Stadium der Fig. 5 auch eine starke Verliingerung und Vergrosserung des Ectoplacentarconus, an dem im mesometralen Telle jetzt Vacuolen auftreten, die in spateren Stadien regelmassig gefunden werden und zwar erfiillt mit miitterlichen Blutextravasaten. Die Ehrnahrung des Embryo mit miitterlichem Hamoglobin * * * * ist jetzt im vollen Gang.

Absorption of maternal hemoglobin by the cells of the ectoplacental cone appears to be established at a relatively earlier period in the rat than in the mouse.


340 G. CARL HUBER

The egg-cylinder presented in figure 29 constitutes the final mesoderm-free stage, the final stage in which no distinct bilaterality may be determined. I assume that the egg-cylinder presented in the figure is cut in the sagittal plane. This assumption is based on the fact that the primary embryonic ectoderm extends slightly farther toward the mesometrial pole on the one side than on the other. In good frontal sections one side of the egg-cylinder in this stage of development should present a mirror picture of the other side. The side on which the primary embryonic ectoderm extends farther toward the mesometrial pole, the left in the figure, is regarded as containing the caudal end of the future embryo. In the primary embryonic ectoderm of this region, it is believed, will develop the primitive streak and groove, and thus the anlage of the mesoderm. Not in all the egg-cylinders of this stage of development found in my series can the caudal end of the future embryonic area be postulated prior to the anlage of the mesoderm, and in cross-sections no such differentiation can be made. The proamniotic cavity of the egg-cylinder shown in figure 29 presents a regular and nearly smooth contour, not divisible into regions such as described for a similar stage for the mouse by Selenka, Melissinos, and Sobotta. A very slight constriction is to be observed only in the region where the primary embryonic and extraembryonic ectoderm are joined in a continuous layer. I am thus wholly in accord with Widakowich, who in describing a similar stage in one of his preparations, states: "Das war die einzige konstante, bald starker, bald schwacher ausgepragte . Einschnurung der Proamnionhohle," as previously quoted.

A series of figures of critical regions taken from a series of cross-sections of an egg-cylinder of a stage nearly identical with that shown in figure 29, though of a slightly smaller egg-cylinder, is given in figure 30, rat No. 42, 8 days, 16 hours, after insemination. The sections chosen for the several drawings, A to D, are from the following regions, as may be ascertained by comparison with figure 29; A, through about the middle of the ectoplacental cone; B, through the proamniotic cavity just below its mesometrial end; C, through the proamniotic cavity just above the


DEVELOPMENT OF THE ALBINO RAT


341


region of the junction of the primary embryonic and extraembryonic ectoderm; D, a Uttle above the middle of the antimesometrial portion of the proamniotic cavity. The levels of the



Fig. SOl Four figures from a series of cross sections of an egg-cylinder of the albino rat in the stage of development shown in figure 29. X 200. Rat No. 42, 8 days, 16 hours after insemination.

The levels at which the several sections drawn were taken is approximately indicated by the several crosses found to the left of figure 29. A, middle of ectoplacental cone; B, ectoplacental end of the proamniotic cavity; C, just above level of junction of the primary embryonic and extraembryonic ectoderm; a little above the middle of primary embryonic ectoderm. The want of any definite bilateral symmetry of albino rat egg-cylinders of this stage of development is shown by this series of sections; p.ect., parietal or transitory ectoderm; ex.ect., extraembryonic ectoderm, surrounding mesometrial portion of proamniotic cavity; 2«-.em6.eei., primary embryonic ectoderm; ji.en^., visceral entoderm; p.enib. ent., primary embryonic entoderm; pr.c, proamniotic cavity.


342 G. CARL HUBER

several sections drawn in figure 30 is approximately indicated by the several crosses found to the left of the egg cylinder drawn in figure 29.

In A of figure 30, there may be observed a vacuolization of the protoplasm of the more peripherally placed cells of the ectoplacental cone, the vacuoles enclosing maternal blood cells. The more centrally placed cells of this ectoplacental cone show a tendency to concentric arrangement. Figures B and C present structural appearances nearly identical. The egg-cylinder is bounded by the thin layer of parietal or transitory ectoderm having scattered masses of nucleated protoplasm on its inner surface. This membrane of apparently homogeneous structure stains sharply in well fixed preparations and may be readily discerned. The cells of the visceral entoderm, somewhat taller in the section taken nearer the antimesometrial pole (C), present clearly the three zones to which attention has been drawn. The cells of the extraembryonic ectoderm bounding the mesometrial portion of the proamniotic cavity, are of cubic, short columnar, or polyhedral form disposed in single or double laj^er, presenting relatively lightly staining protoplasm. In D of figure 30, the cells forming the primary embryonic ectoderm are of di.stinct columnar shape, with relatively deeply staining protoplasm and nuclei arranged nearly in a single layer except for such as show mitotic phases. The cells of the primary embryonic entoderm are of a broad, pavement type for a greater part of the circumference, and may be contrasted with the cells of the visceral entoderm shown in B and C of the figure; the latter are absorptive cells, the former not. This series of figures, more especially B, C, and D, show clearly the absence of bilaterality in the egg-cylinders of the albino rat at this stage of development. The slight compression observed in this egg-cylinder, as shown in the figures, I regard as not of moment.

Fig. 31 Longitudinal sagittal section of egg-cylinder of the alVjino rat showing anhige of the mesoderm. X 200. Rat No. 34, 8 days, 18 hours, after insemination; ect.pl., ectophicental cone or Triiger; ji.ect., parietal or transitory ectoderm; pr.emb.ect., primary embryonic ectoderm; e.r:.ect., extraembryonic ectoderm; pr.emb. e7it., primary embryonic entoderm; mes., mesoderm in anlage; pr.c, proamniotic cavity; v.ent., visceral entoderm.



343


lOCRN'AI. OF MORPHOI.OGV, VOL. 26, XO. 2


344 G. CARL HUBER

Grosser has figured in his figures 68 and 114, an egg-cyhnder of the albino rat which measures nearly 2 mm. in length. The age of this is given as 8| days. So far as may be determined from his figures, the preparation is not described in his text, the age, size, form, and structure of the egg cylinder shown in figure 29 and Grosser's figures 68 and 114, are very similar. In Grosser's figures, I see no evidence of his having differentiated between primary embryonic and extraembryonic ectoderm, while the reference letters for ectoderm and entoderm are reversed. Selenka's figure 31, plate 45, may be of a similar stage. This figure is, however, too diagrammatic to admit of close study. No difference is shown in the shape and structure of the cells bounding the two parts of the proamniotic cavity. Christiani's figure 39 may be of the same stage, but is too schematically drawn. Figure 4 of the article of Widakowich is of a slightly older stage and presents only a part of the egg-cylinder; it is recorded as about 6f days old. The stage under consideration is not figured by Widakowich, although his text description corresponds closely with what has been here presented.

The next stage and the one with which this communication is to be completed is one of importance since it is characterized by the anlage of the mesoderm. My own observations may be introduced with the consideration of an egg-cylinder, a section of which is presented in figure 31, rat No. 34, 8 days, 17 hours, after insemination. This was cut in the sagittal plane and measures 1.1 mm. by 0.2 mm., of which 0.4 mm. fall to the ectoplacental cone. This egg-cylinder is almost an exact duplicate, both in size and form, of that figured in figure 29 of the same age. In the egg-cylinder shown in figure 31, however, there may be observed, to one side, in the region of the junction of the primary embryonic and extraembryonic ectoderm, and between primary embryonic ectoderm and entoderm, a small group of cells which lie in close relation to the ectoderm and constitute early mesodermal cells. The sections of this series pass not exactly parallel to the mid- sagittal plane throughout the whole extent of the egg-cylinder; especially is this true of its antimesometrial portion, in the region of the primary embryonic


DEVELOPMENT OF THE ALBINO RAT 345

ectoderm. This portion in the section figured, passes a little to one side of the niid-sagittal plane. The two sections preceding the one figured enclose the mid-sagittal plane, and in them, the group of cells found between primary embryonic ectoderm and entoderm are in closer relation to the ectodermal layer and at all points distinctly separated from the entoderm. They are regarded as having wandered from the primary embryonic ectoderm to the place they occupy, a fact which is more easily ascertained in cross sections of a similar stage, as will appear from further discussion. From a study of very slightly older stages it can be determined that this region constitutes the primitive streak region of the future embryonic area. It is not my purpose at this time and in this communication to give especial consideration to the much discussed question of the origin of the mesoderm in Mammalia. In the rat, this question is complicated by the question of the anlage of the amniotic fold, which separates the proamniotic cavity into amniotic cavity proper and the ectoplacental cavity, the development of which will be considered in a projected contribution. In anticipation of this second publication, however, the following facts may here receive consideration. Widakowich presents in his figure 4, giving only the antimesometrial end of an eggcylinder obtained the latter part of the 7th day, the anlage of the mesoderm as observed by him. This figure and my own figure 31 present almost identical relations, his figure showing only three mesodermal cells between primary embryonic ectoderm and entoderm. His own words concerning the anlage of the mesoderm in the albino rat, with which I find myself in full accord, except as to the age of the egg-cylinder, read as follows:

Das erste auftreten des Mesoderms beobachtete ich an Keimen vom Ende des 7 Tages. Die ersten Mesodermzellen liegen im Bereiche der vom mesometralen Ende des starker farbbaren primaren embryonalen Ectoderm gebildeten Falte. Es kommt hier eine ganz bestimmte Stelle in Betracht, die dort liegt, wo sich spater das hintere Ende des Primitivstreifens befindet.

There is, however, wide divergence of the views of authors as concerns the anlage of the mesoderm in the rat and mouse.


346 G. CARL HUBER

Selenka, it would seem, in part at least, interpreted correctly the development of the mesoderm in the rat, although a stage showing its anlage was not observed. Duval believes that the mesoderm has origin from a thickened part of the entoderm, probably in the region of the anterior portion of the future embryonic area; the primitive streak was not recognized. Christiani's figures 45 and 47, transverse sections of the egg-cylinder from the eighth day, give correctly the relative position of the mesoderm with reference to the primitive streak; however, they show stages some little time after the anlage of the mesoderm. According to Robinson, in the early part of the eighth day the cavities of the epiblast (primary embryonic ectoderm) and of the trophoblast (extraembryonic ectoderm) meet and fuse to form a hollow cylinder, the proamniotic cavity. He states that "For a time the united cavities of the epiblast and trophoblast increase in size, together with the general growth of the ovum, and this increase continues until in the latter part of the eighth day the mesoblast appears around the margin of the epiblast where it is in apposition with the trophoblast." Robinson was able to differentiate between the primary embryonic ectoderm (epiblast) and the extraembryonic ectoderm (trophoblast) and his figure 14 (plate 23-24), though schematic, shows that he recognized the positions of the anlage of the mesoderm correctly, as also its derivation from the primary embryonic ectoderm. The observations of Melissinos, bearing on the anlage of the mesoderm have been critically reviewed by both Widakowich and Sobotta, and I am wholly in accord with their views when they state that no credence can be given these observations since it is clear that Melissinos has confused sagittal and frontal sections in such a way as to make his observations of no value. According to Melissinos, the mesoderm arises from the outer surface of the middle fold of the egg cylinder, in the region of its union with the antimesometrial ectodermal fold; it is certain that it does not arise from the part of the eggcylinder that has differentiated from the primary embryonic ectoderm; but, if I interpret him correctly, from the extraembryonic portion of the ectoderm. That Melissinos did not


DEVELOPMENT OF THE ALBINO RAT 347

have before him the stages showing the anlage of the mesoderm seems clear. Sobotta's ('11) observations, mouse material, deserve fuller consideration. In interpreting his results, I am mindful of the fact that he was unable to locate the line of union between primary embryonic and extraembryonic ectoderm, as can readily be done in suitable rat material, as has previously been shown by Robinson and Widakowich, and to which attention has constantly been drawn in this communication. I am unable to state from personal observation whether in the white mouse these two types of ectoderm which form the lining of the proamniotic cavity, can be differentiated on ascertaining the right technical method. Sobotta's material seems well fixed. If not, it would seem to me difficult to determine definitely the exact place of origin of the mesodermal cells, whether extraembryonic or embryonic. Sobotta recognized the anlage of the mesoderm in the mouse during the last hours of the seventh day or first hours of the eighth day. This is said to appear at the caudal end of the future embryo as a group of loosely arranged cells lying between the inner and outer layers of the egg-cylinder. At the place where the mesodermal cells arise from the inner layer of the egg-cylinder, there is developed a fold, recognized as the caudal amniotic fold ("Schwanzfalte des Amnios"). After discussing these observations at length, Sobotta concludes as follows:

Was die Deutung dieser frlihen Stadien der Mesodermbildung in der Keimblase der Maus aniangt, so handelt es sich hier nicht um die Bildung des embryonalen Mesoderms, die erst mit der eigentlichen Gastrulation spiiter einsetzt, sondern um Entstehung ausserembryonalen Mesoderms, l^esonder des Teils des mittleren Keimblattes, dass bei der Bildung der primaren Eihaute, Amnios und Chorion in Betracht kommt und des den ausserembryonalen Teil der Leibeshohle, das Exocoelom auskliedet, der Hohle, die eben Amnios und Chorion voneinander trennt. Es erfolgt also, um einen kurzen Ausdruck zu gebrauchen, die Bildung des Amniosmesoderms.

An embryonic anlage is said not to exist at this stage; this is recognized only after the development of the primitive streak. It is not my purpose to enter fully into a discussion of this important question in this communication. This would involve


348 G. CARL HUBER

consideration of older stages, and the making of a number of reconstructions, which it is not contemplated to consider now. It must suffice to state at this time that in the albino rat, as shown by Widakowich and here shown by me, it is possible to delineate clearly the primary embryonic ectoderm and to show that the first evidence of the mesoderm is found antimesometrial to the future amniotic fold and in the region of the future primitive streak; therefore is mesoderm which I would regard as peristomal mesoderm in the sense of C. Rabl, reference to which is made by Sobotta in his discussion of this question. It may be that the rat offers more suitable material for the elucidation of this question than is to be found in the mouse. In the albino rat, the anlage of the mesoderm is from the sagittal portion of the caudal region of the primary embryonic ectoderm, the caudal part of the future primitive streak and antimesometrial to the amniotic fold. Sobotta gives very favorable consideration to the observations of Widakowich, touching this question, which he regards as Bei weitem die beste Darstellung des Gegenstandes." My own observations fully confirm those of Widakowich. These questions will receive fuller consideration in a later publication dealing with the embryology of the albino rat, carrying the development from the time of the anlage of the amniotic fold to the stage of embryo form, the material for which is at hand.

In figure 32 are shown cross-sections of the antimesometrial portion of three egg-cylinders in the region of the developing mesoderm. Sections drawn in A and B, were taken respectively from egg-cylinders obtained from the same uterus as was the one shown in sagittal section in figure 31, rat No. 34, 8 days, 17 hours, after insemination; C, from rat No. 41, 8 days, 16 hours, after insemination. It is very probable that the series from which A of this figure was drawn, is not cut in exactly the cross plane. A study of the series shows, however, that the deviation from this plane is not marked. The sections from which this figure was drawn pass a little below (antimesometrial) to the region of junction of the primary embryonic and extraembryonic ectoderm. To one side, the lower in the figure, the primary embryonic ectoderm shows a slight thickening and evidence of


DEVELOPMENT OF THE ALBINO RAT


349



pr.o-nb.ent-V-f^



Fig. 32 Three cross sections from egg-cylinders of the albino rat, showing early stages in the development of the mesoderm. X 200. A and B, rat No. 34, 8 days, 17 hours; C, i"at No. 41, 8 days, 16 hours, after insemination.

These sections taken from three egg-cylinders are through the primary embryonic ectoderm, near its junction with the extraembryonic ectoderm, thus through the antimesometrial portion of the proamniotic cavity. A, early stage, in anlage of the mesoderm; B, anlage of the primitive streak and groove; C, well developed primitive streak and groove, with lateral wings of mesoderm; pr.e/?i6. ect., primary embryonic ectoderm; pr.emb.ent., primary embryonic entoderm; ines., mesoderm; pr.str., primitive streak; pr.gr., primitive groove; p. ect., parietal or transitory ectoderm; pr.c, proanmiotic cavity.


cell proliferation. The cells of this region have not the form of tall columnar cells, such as seen in the greater part of the remaining primary embryonic ectoderm, but are of polyhedral


350 G. CARL HUBER

form and are continuous, in the mid sagittal plane, with cells that have wandered between the primary embryonic ectoderm and entoderm, cells regarded as constituting the mesoderm. In all of the sections of this series, so far as the mesoderm extends, this is distinctly separable from the entoderm, and is continuous with the primary embryonic ectoderm only along a narrow region of thickened primary embryonic ectoderm, situated in the mid-sagittal plane, and which may in this series be regarded as the anlage of the primitive streak. From the sides of this region of slightly thickened primary embryonic ectoderm, the extent of which is evidenced by the absence of an external limiting membrane, cells wander laterally to form the mesoderm. B, of figure 32, presents essentially the same appearance, although representing a slightly older stage. The sections of this series I regard as cut fairly well in a plane at right angles to the long axis of the respective egg-cylinder. The section taken for the sketch is situated a very little further away from the line of junction of the primary embryonic and extraembryonic ectoderm, than is the section the drawing of which is shown in A of this figure, as may be judged from the more uniformly pavement type of the entodermal cells. The triangular form of the proamniotic cavity is regarded as normal, and as indicating an early stage in the anlage of the primitive groove. In this figure, in its lower portion, the region of the primitive streak is readily discernible by reason of the fact that there is wanting here an external limiting membrane, and further by reason of the form of the cells and the form and relative position of their nuclei; certain of these cells indicating, both by their form and their position, the source and the direction of the wandering of the cells which constitute the anlage of the mesoderm. The wandering of the mesodermal cells between the primary embryonic ectoderm and entoderm, to form the lateral mesodermal wings, is clearly shown in this figure, especially to the left. The antimesometrial ends of the egg-cylinders, sections of which are shown in A and B of this figure, are as yet free from the invading mesoderm, as is also the part of the egg-cylinders lying opposite the region of the primitive streak, the upper portions of the


DEVELOPMENT OF THE ALBINO RAT 351

respective figures, these forming the region of the future anterior ends of the respective embryos. In C of figure 32 is shown a drawing of one of the sections of a series of cross-sections of an egg-cyhnder taken from rat No. 41, 8 days, 16 hours, after insemination, presenting a stage in which the primitive groove may be definitely made out. This figure is not unhke figure 6 of the article of Widakowich, obtained from an egg-cyhnder secured on the eighth day. Concerning this figure he states: Das Ectoderm steht in direktem Zusammenhange mit zwei Mesodermzungen die gegen die der Primitivrinne gegeniiberliegende Seite zu auswachsen." The section drawn in C of this figure is taken from the region very near the junction of the primary embryonic and the extraembryonic ectoderm, as may be observed from the character of the entodermal cehs, in the lower part of the figure. The increase in the thickness of the mesodermal wings, the result, in part at least, of proliferation of mesodermal cells, as evidenced by the presence of mitotic figures, is clearly shown in this figure. The mesoderm is distinctly separable from the entoderm as also from the primary embryonic ectoderm except in the region of the primitive streak and groove. The growth of the mesoderm after its anlage has been correctly shown for the albino rat by Selenka, Robinson, and Widakowich; the latter especially giving excellent figures. His figure 5 is especially instructive. In this, he represents the appearances shown by two views of an isolated egg-cylinder, with the primitive groove in anlage, showing the lateral extensions of the mesoderm. Sobotta ('11) has given the best and most comprehensive account of the anlage and growth of the mesoderm in the mouse. An excellent cross-section of a mouse eggcylinder in the primitive streak stage is presented in his figure 6, which presents very similar appearances to my C of figure 32. None of the figures of cross-sections of egg-cylinders included by me show the very beginning of the anlage of the mesoderm, though A of figure 32 approaches this very closely, as does also figure 31, presenting a sagittal section. The evidence at hand warrants the conclusion that in the albino rat, the mesoderm has its anlage in the caudal region of the primary embryonic


352 G. CARL HUBER

ectoderm, from a narrow zone of cells situated in the region of the future primitive streak. From this region there is an outwandering of cells which invade the potential cleft between primary embryonic ectoderm and entoderm, spreading laterally in wing-like sheets. This I would regard as prostomial mesoderm in the sense of C. Rabl. The anlage of the mesoderm in the albino rat, and the early stages of its lateral extension, with the anlage of the primitive streak and groove, falls to the latter part of the ninth day after insemination.

Beginning with the pronuclear stage, found at the end of the first day, 8 days are required for the completion of the process of segmentation, blastodermic vesicle formation and the formation of the primary germ layers — ectoderm, mesoderm, and entoderm — in all, 9 days out of a possible 21 to 23 days, the normal gestation period of the albino rat.

CONCLUSIONS

Early stages of mammalian development may readily be obtained from the albino rat (Mus norvegicus albinus). When care is exercised, mating may be observed and the age of the embryo, reckoned from the time of mating (insemination), determined with a fair degree of accuracy. Ovulations occur about the time of parturition and again 29 to 30 days post partem. This latter period is more favorable for obtaining insemination and semination, thus fertilized ova. The process of fertilization probably takes place during the latter half of the first day after insemination.

The pronuclear stage, a stage which extends through a period of perhaps 12 to 15 hours, in the middle phase, is observed at the end of the first day after insemination; the fertilized ova having wandered about one-fourth of the length of the oviduct by that time. Of the two pronuclei, the female pronucleus is slightly the larger. The two pronuclei lie near the center of the ovum, are distinctly membraned, and do not fuse prior to the formation of the first segmentation spindle.

The formation of the first segmentation spindle and the first segmentation occur during the early part of the second day after


DEVELOPMENT OF THE ALBINO RAT 353

insemination. The resulting 2-cell stage extends for a period of about 24 hours and is found in about the middle of the oviduct. The first two blastomeres are equivalent cells. One of these segments before the other, resulting in a 3-cell stage, present for each ovum for only a relatively short period.

The 4-cell stage is observed at the end of the third day after insemination. The ova have by this time traversed about ninetenths of the length of the oviduct.

The 8-cell stage is observed the latter half of the fourth day after insemination and at the end of the fourth day the ova pass from the oviduct to the uterus in the 12-cell to 16-cell stage. The oolemma is lost usually in the 4-cell stage, the segmenting ova conforming in shape to the general form of that portion of the oviduct in which they are found.

Three successive segmentation stages, spaced at intervals of about 18 hours, resulting in 2-, 4-, and 8-cell stages occur during transit through the oviduct. During the fourth segmentation the ova pass from the oviducts to the uterine horns, at the end of the fourth day.

The mass increase of the ova during the first three segmentations is approximately from 0.15 c.mm. in the pronuclear stage to 0.18 c.mm. in the 8-cell stage. The slow rate of segmentation and the relatively small mass increase may be attributed to the relative scarcity of the embryotroph during transit through the oviducts.

During the early hours of the fifth day after insemination, all of the segmenting ova are found lying free in the lumen of the uterus, spaced about as in the later stages of development, the fifth series of segmentations having been completed by this time, the resulting morula masses having ovoid form, measuring approximately 80 ix by 50 m and consisting of from 24 to 32 cells. The mechanism operative in spacing the ova in the uterine horns has not been determined.

The early stages of blastodermic vesicle formation are observed during the middle and latter half of the fifth day. The segmentation cavity begins as a single, irregularly crescentic space, eccentric in position, and arising between the cells of the morula.


354 G. CARL HUBER

By the end of the fifth day after insemination, all fertilized, normal ova are found in the blastodermic vesicle stage. One pole of each vesicle, its floor, consists of a relatively thick mass of cells, in which there is no differentiation in layers and no evidence of ectodermal and entodermal cells. The other pole of each vesicle, its roof, consists of a single layer of flattened cells, bordering the segmentation cavity.

During the sixth day, the blastodermic vesicles which still lie free in the lumen of the uterus, increase in size, partly as a result of extension of the roof cells, partly owing to rearrangement and flattening of the cells of the floor. This portion of the vesicle now presents the form of a concavo-convex disc, forming about one-sixth of the vesicle wall and consisting, as a rule, of three layers of cells, the inner of which is now differentiated to form the yolk entoderm.

During the seventh day after insemination the blastodermic vesicles become definitely oriented in a decidual crypt, the thicker portion, its floor, being directed toward the mesometrial border. The phenomenon of the inversion of the germ layers" or "entypy of the germ layers" is initiated, the result of cell rearrangement and cell enlargement in the germinal disc, manifested as an outgrowth to form the ectoplacental cone or Trager and an ingrowth into the vesicle, the anlage of the egg-plug or egg-cylinder. In the egg-plug there is recognized a circumscribed, compact mass of cells, staining more deeply than surrounding cells, which constitute the ectodermal node, the anlage of the primary embryonic ectoderm of the future embryo. This ectodermal node, so far as it extends into the cavity of the blastodermic vesicle, is surrounded by yolk entoderm.

During the eighth day after insemination, the egg-cylinder comes in definite relation with the maternal decidua and receives as embryotroph maternal hemoglobin, partly through phagocytic action of the cells of the ectoplacental cone, partly through absorption of maternal hemoglobin by the cells of the entoderm, initiating a period of very active growth as evidenced by active mitosis. The egg-cylinder increases in length, and entypy is completed. A cavity develops in the ectodermal


DEVELOPMENT OF THE ALBINO RAT 355

node, the antimesometrial portion of the proamniotic cavity. A little later a second cavity develops in the extraembryonic ectoderm, the mesometrial portion of the proamniotic cavity, the two cavities fusing by the end of the eighth day to form a single proamniotic cavity, lined in its antimesometrial portion by primary embryonic ectoderm, and in its mesometrial portion by extraembryonic ectoderm, the two types of ectoderm forming a continuous layer with the line of junction readily distinguishable. No evidence of bilateral symmetry is at this stage observed in the egg-cylinder.

During the ninth day after insemination there is observed the anlage and the early developmental stage of the mesoderm and the anlage of the primitive streak and groove. The mesoderm has its anlage in the caudal portion of the primary embryonic ectoderm in the sagittal region and is of the nature of prostomial mesoderm, extending laterally in wing-like extensions between the ectoderm and entoderm.


356 G. CARL HUBER

LITERATURE CITED

AssHETON, R. 1895 A re-investigation into the early stages of the development of the rabbit. Quart. Jr. Mic. Sc, vol. 37, N. S.

1899 a The development of the pig during the first ten days. Quart.

Jr. Mic. Sc, vol. 41, N. S.

1899 b The segmentation of the ovum of the sheep, with observations

on the hypothesis of a hypoblastic origin for the trophoblast. Quart.

Jr. Mic. Sc, vol. 41, N. S. BiscHOFF, Th. L. W. 1845 Entwicklungsgeschichte des Hundeeis. Braunschweig.

1852 Entwickelungsgeschichte des Meerschweinchens. Giessen. BuRCKHARD, G. 1901 Die Implantation des Eies der Maus in die Uterus schleimhaut und die Umbildung deselben zur Decidua. Arch. f.

mikr. Anat., Bd. 57. Christiani, H. 1892 L'inversion des feuillets blastodermiques chez le rat

albinos. Arch, de Phys. norm, et pathol., vol. 24 (S. 5, T. 4). CoE, W. R. 1908 The maturation of the egg of the rat. Science, N. S., vol 27. Daniel, J. F. 1910 Observations on the period of gestation in white mice.

Jour. Exper. Zool., vol. 9. d'Erchia, F. 1901 Ueber die Einbettung des Eies und die Entwicklung und

den Bau der AUantois-und Dottersackplacenta bei der weissen Maus.

Zeitsch. f. Geburtshilfe und Gynaecologie, Bd. 44. Donaldson, H. H. 1912 The history and zoological position of the albino

rat. Jour. Acad. Nat. Sc, Philadelphia, vol. 15, second series. DuESBERG, J. 1908 La spermatogenese chez le rat. Arch. f. Zellforschung,

Bd. 2. Duval, M. 1891 Le placenta des rongeurs (Suite I). Troisieme partie.

Jour, de I'anat. et de la phys. Fraser, a. 1883 On the inversion of the blastodermic layers in the rat and

mouse. Proceed, of the Royal Society, vol. 34. Grosser, O. 1909 Vergleichende Anatomie und Entwicklungsgeschichte der

Eihaute und der Placenta mit besonderer Berlicksichtigung des Men schen. Wien und Leipzig. Heape, W. 1886 The development of the mole (Talpa europea), the ovarian

ovum, and the segmentation of the ovum. Quart. Jr. Mic Sc, vol.

26, N. S. VON Hensen, v. 1876 Beobachtung iiber die Befruchtung und Entwicke lung des Kaninchens und Meerschweinchens. Zeitsch. f. Anat. und

Entwick., Bd. 1. Hertwig, O. 1906 Die Lehre von den Keimblattern. In O. Hertwig's Hand buch der vergleichenden und experimentellen Entwickelungslehre

der Wirbeltiere. Bd. 1, Fischer, Jena. Hertwig, R. 1906 Der Furchungsprozess. In O. Hertwig's Handbuch der

vergleichenden und experimentellen Entwickelungslehre der Wirbeltiere. Bd. 1, Fischer, Jena.


DEVELOPMENT OF THE ALBINO RAT 357

HuBRECHT, A. A. W. 1895 Die Phylogenese des Amnios unci die Bedeutung

des Trophoblast. Verh. Kon. Akad. Wetensch. Amsterdam, Ser. 2

(quoted from O. Hertwig). Jexkinson, J. W. 1900 A reinvestigation of the early stages of the development of the mouse. Quart. Jr. Mic. Sc, vol. 43, N. S. Keibel, F. 1888 Zur Entwickelungsgeschichte des Igels. (Erinaceus euro paeus). Anat. Anz., Bd. 3. King, H. D. 1913 Some anomalies in the gestation of the albino rat (Mus

norvegicus albinus). Biol. Bull., vol. 24. KiRKHAM, W. B., and Burr, H. S. 1913 The breeding habits, maturation

of the eggs and ovulation of the albino rat. Am. Jour. Anat., vol. 15. KoLSTER, R. 1903 Zur Kenntnis der Embryotrophe beim Vordhandensein

einer Decidual Capsularis. Anat. Hefte, vol. 22. KuPFFER, C. 1882 Das Ei von Arvicola arvalis und die vermeintliche Um kehr der Keimblatter an demselben. Sitz. Ber. d. K. b. Akad. d.

Wiss., II CI, Bd. 5. Lee, T. G. 1903 Implantation of the ovum in Spermophilus tridecemlineatus,

Mitch. Mark Anniversary Volume. Henry Holt and Co., New York. Long, J. A., and Mark, E. L. 1911 The maturation of the egg of the mouse.

Carnegie Institute of Washington, Publication No. 142. Long, J. A. 1912 Studies on early stages of development in rats and mice,

No. 3, by Mark and Long. The living eggs of rats and mice with a

description of apparatus for obtaining and observing them. L'niver sity of California Publications in Zoology, vol. 9. INIandl. 1908 tjber das Epithel im geschlechtsreifen Uterus. Zentralbl. f.

Gynakologie. Melissinos, K. 1907 Die Entwicklung des Eies der Mjiuse (Mus musculus

var. alba u. Mus rattus albus) von der ersten Furchungs-phiinom enen bis zur Festsetzung der Allantois an der Ectoplacentarplatte.

Arch. f. mikr. Anat., Bd. 70. PuJiULA, D. 1909 Die Frage der Riesenzellen bci der Entwicklung der Maus.

Actos y memorias Primer Congreso de Naturalistas Espanolas. Zara goza (quoted from Sobotta, 1911). Reichert, C. B. 1861 Beitrage zur Entwickelungsgeschichte des Meer schweinchens. Abhandl. d. K. Akad. d. Wissensch. Berlin, Bd. 182. Robinson, A. 1892 Observations upon the development of the segmentation

cavity, the archenteron, the germinal layers, and the amnion in mammals. Quart. Jr. Mic. Sc, vol. 33, N. S. Selenka, E. 1883 Studien iiber die Entwickelungsgeschichte der Thiere.

I Heft, Keimblatter und Primitivorgane der Maus. Wiesbaden.

1884 Studien iiber Entwickelungsgeschichte der Thiere. 3 Heft, Die

Blatterumkehrung im Ei der Nagethiere. Wiesbaden.

1901 Die Placentaranlage des Lutung. Sitzber. d. path. phys. Classe

d. Kgl. Bair. Akad. d. Wissensch., Heft I. SoBOTTA, J. 1895 Die Befruchtung und Furchung des Eies der Maus. Arch.

f. mik. Anat., Bd. 45.

1903 Die Entwicklung des Eies der Maus vom Schlusse der Furchung speriode bis zum Auftreten der Amniosfalten. Arch. f. mik. Anat.,

Bd. 61.


358 G. CARL HUBER

SoBOTTA, J. 1908 Weitere Mitteilung fiber die Entwickelung des Eies der

Maus. Verhandl. der Anat. Gesellschaft.

1911 Die Entwicklung des Eies der Maus vom ersten Auftreten des

Mesoderms an bis zur Ausbildung der Embryonalanlage und dem

Auftreten des Allantois. I Teil: Die Keimblase. Arch. f. mik.

Anat., Bd. 78. SoBOTTA, J., and Burckhard, G. 1911 Reifung und Befruchtung des Eies

der weissen Ratte. Anat. Hefte, Bd. 42. VON Spee, Graaf F. 1901 Die Implantation des Aleerschweincheneis in die

Uteruswand. Zeitschr. Morph. Anthrop., Bd. 3. Van Beneden, E. 1899 Recherches sur les premiers stades du developpe ment du Murin. (Vespertilio murinus.) Anat. Anz., Bd. 16. WiDAKOwicH, V. 1910 tJber die erste Bildung der Korperform bei Entypie

des Keimes. Beitrage zur Entwicklungsgesohichte der Ratte. Zeitsch.

f. wissensch. Zool., Bd. 94.


Huber GC. The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova: end of the first to the end of the ninth day. (1915) J Morphol. 26: 359-391.

The Development Of The Albino Rat, Mus Norvegicus Albinus II. Abnormal Ova; End Of The First To The End Of The Ninth Day

G. Carl Huber

From the Department of Anatomy, University of Michigan, and the Division of Embryology, Wistar Institute of Anatomy and Biology, Philadelphia

TEN FIGURES

CONTENTS

Introduction 359

Half embryos in Mammalia 361

Degeneration of ova at the end of segmentation 364

Incomplete or retarded segmentation 365

Abnormal segmentation cavity formation 370

Degeneration of ova as a result of pathologic mucosa 373

Imperfect development of ectodermal vesicle 376

Two egg-cylinders in one decidual crypt 382

Conclusions 384

Literature cited 386

INTRODUCTION

In the course of my study of the normal development of the albino rat, from the end of the first to the end of the ninth day after insemination, as recorded in Part I of this series of contributions, there were encountered from time to time ova which appeared to deviate both in rate and type of development from what, as a result of extended study, came to be regarded as the normal developmental cycle of the albino rat. When taken collectively, the number of these abnormal ova is not large, although they embrace nearly all of the developmental stages studied. When taken singly, it may be stated that while it is comparatively easy to record the points of deviation from the normal, it must be admitted that the probable fate of the respective stages can only be conjectured. Nevertheless, a record of

359

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


360 G. CARL HUBER

the abnormal stages met with seems warranted, especially in view of the fact that the literature is very meager in its account of early stages of mammalian ova presenting abnormal development.

The excellent and comprehensive studies of Mall on pathologic human ova, extending over many years, may be interprete as leading to the general conclusion that pathologic ova and monsters "^are produced from normal eggs by conditions which either interfere with their nutrition or poison them." There is evidence to show that defective implantation, using the term in its broadest sense so as to include relation to the embryotroph or pabulum, is directly associated with abnormal development. Comparative experimental teratology so successfully followed by a number of European and American experimental embryologists warrants the conclusion that all of the abnormalities or malformations observed in the human embryo may be brought forth by the application of suitable mechanical interference or chemical solutions. Experimental teratology possesses the very great advantage of enabling the observer to follow the pathologic process from step to step, admitting more readily of their interpretation, than when single stages are obtained from nature. The evidence appears to be accumulating that the primary causes which produce pathologic ova lie not in the germ cells, but are rather to be sought in the environs of the germ cells in the course of their development.

I am cognizant of the fact that the interpretation of the chance findings of abnormal stages of mammalian ova is much more difFcult than of abnormal ova produced experimentally. The fact, however, that nearly all of the abnormal ova observed by me in my albino rat material were found in tubes and uteri containing normal ova also, tubes and uteri which so far as observable appear in most instances to be normal, and the further fact 1 hat certain of the abnormal ova are of stages prior to what may be regarded as showing implantation, stages concerning which we possess no data as far as human ova are concerned, has lead to the tentative conclusion that certain of the abnormal ova may be the resultant of abnormal germ cells, perhaps of an abnormality which may not show a structural expression.


PATHOLOGIC OVA, ALBINO RAT 361

It is my primary purpose to make records of the abnormal ova observed in the material at hand ; and to follow these records with a brief consideration of the observations made. There is no literature dealing with the problem immediately at hand — abnormal rat ova. It is not my purpose at this time to enter into the extensive literature of comparative experimental teratology. This has been critically summarized relatively recently by O. and R. Hertwig, and by ]\Iall, in his several contributions dealing with human pathologic ova.

HALF EMBRYOS IN MAMMALIA

The first preparation to which attention is called is one taken from the oviduct of rat No. 60, 1 day, 18 hours, after insemination. The two oviducts of this rat contained seven ova in the 2cell stage, to one of which especial attention was drawn in Part I (page 271). As there recorded, in one of the 2-cell stages, the first two blastomeres were separated by an appreciable distance. There is loss of oolemma. The possibility of half embryos in Mammalia was suggested. The preparation under consideration is figured in figure 1, A and B. In A of this figure there is presented a portion of the wall of the oviduct, its epithelial lining and the immediately adjacent mucosa, including the fourth of a series of six sections (10 m) passing through the two blastomeres. In this region, the cilia of the epithelium are clearly observable, as may be seen from the figure. In B of this figure there are sketched in approximately relative position the several sections of the series passing through the two blastomeres, the relative position of which, with reference to the w^alls of the tube, is shown in A of the figure. The six drawings were made from a well ribboned series; the slide was moved from section to section by means of a mechanical stage, and the perpendicular indicated on each drawing as made. The relative position of the several drawings, therefore, is quite correct. It may be observed that throughout the series the two blastomeres are separated by an appreciable space, and that one of the cells has rotated slightly on its axis. If these two blastomeres had remained in close apposition, they would present the appearance of a normal 2-cell stage


362


G. CARL HUBER




Fig 1 Oviduct and ovum of albino rat, in 2-cell stage, with first two blastomeres separated. Rat No. 60, 1 day, 18 hours, after the beginning of insemination. X 200. A, epithelial wall of oviduct with adjacent mucosa, and the fourth of a series of six sections of the 2-cell stage with separated blastomeres, showing them in their relation to the epithelium. B, the series of six sections which pass through the separated blastomeres, the fourth of which is shown in A. The series reads from right to left.

as shown in B and C of figure 1, Part I. There is here clearly a separation of the first two blastomeres and not a close approximation of two unfertilized ova. In all of the unfertilized ova met with in the oviducts in the series at my disposal, these present the second maturation spindle and oolemma and are not to be confused with the blastomeres of the 2-cell stage, either as to size or structure. Both of the blastomeres in the preparation under consideration present normal protoplasmic structure, having a finely granular protoplasm. Their nuclei, as may be seen from the figures, are of normal size and structure. They present regular form, are distinctly membranated, have large chromatoid nucleoli, and chromatin scattered in fine granules and threads. However, attention needs to be drawn to the presence of two micro-nuclei, one in each of the two blastomeres, showing in the third and fourth section of the series respectively (B, fig. 1). These micro-nuclei are nearly free from chromatin, each presenting a small chromatoid nucleolus. They are not to be regarded as cell inclusions, as perhaps representing phagocytic leucocytes. It may be conjectured that they were formed by amitotic division, by budding and constriction from the parent


PATHOLOGIC OVA, ALBINO RAT 363

nuclei, perhaps indicating altered metabolism in the two blastomeres. I am inclined to think that both of these cells would have degenerated in the course of further development; however, their fate can only be guessed and not predicted. The possibility of their developing into half embryos is suggested. Half embryos developing as a result of a separation of the first two blastomeres has not been observed in the Mammalia, and an experimental test of the question is for the present not a probability.

As a result of experimental embryology it has been clearly shown that through mechanical interference polysomatous monsters may be produced from normal ova. The first two blastomeres are totipotent, as expressed by Driesch. Driesch was able to produce polysomatous forms by mechanical separation of the first two blastomeres in sea urchin eggs; Wilson, by separating through shaking of 2- and 4-cell stages in Amphioxus; O. Hertwig, Herlitzka and Spemann, by separating the first two cells in amphibian eggs; O. Schultze and others, by use of gravity and compression; and Loeb and others by use of chemical agents. By various means, then, when suitably applied and at the right time, hemiembryos have been produced by separating or potentially separating the first two blastomeres in certain forms. 0. Hertwig states:

Bei den kleinen, mit geringen Mengen von Dotter ausgestatteten Eiern der Wirbeltiere sind spontan entstandene, das heisst, ohne experimentelle Eingriffe veranlasste Mehrfachbildungen ausserordentlich selten, bei manchen Klassen iiberhaupt noch nie beobachtet worden, dagegen sind sie relativ haufige Befunde bei manchen untersuchten Arten von Knochenfischen und Vogeln, besonders bei der Forelle und beim Hiihnchen.

So far as I am aware, the possibility of hemiembryos in Mammalia has not been shown. In the albino rat, the oolemma may be lost as early as the 2-cell stage. In forms with early loss of oolemma, the separation of the two first blastomeres does not appear to me as an impossibility. The probable fate of separated mammalian blastomeres can only be conjectured, since it is manifestly impossible, for the present, to follow them in further development.


364 G. CARL HUBER

DEGENERATION AND DEATH OF OVA AT THE END OF

THE SEGMENTATION STAGES

In figure 2, A and B, are presented drawings of typical sections of two morula masses showing complete degeneration and death. The degenerated ovum shown in A, of this figure was obtained from rat No. 52, 4 days, 15 hours, after insemination. In all, eight normal ova were found in the uterus of this rat,


^


Fig 2 Ova of the albino rat in late segmentation stages, showing death and dissolution of the constituent cells. X 200. A, rat No. 52, 4 days, 15 hours, after the beginning of insemination. B, rat No. 68, 4 days, 16 hours, after the beginning of the insemination. Tliis figure shows an imperfectly developed morula with probable retention of oolemma.

these showing late morula stages and stages of early blastodermic vesicle formation, three of which were sketched and are shown in A, B, and C of figure 20, Part I. The degenerated ovum here under consideration lies in very close proximity to the normal blastodermic vesicle shown in C of figure 20, Part I. The shallow mucosal pits harboring the two ova are in contiguity. The two contiguous pits resemble each other very much; the mucosa underlying them is in every respect the same, indicating, it would seem, that to a certain stage in development — to the end of segmentation^ — the development of the degenerated ovum proceeded normally. The degenerated egg-mass measured approximately 80 ^ by 50 Ai by 40 ju. In reaction to stains, it differs markedly from the adjacent normal vesicle. The staining is very pale; cell boundaries are indistinct or lost, and the nuclei scarcely retain any coloring matter. Scattered through the protoplasm are found small globular masses, perhaps of lipoid character. Protoplasm and nuclei present evidences of cytolysis and chromatolysis, and have the appearance presented by necrotic tissue. Had normal development supervened, both ova (the pathologic and the adjacent normal one) would in all probability have been enclosed within the same decidual crypt, a condition exceedingly rare, judging from the material at hand. Whether the very close proximity of these two ova bears causal relation to the death of one, by reason of the consequent lessening of the available pabulum or embryotroph, can only be conjectured. There is at this stage no question of faulty implantation, the ova, though presumably permanently lodged, lie free in the lumen of the uterus. Whether on the other hand, the death of this ovum was the result of some inherent nutritional deficit must also remain unanswered. However, this preparation may serve to show that ova of the albino rat, after reaching the uterine tube, and after apparently normal segmentation, may undergo death and dissolution, for reasons which are not structurally discernable.

B of figure 2, rat No. 68, 4 days, 16 hours, after insemination, is from the uterus of a rat containing four ova in early stages of blastodermic vesicle formation, three of which were sketched under D and E of figure 20, and the series of figure 21, Part I. The preparation here described lies free in the lumen of the uterus, and appears to represent an uncompleted segmentation, with cells and nuclei showing cytolysis and chromatolysis. The mass is surrounded by a thin membrane regarded as an oolemma. Normally the oolemma of the segmenting ova of the albino rat is lost in the 4-cell stage, now and again in the 2-cell stage. Whether the retention of the oolemma may be brought in causal relation to the death and dissolution of the enclosed cells is problematic. That such causal relation may exist for the ova of the albino rat, appears to me as not impossible. This degenerated egg-mass presents the only instance of the laie retention of the oolemma in the albino rat material at my disposal.

INCOMPLETE OR RETARDED SEGMENTATION

The blastodermic vesicles presented in figures 3 and 4 have been interpreted as showing incomplete or retarded division of certain of the cells of early stage morula masses. The probable fate of such blastodermic vesicles in further development cannot be projected with any degree of certainty. The most characteristic vesicle showing this phenomenon is presented in figure 3, and is taken from rat No 53, 5 days after insemination, the uterus of which contained seven blastodermic vesicles showing early stages of development, four of which are reproduced in figure 22, Part I. In A and B of figure 3 are reproduced two consecutive sections of a series of five sections of 10 /x thickness, includng this ovum. In the lower part of this ovum there is found a small segmentation cavity, bounded by cells which present normal appearances. The roof of this vesicle is slightly




Fig. 3 Early stages of the blastodermic vesicle of the albino rat, presenting evidence of irregular or retarded segmentation. X 200. Rat No. 53, 5 daj's after the beginning of insemination.

Fig. 4 Three ova of the albino rat, showing early blastodermic vesicle stages, in each of which certain of the cells suggest irregular or retarded segmentation. X 200. A, rat No. 64, 4 days, 14 hours, after the beginning of insemination. B, rat No. 68, 4 days, 15 hours, after the beginning of insemination. C, rat No. .54, 6 days, 16 hours, after the beginning of insemination.

folded and compressed, as a consequence of which the roof wall in the sections figured is presented in part as seen in surface view. In the floor of this vesicle there is to be observed, surrounded by other smaller cells, one large cell, of nearly spherical shape, having a diameter which is three or four times as great as that of the majority of the surrounding cells. The protoplasm of this large cell stains less deeply than does that of the majority of the other cells constituting the floor of the vesicle. Its nucleus is relatively large and slightly lobulated, so much so that in the section of it shown in A of this figure, in the optical section sketched, the nucleus appears as three separate nuclei, in reality,


PATHOLOGIC OVA, ALBINO RAT 367

lobules of the same nucleus. In A of this figure there is shown to the lower left of the large cell another relatively large cell, enclosing a globular inclusion, which stained faintly, and the nature of which was not fully determined. In the upper part of each of the two figures are seen cells which show cytolysis and loss of nuclei; regarded as degenerating cells. When compared with the normal blastodermic vesicles obtained from the same uterus, the ovum here described presents a unique appearance, and was readily recognized as showing development and structure which deviated from the normal. At this stage of development, the blastodermic vesicles of the albino rat are still found lying free in the lumen of the uterus, showing no structural relation to the uterine mucosa. This vesicle has been interpreted as showing irregular or retarded segmentation. It is conjectured that one of the cells, perhaps of the 8-cell stage, did not undergo further cleavage. The large cell presents an appearance evidencing beginning stages of degeneration, and in further development, would probably have undergone dissolution. The majority of the smaller cells of the roof appear as if normal, as do also the cells of the floor, certain of the smaller cells of the floor presenting mitoses as evidence of further proliferation.

In figure 4, A, B, and C, there are presented typical sections of three ova of the albino rat showing what has been regarded as irregular segmentation. A of this figure represents an ovum taken from rat No. 64, 4 days, 14 hours, after insemination, in the uterus of which there were found five normal ova showing early stages of blastodermic vesicle formation, four of which are cut longitudinally, one in a series of cross-sections. In each of the four longitudinally cut series the floor of the respective vesicles is markedly folded, owing to fixation contractions; therefore, none were sketched as normal stages. ' In appearance, they resemble closely the vesicles sketched under C, D, and E of figure 20, Part I. In the pathologic ovum, shown in A of figure 4, there is no evidence of segmentation cavity formation. However, the ovum cannot be regarded as presenting a late morula stage such as is figured in A of figure 20, Part I, since it shows distinct departure from the normal. The marked constriction


368 G. CARL HUBER

seen to the lower left of the figure passes through the series of four 10 M sections including this ovum, and in part separates a portion composed of relatively small cells from a larger portion composed of larger cells. The rate of segmentation of certain of the cells composing the upper larger portion of this cell mass appears to have been retarded, thus retarding the development of the whole mass. This pathologic ovum rests normally in a shallow pit of the mucosa, very similar in form and structure to the shallow pit lodging the five normal vesicles found in this uterus.

The ovum shown in B of figure 4 was obtained from the uterus of rat No. 68, 4 days, 16 hours, after insemination, with four normal vesicles showing early stages of blastodermic vesicle formation. From this uterus was also taken the completely degenerated cell mass with persistent oolemma shown in B of figure 2. This vesicle on superficial observation does not appear to depart markedly from the normal appearance for this stage. In form and size it corresponds closely to the normal ova taken from this uterus. The segmentation cavity seems to have developed normally. The slight folding of the roof seen to the left of the figure is accidental, due to fixation shrinkage, and is very similar to folding of the roof to be observed in many of the normal preparations of the series. In the floor of the vesicle there may be observed three relatively large cells, partly enclosed by smaller cells of a size comparable to that of the cells forming the floor of the normal blastodermic vesicles of this stage of development. The three relatively large cells, clearly distinguished in the figure, are interpreted as showing a retarded segmentation. So far as may be determined, their protoplasm and nuclei present normal structure, the lowest of the three cells showing an early mitotic phase. I am inclined to the opinion that this ovum would have continued in development, perhaps in later stages showing distinct arrest in development. This hypothesis seems warranted on the basis of the study of a vesicle shown in C of figure 4, taken from rat No. 54, 6 days, 16 hours, after insemination. Normal stages for the albino rat, taken about the middle of the seventh day after insemination, are shown in figure


PATHOLOGIC OVA, ALBINO RAT ' 369

24, Part I. Reference to this figure may serve to show that during the early hours of the seventh day after insemination, the phenomenon of inversion or entypy of the germ layers is initiated in the albino rat. The ova are, on reaching this stage of development, enclosed within a well differentiated decidual crypt which communicates as yet freely with the lumen of the uterus. These crypts present a continuous lining of uterine epithelium; the contained ova are thus not as yet in direct relation with the maternal decidua. In the normal blastodermic vesicle of this stage, the ectoplacental cone is in anlage, and in the cell mass which extends into the cavity of the vesicle — the egg-plug or egg-cylinder — there is evident a clearly circumscribed nodule of cells, which has been designated the ectodermal node and recognized as the anlage of the primary embryonic ectoderm ; this node is in part surrounded by the yolk entoderm. In the uterus of rat No. 54, there are contained nine blastodermic vesicles, one of which is sketched in C of figure 24, Part I. Not nearly all of these vesicles are so favorably cut as that shown in this figure, the majority being cut in a plane which is oblique to the long axis of the vesicle. However, in all of them the ectoplacental cone and the ectodermal node may be determined except in the one shown in C of figure 4. This vesicle was obtained from a series of sections passing at right angles to the plane of the mesometrium. It lies free in a deep decidual crypt and passes through six sections of 10 IX thickness; thus is compressed from side to side. This vesicle is distinctly smaller than the normal ones taken from this series, especially so as concerns its cavity. An ectoplacental cone is not clearly differentiated, and it is not possible to determine an ectodermal node, nor is it clear that the yolk entoderm has differentiated. In the cell mass from which ectoplacental cone and ectodermal node should have developed, the upper portion of this figure, there are evident, in the sections figured, four relatively large cells with relatively large nuclei, cells which have been interpreted as evidencing retarded segmentation with consequent retardation in the normal differentiation of the vesicle. On tracing this vesicle through the series of six sections it would seem that the direction of section is favor


370 G. CARL HUBER

able. The uterine mucosa appears to have reacted normally; the decidual crypt in which this vesicle is lodged presenting normal size and form, and the surrounding decidua normal structure. The vesicle itself is retracted from the uterine epithehum, intact throughout the crypt, thus, does not appear to have attained the normal adhesions observed in normal vesicles of this stage. The four ova depicted in figures 3 and 4, appear to present a distinctive type of abnormal development, a type which is interpreted as showing retarded segmentation in certain of the cells of the 8-cell and perhaps 16-cell stage. All are found in




Fig. 5 Four consecutive sections of the ovum of the albino rat showing abnormal development of the segmentation cavity X 200. Rat No. 46, 6 clays, 14 hours, after insemination.

uteri containing normal stages. The appearances presented, if correctly interpreted, speak in favor of a structural or metabolic defect inherent in the cells themselves and not primarily dependent on environment, pabulum, or embryotroph.

ABNORMAL SEGMENTATION CAVITY FORMATION

The following three ova have been grouped as showing irregularity in the formation of the segmentation cavity.

In figure 5 are reproduced four consecutive sections passing through an abnormal ovum obtained from rat No. 46, 6 days, 14 hours, after insemination. There were obtained from the uterus


PATHOLOGIC OVA, ALBINO RAT 371

of this rat ten blastodermic vesicles, two of which are reproduced in A and B of figure 24, Part I, as showing typically early stages of the anlage of the ectoplacental cone and entypy of the germ layers. The ovum shown in figure 5 is found in a decidual crypt which is in very close proximity to the one containing the vesicle figured under B of figure 24, Part I, the two crypts being separated by a distance of approximately 1.3 mm., while the distance between decidual crypts is normally 1 cm. to 1.5 cm. The decidual crypt lodging the abnormal ovum presents a normal appearance, resembling very closely in form, depth and structure



Fig. 6 Two ova of the albino rat, interpreted as evidencing retarded or irregular formation of the segmentation cavity. X 200. A, rat No. 90, 6 days, 17 hours, after the beginning of insemination. B, rat No. 90, 6 days, 17 hours, after the beginning of insemination, p.ect., parietal or transitory ectoderm; y.ejil., yolk entoderm; p.ent., parietal entoderm.

of the surrounding decidua, the crypt and decidua enclosing the adjacent normal vesicle figured in B of figure 24, Part I. The abnormal ovum in question appeared to have proceeded normally in segmentation, its constituent cells being of about the size and structure of the cells of normal vesicles taken the early part of the seventh day after insemination. The cell-mass encloses a relatively small cavity which may be regarded as an abnormally placed segmentation cavity, in that its position is not eccentric, and that it is surrounded on all sides by more than one layer of cells. There is thus no differentiation of floor and roof as in normal blastodermic vesicles, and no development of ectoplacental cone and egg-cylinder as in the other ova obtained from


372 G. CARL HUBER

this uterus. I am for the present unable to offer any plausible explanation or give reasons for such abnormal development of the segmentation cavity. The fate of such a structure may perhaps be conjectured from a study of the abnormal ovum shown in A of figure 6, interpreted as showing a similar abnormality, but obtained in early stages of degeneration. This ovum and that shown in B of the same figure was obtained from the uterus of rat No. 90, 6 days, 17 hours, after insemination. In the uterus of this rat there are found six ova, only one of which was developed to a stage comparable to that shown in figure 24 (Part I) of about the same age. Three other vesicles present a sHghtly younger stage and may be compared with vesicles shown in D and E of figure 23, Part I. None of these four vesicles is favorably cut, but so far as may be determined, are of normal structure for the respective stages represented, A of figure 6 is also cut slightly obliquely, not sufficiently so, however, to make difficult its interpretation. The figure drawn is that of the third of a series of seven sections having 10 ^ thickness, and depicts what is regarded as representing an ovum with abnormal segmentation cavit}^ formation. In this ovum, the segmentation cavity is slightly more eccentric than is that shown in figure 5, and contains a granular detritus which in the preparations is distinctly stained with Congo red. The roof of this vesicle is composed almost throughout of more than one layer of cells. There is no differentiation of ectoplacental cone and ectodermal node, nor of yolk entoderm. Two cells regarded as phagocytic leucocytes, staining much more deeply in Congo red than do the cells of the ovum, have, in the section figured, penetrated the egg-mass, indicating early degenerative changes.

The vesicle shown in B of figure 6, obtained from the same rat, is favorably cut, and is readily followed through the series. The structural appearance presented by this vesicle is not explained by supposing it due to very oblique plane of section of a normal vesicle, a plane of section which might include the roof of the vesicle while avoiding its floor. The vesicle is abnormal in that it presents a want of development of the thickened germ disc, and a hyperdevelopment of the yolk entoderm. In none of the


PATHOLOGIC OVA, ALBINO RAT 373

sections of the series which includes this vesicle, which is cut in very favorable longitudinal direction, and is thus readily oriented with reference to mesometrial and antimesometrial portion, is there seen any thickening of the outer layer of cells, to form the part known as the floor of the vesicle, which at this stage of development is uniformly directed toward the mesometrial border. In A and B of figure 23, Part I, are shown vesicles with which the ovum here discussed may be compared. In the preparation under discussion, the yolk and parietal entoderm form almost a continuous layer, one of the detached cells showing a mitotic phase. In the normal vesicles of this stage of development the parietal entoderm is represented by a few scattered cells, as may be observed by a study of the figures to which reference is above made. Whether this vesicle is to be regarded as showing a later stage of an ovum in which there was irregularity in the formation of the segmentation cavity, I must for the present, leave as problematic. It has occurred to me that by enlargement of the segmentation cavity of an ovum such as shown in figure 5, with centrally placed segmentation cavity, there might result in further development the formation of a vesicle such as shown in B of figure 6.

It is freely admitted that the deductions here made, relative to irregularity in the formation of segmentation cavity, are not supported by conclusive evidence. It has seemed to me, however, that the interpretations given to the appearances presented are less open to criticism than others that might be suggested. These abnormal ova also suggest an inherent defect in the ova, leading to abnormal development, rather than abnormal development resulting from defective environment.

DEGENERATION OF OVA AS RESULT OF PATHOLOGIC UTERINE MUCOSA

In figure 7 are reproduced two ova which seem to me to show the primary stages of degeneration owing to pathologic condition of the uterine mucosa. Vesicle A was taken from the uterus of rat No. 91, 5 days, 16 hours, after insemination. In the uterus of this rat there were found only two ova. Vesicle B was taken


374 G. CARL HUBER

from rat No. 104, 6 days after insemination. In the uterus of this rat there were found six ova. In both of these rats, the ova present essentially the same stage of development, comparable to that shown in A and B of figure 23, Part I. As may be observed from the text of Part I (page 301) the stages obtained at the end of the sixth day and early hours of the seventh day, were found very difficult to fix. At this stage the ovum consists of a relatively large, thin walled vesicle, very prone to fixation shrinkage. All of the ova or vesicles obtained from rats Nos. 91 and 104, are very badly folded in their roof portion. Those shown



Fig. 7 Two ova of the albino rat partly surrounded by maternal blood with many phagocytic leucocytes. The folding of the roof of the vesicles is due to fixation shrinkage. X 200. A, rat No. 91, 5 days, 16 hours, after the beginning of insemination. B, rat No. 104, 6 days after the beginning of insemination.

in A and B, figure 7, are representative. This folding, a result of imperfect fixation, is present in all of the vesicles of this stage, even though the respective vesicles present normal structure. The ova here figured may be regarded as having fairly normal structure, both as to rate of development and as to arrangement, form, and structure of constituent cells. All of the eight vesicles obtained from these two rats (No. 91, 2 ova; No. 104, 6 ova) are in part surrounded by exudated maternal blood, containing numerous leucocytes. Small masses of blood with leucocytes are found here and there in different parts of the uterine lumen of both rats, lodged in mucosal folds other than the characteristic decidual crypts enclosing the respective ova. These


PATHOLOGIC OVA, ALBINO RAT 375

decidual crypts are relatively shallow when compared with those of normal uteri of similar stages with normal ova. The uterine mucosa of the two rats under discussion does not appear to have reacted in a normal manner. In these preparations, attention is especially drawn to the presence of maternal blood with numerous phagocytic leucocytes found in relation with the ova, a condition never observed in normal development of ova and uterine mucosa. In A and B, figure 7, the red and white blood cells with granular detritus may be observed as found in relation with the respective vesicles, these presenting essentially the same appearances as do the other six ova obtained from these two rats; the one figured having been more favorably cut than any of the others. The appearances presented in these two rats are interpreted as showing a probable degeneration of the eight ova, and probably complete dissolution and removal. The vesicles appear to have developed normally to the stage at which they were obtained. As a result, however, of pathologic condition of the uterine mucosa, maternal blood, especially leucocytes, have entered the lumen of the uterus, the leucocytes being destined to play the role of phagocytes. In normal development of the albino rat, maternal blood does not enter the lumen of the uterus — decidual crypts — until after the uterine epithelium has become detached from the mucosa of the wall of the decidual crypt, in the region of lodgment of the enclosed ovum. Normally, very few leucocytes are met with in the lumen of the uterus, even in later stages of development, stages in which maternal red blood cells are met with in the decidual crypts. After experience had accumulated, uterine tubes supposed to contain developmental stages aging from the fourth to the sixth day, which on examination revealed blood and especially leucocytes in the lumen of the uterus, were regarded as not favorable specimens for finding ova. In a number of such uteri, cut completely in serial sections, no ova were found. It is possible that, owing to phagocytic action of the leucocytes present, the ova may have been completely removed prior to killing and fixing the tissues. In such condition, it would seem to me as pertinent to speak of faulty implantation, due to abnormal uterine mucosa. It seems to me signifi JOURXAL OF MORPHOLOGY, VOL. 26, NO. 2


376 G. CARL HUBER

cant that in the two rats in which the pathologic condition affects primarily the maternal tissue, the uterine mucosa, all of the contained ova are prone to degeneration. In the abnormal ova previously described, for which it was suggested that the causes for the abnormality were to be sought in the ova themselves, in the great majority of instances, only one abnormal ovum was found in each uterus along with a variable number of ova which are to be regarded as normal for the respective stage.

IMPERFECT DEVELOPMENT OF THE ECTODERMAL VESICLE

The series contains two ova, very favorably cut, ova in which the ectodermal vesicle with the antimesome trial portion of the proamniotic cavity does not seem to have developed normally. Stages showing the differentiation of the egg-cylinder, the formation of the ectodermal vesicle with the antimesometrial portion of the proamniotic cavity, the formation of the mesometrial portion of the proamniotic cavity in the extraembryonic ectoderm, the union of the two primary proamniotic cavities to form a single space, are clearly shown in figures 26 and 27, Part I, in the series of closely approximated stages there portrayed. From a study of these figures, it will be observed that the antimesometrial portion of the proamniotic cavity develops within the ectodermal node before the mesometrial portion of this cavity develops in the extraembryonic ectodermal portion of the egg-cylinder. In the egg-cylinder shown in figure 8, rat No. 94, 8 days after the beginning of insemination, such is not the case. In the uterus of this rat there were found seven egg-cylinders, one of which, very favorably cut, is shown in C, figure 27, Part I. The other egg-cylinders obtained from this uterus, except the abnormally developed one to be discussed, though not favorably cut, present essentially the same form and structure as that figured under C of the figure above referred to. The egg-cylinder portrayed in figure 8 compares in size and form with those regarded as normal and taken from the same uterus. For the greater part it presents normal structure and normal relations of cells. The ectoplacental cone, only in part included in the figure, and the parietal


PATHOLOGIC OVA, ALBINO RAT


377



Fig. 8 Egg-cylinder of albino rat showing retarded development of ectodermal node and of the formation of the antimesometrial portion of the proamniotic cavity. X 200. Rat No. 94, 8 days after the beginning of insemination. ect.pl., ectoplacental cone or Triiger; v.ent., visceral entoderm; met.pr., mesometrial portion of the proamniotic cavity; p.ect., parietal or transitory ectoderm; pr.emb.ent., primary embryonic entoderm; ect.n., ectodermal node; a.met.pr., imperfectly developed antimesometrial portion of proamniotic cavity; ex.ect., extraembryonic ectoderm.


ectoderm, in structure and relation to decidual crypt, are to be regarded as of normal development. The visceral entoderm, surrounding the extraembryonic ectodermal portion of the eggcylinder, is of normal structure, showing the three zones evidenc


378 G. CARL HUBER

ing its absorptive function. The extraembryonic ectoderm, enclosing the mesometrial portion of the proamniotic cavity, presents normal structure and relations of cells. The only abnormality observed is in the region of the ectodermal node, the anlage of the ectodermal vesicle with the enclosed antimesometrial portion of the proamniotic cavity. With this stage of development of the egg-cylinder (see figs. 26 and 27, Part I) the ectodermal node presents a well formed cavity, surrounded by the cells of the primary embryonic ectoderm, radially arranged. In the egg-cylinder under discussion (fig. 8) there is distinctly a retardation in the development of the ectodermal vesicle with full differentiation of the primary embryonic ectoderm. An imperfectly developed antimesometrial portion of the proamniotic cavity is evident. This small cavity, indistinctly bounded, extends obliquely through several sections of the ectodermal node, and contains amorpjious granular detritus, which in the preparations is stained by Congo red. The cells destined to form the primary embryonic ectoderm show no definite arrangement, especially as concerns the more centrally placed cells of the node. Since the primary embryonic ectoderm is the anlage for the ectoderm of the embryo, an arrest in its differentiation would of necessity profoundly affect further development of the embryo. Antimesometrial to the ectodermal node (just above it in the figure) there is found a small vesicle the walls of which are not distinctly delimited and composed of extraembryonic ectodermal cells, surrounding a small, completely bounded cavity. I am not prepared to say whether this small vesicle is to be regarded as developing from cells of the extraembryonic ectoderm, or from a displaced, accessory ectodermal node, in which a discrete portion of the proamniotic cavity has developed. If the latter, the possibility of a double anlage for the embryonic ectoderm is to be considered. My interpretation of this eggcylinder as showing a retardation of the development of the ectodermal node and differentiation of the primary embryonic ectoderm, is confirmed from a study of a slightly older stage showing essentially the same condition. This ovum is presented in figure 9, and is taken from rat No. 41, 8 days, 16 hours, after the



Fig. 9 Egg-cylinder of albino rat, in which the antimesometrial and mesometrial portions of the proamniotic cavity have failed to unite to form a single or definite proamniotic cavity. X 200. Rat No. 41, 8 days, 16 hours, after the beginning of insemination, ect.pl., ectoplacental cone or Triiger; p.ect., parietal or transitory ectoderm; v.ent., visceral entoderm; met.pr., mesometrial portion of the proamniotic cavity; ex. ec<., extraembryonic ectoderm ; a.met.pr., antunesometrial portion of the proamniotic cavity; pr.emb.ect., primary embryonic ectoderm; +» region at which, in normal development, by the end of the eighth and beginning of the ninth day, the two portions of the proamniotic cavity would have united to form a single space, the definite proamniotic cavity.


ect pi. f

//


p. ect


V. ent


met. pr




pr. smb. 6


379



ex. ect.


a. met. pr.


--H---pr.emb.ect>


380 G. CARL HUBER

beginning of insemination. The uterus of this rat contains eight egg-cyhnders, all of which, except the one here figured, show normal structure, though presenting quite different stages of development. One of these, cut serially in cross-section, is figured in C, figure 32, Part I, as showing anlage of mesoderm with primitive streak and groove. Two of the other egg-cylinders show the anlage of the mesoderm, two others show late premesoderm stages of the egg-cylinder, the remaining egg-cylinders are less fully developed, one showing a development which may be compared to B of figure 26, Part I, thus a much younger stage. By the end of the eighth day and with the early hours of the ninth day after the beginning of insemination in the albino rat, the two parts of the proamniotic cavity, which develop discretely, have joined to form a single space (C, fig. 27, Part I). The egg-cylinder shown in figure 9, presents normal development in all parts, except that there is as yet no union of the two parts of the proamniotic cavity. This egg-cylinder is most favorably cut, in longitudinal direction; the plane of section being almost parallel to the mid-sagittal plane. This eggcylinder, therefore, is easily followed through the several sections of the series into which it was cut. The irregularity of outline of the ectodermal vesicle, lower right of figure, it is believed, is not due to fixation shrinkage. Judging from size and structural differentiation of this egg-cylinder, union of the antimesometrial and mesometrial portions of the proamniotic cavity should have been completed before this stage of development was reached, with the primary embryonic ectoderm and the extraembryonic ectoderm forming a continuous layer, as shown in figure 29, Part I. The folding of the wall of the antimesometrial portion of the egg-cylinder, lower right of figure, evident in nearly all of the sections of the series, is regarded as indicating an abnormal growth of the primary embryonic ectodermal cells composing the wall of the ectodermal vesicle, as a result of retarded extension of the antimesometrial portion of the proamniotic cavity, perhaps an adjustment to meet the altered mechanical stress resulting from abnormal development. The condition here seen, it would seem, is foreshadowed in the egg-cylinder shown in figure 8.


•■•'■:^k


ect pi -^^ ^^'C te Q 4, 0-:C^


ect pi.



pr. emb. ect.


Fig. 10 Two egg cylinders of the albino rat found within the same decidual crypt, with in part common ectoplacental cone. X 150. Rat No. 87, 9 days after the beginning of insemination, ect.pl., ectoplacental cone or'Trager; p.ect., parietal or transitory ectoderm; v.eiU., visceral entoderm; e.v.ect., extraembryonic ectoderm; pr.c, proamniotic cavity; pr.emb.ect., primary embryonic ectoderm; pr.emh.ent., primary embryonic entoderm; mes., mesoderm.

381


382 G. CARL HUBER

The causes operative in this retardation of development and differentiation of the ectodermal vesicle and primary embryonic ectoderm, I have been unable to determine. They would appear to be inherent in the egg-cylinder, since ectoplacental cone and visceral entoderm, so far as may be determined from a study of sections, appear to have functioned normally, in furnishing the necessary embryotroph in the form of maternal hemoglobin, as is normal for egg-cylinders of the albino rat of this stage of development:

TWO EGG-CYLINDERS IN ONE DECIDUAL CRYPT

The ova portrayed in figure 10 present a condition which must be regarded as exceedingly rare, since it represents the only instance of this condition observed in the extended series of preparations of the various stages of the development of the albino rat from the end of the first to the end of the ninth day after insemination, in my possession. This preparation is from rat No. 87, 9 days after the beginning of insemination. The uterus of this rat contained, other than the preparation here considered, six eggcylinders of normal development, all showing a stage which is slightly older than that shown in figure 31, Part I, in that the mesoderm shows further development than is shown in that figure. In the preparation here figured there are found two egg-cylinders enclosed within the same decidual crypt. This figure, which is drawn by combining the drawings made from two sections, is reproduced at a magnification of 150 diameters, while all of the other figures portraying sections of ova, both in Part I and in Part II of this communication, are reproduced at a magnification of 200 diameters. This should be borne in mind when comparing this figure with the others. In figure 10, the lower portion of the large egg-cylinder to the level of the lower end of the smaller one was drawn from one section, while the remainder of the figure was drawn from the fourth following one. The adjustment was made by overlapping in the camera lucida drawing ( X 600) the sharp mesometrial border of the primary embryonic ectoderm of the larger egg-cylinder. Scarcely any


PATHOLOGIC OVA, ALBINO RAT 383

adjustment was found necessary, none of the right wall of the larger egg-cylinder, and only very slightly so of its left wall. The slight deviation from the longitudinal axis of the larger eggcylinder made the procedure desirable. It is thought that the figure as presented gives correctly the size of the respective eggcylinders, and in all essentials, their relations; the greater part of the figure having been drawn from one section. Both of the egg-cylinders reveal normal structure for the stages of development attained. The larger one is cut in the coronal plane, as is readily determined by the distribution of the mesoderm, one side representing a mirror picture of the other. The direction of section in the smaller egg-cylinder, except that it is longitudinal, is not to be determined, since before the anlage of the mesoderm, a bilateral symmetry cannot be recognized in sections. Since these two egg-cylinders are in all essentials of normal form and structure, and since their structure is clearly brought out in the figure, an extended description of them at this place seems uncalled for. For respective stages the reader is referred to Part I. Attention may be drawn, however, to the fact that the visceral entoderm on the contiguous surfaces of the two egg-cylinders is less fully differentiated, and shows less absorption of the maternal hemoglobin than is seen on the exposed or free surfaces, this, no doubt, for mechanical reasons. Further, that in the region where the two egg-cylinders are in contact, the parietal ectoderm of each can be traced as a distinct layer to the bases of the respective ectoplacental cones, showing that each developed from a separate ovum. The ectoplacental cones are for a short distance distinct. In tracing the sections through the series the impression is gained that the ectoplacental cone of one of the eggcylinders overlaps that of the other in such a way that in the plane of the sections obtained, one seems continuous with the other, as represented in the figure. The boundary between the two is not distinct, and it would seem that as a result of pressure, partial fusion of the two had taken place. The presence of two egg-cylinders, enclosed within a single decidual crypt, as shown in this figure, with one of them ha^'ing much smaller size and representing a younger stage of development, I believe is


384 G. CARL HUBER

not to be explained on the supposition of superfecundation or superfoetation. The record for this rat does not show insemination on successive days. At The Wistar Institute, after all of the supposedly successful matings of albino rats, the females rats are caged apart from the males. The smaller egg-cylinder, though appreciably smaller, is in stage of development separated from the other by a time interval of perhaps less than 24 hours. It presents a stage of development which is comparable to C of figure 27 (8 days) and except for size, to the one figured in figure 29 (8 days, 17 hours) of Part I. It is believed that in this case both ova were seminated at about the same time, and proceeded through normal segmentation and that on reaching the lumen of the uterus during the fifth day they became lodged in close proximity in the same mucosal fold. With the development of the decidual crypts, both became enclosed within the same crypt, at perhaps slightly different levels. In further development one blastodermic vesicle dominated the other and from about the seventh day on, one developed and differentiated more rapidly than the other. Had development continued, two distinct embryos, \/ith separate amniotic cavities, attached to the same placenta, would have been formed, with one embryo large and more fully developed than the other. From mere difference in size and of development of embryos in the same litter it is not warranted to postulate superfecundation nor superfoetation. I am of the opinion that usually when two morula masses are lodged in close proximity in the same mucosal fold, one or the other degenerates (fig. 2, A) and that the normal development of both, as in the preparation shown in figure 10, is of very rare occurrence.

CONCLUSIONS

A study of the abnormal or pathologic ova met with in the extended series of preparations covering the first ten days of the development of the albino rat, enables grouping them in two main classes:

a. Such in which all of the ova of a given rat show, or are associated with, abnormal development.


PATHOLOGIC OVA, ALBINO RAT 385

b. Such in which a single abnormal or pathologic ovum is found in the same uterus along with an average number of normally developed ova.

When all the ova in a given uterus show abnormality, the presumption seems warranted that the underlying cause of the abnormality is to be sought in an altered or pathologic condition of the uterine mucosa. In the instances observed, the presence of maternal blood with many phagocytic leucocytes was noted in the lumen of the uterus, adhering to and surrounding the ova. From the study of sections of the uteri of an appreciable number of albino rats, in which insemination and supposedly semination seemed normal, but in which on complete serial sectioning of the uterine tubes no ova were found, but in the lumen of the uterine tubes of which the presence of maternal blood and phagocytic leucocytes was noted, the conclusion seems warranted that death and complete absorption of ova, after a given stage of normal development has been reached, may occur. In such cases, one may with propriety speak of faulty implantation, due to altered or pathologic condition of the uterine mucosa, even in cases where no actual implantation would have occurred in corresponding normal stages. In the two rats (Nos. 91 and 104) in which this condition was observed, the decidual crypts were shallow and not developed to the extent normal for the respective stages, evidencing the abnormal condition of the mucosa.

In cases in which a single abnormal or pathologic ovum is found in the uterus along with several normal ova, the presumption seems justified that the underlying cause responsible for the abnormal development is to be sought in the ovum itself, and not in its environs.

Abnormal developmental stages, interpreted as due to irregular or retarded segmentation, irregular or abnormal segmentation cavity formation, and retarded development of the ectodermal node and primary embryonic ectoderm, where only a single ovum shows abnormal development in a uterus containing the average number of ova presenting normal development, are difficult to explain on the assumption that extraneous influences affecting a single ovum are operative. Practically all


386 G. CARL HUBER

of the abnormal ova of the class described, and especially is this true for older stages, present normal relations to the uterine mucosa and the walls of the decidual crypt after implantation, and so far as may be determined by structure, give evidence of normal absorption of maternal hemoglobin in stages in which such absorption is pertinent. It may be argued that a single ovum may be less favorably placed in relation to embryotroph or pabulum, and as a result of unfavorable nutrition, develop abnormally. This is difficult to conceive for stages in which the ova lie free in the lumen of the uterus, namely, to about the beginning of the seventh day after the beginning of insemination, when embryotroph or pabulum must be relatively evenly distributed. The presumption, it would seem to me, in such cases is in favor of regarding the primary cause of the abnormal development as inherent in the ovum.

Separation of the first two blastomeres and the presence of tw^o egg-cyhnders in a single decidual crypt are regarded as chance findings and as of rare occurrence, since each was met with only once in the material at hand.

LITERATURE CITED

Literature on pathologic ova of the albino rat is lacking. For the literature of all but the more recent work, dealing with comparative experimental teratology, the bibliographies accompanj-ing the chapters of O. and R. Hertwig may be consulted; for that dealing with the pathologj- of human ova, the bibliograpliies accompanying the contributions of F. P. Mall may be consulted.

Hertwig, O. 1906 Missbildung und ^lehrfachbildung, die durch Storung dcs ersten Entwicklungsprozesse hervorgerufen werdeh. Hertwig's liandbuch der vergleichenden und experimentellen Entwickelungslehre der Wirbeltiere, Bd. 1, Part 1; Fischer, Jena.

Hertwig, R. 1906 Der Furchungsprozess. Hertwig's Handbuch,Bd. 1, Part 1.

Mall, F. P. 1900 Welch Festschrift, Johns Hopkins Hospital Reports, vol. 9. 1903 Vaughan Festschrift, Contributions to medical science, G. Wahr, Ann Arbor.

1908 A study of the causes underlying the origin of human monsters. Jour. Morpli., vol. 19.

1910 The pathology of the human ovum. Keibel and Mall, "^lanual of Human Embryology." Lippincott Company, Philadelphia.


Comment On Miss Beckwith's Paper On "The Genesis Of The Plasma-Structure In Hydractinia Echinata"

Marianna Van Herwerden

From the Physiological Laboratory, Utrecht, Holland

In a recent article' discussing the origin and relationships of the protoplasma granules in the egg of Hydractinia echinata, Miss Cora J. Beckwith gives a totally erroneous idea of the results of a study on the eggs of Strongylocentrotus lividus and some other Echinoderms which I published in the Archiv flir Zellforschung, Band 10, 1913.

It seems probable that she did not see my original article, otherwise her conclusion (p. 215) that I consider the basophihc granules in the cytoplasm as an artefact (because I could not have seen them in the living egg) would have been utterly impossible. In reahty, I emphasize in my paper that I have seen these granules in young, living eggcells (Arch. Zellforsch, Bd. 10, p. 438) and that they later form the building-stones of the so-called chromidia of the German authors. All that I considered as an artefact was the 'particular ivay in which the chromidia are arranged around, the nucleus, this appearing in a most pronounced manner after insufficient fixation methods. Had she read pages 438 and 439, she would have seen that I agree with her (p. 200) that with good fixation there is a uniform distribution of the gi'anules.

On page 215 Miss Beckwith says: "Unhke Schaxel, however, he (van Herwerden) holds the mitochondria to be developed from this basophilic substance, since it is also a nucleinic acid compound;" and (p. 216) "the nuclease digestion indicates nucleinic acid present in the granules and mitochondria." Anyone will search my paper in vain for a chemical test for mitochondrial constitution. I have never attempted to give one. Having demonstrated the nucleinic-acid nature of the graiiules composing the particular structures called chromidia, I only said that, with the Benda stain for mitochondria, I obtained violet-colored granules on the alveolar walls which, located as they are, probably are to be considered as the same elements we recognized in the alcohol-fixed preparations as containing nucleinic acid. The doubts I expressed in my paper on the specificity of the Benda-stain reaction (see, also, Ciona intestinahs) was reason enough for avoiding the use of the term mitochondria instead of basophihc granules. I only stated that, using Benda, the deep stain of the granules situated

'Jour. Morph., vol. 25, no. 2, 1914

on the alveolar walls, suggests that they may be identical with the building stones of the chromidia. But every reader of my paper will understand that this is not the essential point of my argument in discussing Schaxel's emission hypothesis.

I quite agree with Miss Beckwith that similarity in staining reactions is not sufficient for the identification of materials. Emphasizing this very point in my paper, I introduced the nuclease digestion as a convincing microchemical test, and I only regret that Miss Beckwith did not make use of this method, for I believe that for this special case it would have had a greater value than her attempts to accept or reject the identity of the chromatin and the basophilic granules in the cytoplasm.

To avoid any misconception, I must add, further, that the demonstration of nucleinic-acid compounds in the cytoplasm of the egg of the sea-urchin does not in any way imply their introduction from the nucleus; it is possible that they have been generated in loco by chemical changes.

THE AUTHOR'S REPLY

In reply to Dr. van Herwerden's comments may I say that I regret exceecUngly having, in my brief summary, in any degree misinterpreted her article. That the difficulty is due to a misunderstanding both on my part and hers, I shall hope to show. I have reread with great care Dr. van Herwerden's article (Arch, fiir Zellforsch., Bd. 10, 1913) to see wherein I have failed to understand it. In regard to the first point mentioned, i.e., that I quoted her as saying that the basophilic granules are artifacts, it is evident that my summary, as well as her original statement, is none too clear. I find no reference to an earher article in which she believes that that point is evident. That I understood basophilic granules to be visible in the living mature egg, and so stated, is seen from a sentence preceding the one she quoted (p. 215) in which I said that she had described the mitochondria (basophilic granules) as visible in life in the mature egg. May I correct here her impression regarding a minor point. I did not say, as she quotes me as saying, that she "could not have seen them' in the living egg," l)ut that she did not see them in the young living egg. This last statement of mine, that she did not see the basophilic granules in the young living oocyte, she may question with justice. I evidently mistook her statement to the effect that some young cells showed no granules to mean all cells (p. 440) and my conclusion is therefore erroneous on that point.

In regard to my further statement that the chromidia of fixed material are artifacts, in view of Dr. van Herwerden's more recent comment, I see that I misinterpreted the following sentence (p. 439); "Ichhalte also die ChromicUenstruktur dieser Zellen fur ein Kunstproduct. Die Herkunft und die Bedeutung der basophilen Korner in den Eizellen welche die Baustein dieser Chromidien bilden etc.," having understood 'Chromidienstruktur' to mean chromidial structure rather than chromidial arrangement as was evidently intended.

Again, I have not intended to indicate and cannot see that I indicated that Dr. van Herwerden made a chemical test of mitochondria. As to her taking exception to my statement that she said that "nuclease digestion incUcates nucleinic acid present in the granules and mitochrondria" I can only say that I based that conclusion on the sentences (p. 446): "Untersucht man das reife Ei von Strongylocentrotus lividus und Holothuria tubulosa nach der Bendaschen Methode, so trifft man violette Korner auf den Wabenwanden cles Zellplasma zwischen den blassen Dotterkornern gelagert an, welche hochst wahrscheinlich dieselben Elemente sind, die wir im Alkoholpraparat als nucleinsaurehaltende Korner kennen lernten, welche nach der Nucleasawirkung verschwanden. Ich meine also in Gegensatz zu Schaxel, dass in cliesem Fall die als Mitochondrien im reifen Ei dieser Tiere beschreibenen Elemente mit den nucleinsaurehaltenden Bausteinen der sogenannten Chromidien identisch sind." If I have taken her 'hochst wahrscheinlich' to mean more than she intended, I can but apologise.

I can only be glad that the difficulty has been largely one of misinterpretation and that on the whole our results substantiate one another.

Cora Jipson Beckwith


The Development Of The Hypophysis In Squalus Acanthias

E. A. Baumgartner

Institute of Anatomy, University of Minnesota and Washington University Medical

School^

FORTY-THREE FIGURES

CONTENTS

Introduction 391

Literature 392

1. Embryology 392

2. Anatomy and histology 396

Morphology and morphogenesis 400

1. Description of the hypophysis in the pup stage 400

2. Early development of the hypophysis 407

3. Later development of the hypophysis 412

4. The hypophysis of the adult • • • •. 417

Histology and histogenesis '. 425

1. Histology of the adult hypophysis 425

2. Histogenesis of the hypophysis 431

3. Development of the interhypophyseal canal 438

4. Development of the hypophyseal stalk 440

Summary 442

Bibliography 445

INTRODUCTION

Although considerable work has been done on the hypophysis of elasniobranchs, many points still remain concerning which there has been much discussion and which evidently require further investigation. It was thought that an intensive study of the history of this organ in one form might be of value.

The embryos used are from the collection of Dr. R. E. Scammon and from the large series which form a part of the embryo ^ This work was begun while at the University of Minnesota and completed at Washington University Medical School.

logical collection of the Harvard Medical School.- For the pup and adult stages special sections and dissections were made. The difficulties in making a dissection of the hypophysis in selachians have been noted by other investigators. In the present work, it was possible only after numerous attempts to obtain a dissection showing the ventral lobes connected with the rest of the hypophysis. Graphic and wax reconstructions of different embryos and parts of the adult Squalus were made.

LITERATURE

/. Evibryology

The literature on the development of the elosmobranch hypophysis may properly be divided into two categories, one on the embryology and the other on the adult anatomy and histology of this organ. Although the earlier work on the hypophysis in elasmobranchs concerned its adult anatomy, probably investigators have occupied themselves more with its development. Some, of course, have given attention to both the adult anatomy and the development in the same paper or in a series of articles.

Miiller (71) confirmed Rathke's earlier observations that the hypophysis is developed from the mouth. In Acanth'as vulgaris and other selachians he found that it was composed of a principal posterior part and a secondary anterior part. He described the position and gave measurements of the size of the hypophysis and of the thickness of different parts of the wall in an Acanthias embryo 30 mm. long. In a 10 cm. embryo of Mustelus he described the tubular masses forming the hypophysis.

Balfour (74) briefly stated that the hypophysis is an outpouching from the mouth. In 1878 he described the hypophysis as being somewhat constricted from the mouth by the close of

^ Through the courtesy of the late Dr. Charles S. Minot I was able to make use of the extensive Harvard Embryological Collection and had the privileges of his laboratory during the summer of 1914. I also wish to thank Dr. J. S. Kingsley and others of the South Harpswell Laboratory for the courtesies of that laboratory for a part of the summer.

'Stage K.' Soon after this its terminal part dilates and the hypophysis is completely constricted from the mouth.

Reichert ('77) in a description of some cleared embryos of Acanthias remarked on the position of the hypophysis and its relation to the notochord. He opposed the theory of the ectodermal origin of the hypophysis in Acanthias.

Rabl-Riickhard ('80) in his work on the relations of the notochord, confirmed Mliller's observations concerning the hypophysis. He stated that there is a ventral secondary outpouching from the hypophysis in a 60 mm. Acanthias. In an embryo corresponding to Stage K of Balfour he has perhaps mistaken the connection between the premandibular somites for the hypophysis. This seems probable from the position as well as from the fact that he found no connection between this structure and the mouth at this time.

In 1888 E dinger published a paper on the comparative anatomy of the forebrain. He stated that in Torpedo the hypophysis is at first a simple outpouching of the mouth which later develops secondary outpouchings.

Sedgwick ('92) in his work on Scyllium and Raia made the statement that the first rudiment of the mouth extends into the pituitary body.

Von Kupffer ('94) reviewed the literature on the development of the hypophysis and gave some results of his own work. He thought that the hypophysis is not derived entirely from ectoderm, and his observations on other vertebrates supported his view that the hypophysis is partly of entodermal origin.

Hoffmann ('96) described the early stages in Acanthias development. He stated that the hypophysis develops entirely from ectoderm. He found the first definite outpouching anterior to the buccopharyngeal membrane in 13 to 14 mm. embryos. He also described a yellowish pigment in the buccopharyngeal membrane which could still be seen in the stalk of 25 mm. embryos.

Ha-ler ('96) gave an account of the development of the hypophysis in Mustelus. He described the position of the organ in a 22 mm. embryo and called attention to the low epithelium found at the opening of the hypophysis into the mouth. The


394 E. A. BAUMGARTNER

connection with the mouth is soon lost and in a 90 mm. stage the whole structure has come into closer relation with the brain floor and vascular sac, and has grown forward. The roof of the hypophysis has become thickened, as have the anterior and posterior ends. From the floor of the caudal end two lateral outpouchings have developed which later form the inferior sacs. In a 20 cm. Mustelus all parts of the hypophysis are quite well developed. A vascular layer separates the roof from the brain floor. The roof of the inferior sacs becomes thinner. Haller also described the interhypophyseal canal joining the inferior sacs to the superior part. He figured glandular outgrowths extending forward from it. From the floor of the anterior sac are several prolongations extending anteriorly while from the roof are many small outpouchings. The extreme anterior end shows many mitotic figures and is broken up into a network by many capillaries. The floor at the cephalic end of the anterior lobe is very thin — this, Haller stated, is where the hypophysis is constricted from the mouth and here the epithelium has remained of a low cuboidal type. The head of the organ is composed of many closely-crowded tubules. These develop first as solid evaginations, then the nuclei separate and rearrange themselves, later a cleft appears in the protoplasm, which cleft ultimately connects with the main lumen.

Chiarugi ('98) briefly considered the hypophysis in a paper on the description of a" prehypophyseal body and the hypophyseal area in Torpedo ocellata. He described a connection between the premandibular somites and the hypophysis and figured a median sagittal section of a 15 mm. embryo which showed a constriction of the hypophysis from the mouth.

Nishikawa ('99) noted that the hypophysis is present as a simple outpouching in 32 mm, Chlamydoselachus embryos. He made a series of drawings of transverse sections which show its position at this stage. Sewertzoff ('99) in studying the development of the selachian skull took up the interrelation of development of the skull and brain. The trabeculae and parachordal plates in Acanthias and Pristiurus are almost at right angles to each other in early


DEVELOPMENT OF THE HYPOPHYSIS 395

stages (figs. 25 and 29). Later a bend in the medulla and a corresponding one in the parachordal plate changes the relation of the two cartilages. Also the forebrain has shifted from a position ventral to the medulla to a more dorsal and rostral one (fig. 1). Sewertzoff showed the change in position of the hypophysis occurring with the change in position of the skull and brain (fig. 23).

Rossi ('02) described the development of two lateral lobes in Torpedo. These, he stated, develop very early from the lateral walls of the evagination forming the hypophysis. He homologized the different portions of the hypophysis as he found them with those described by Haller, but described two special lateral lobes which he stated have no homologous parts in Mustelus according to Haller's description.

Gentes ('06, '07) found a close relationship between the vascular sac and the underlying hypophysis. In several short reports ('08) he described the lateral lobes and the development of the inferior lobes of the hypophysis. These two parts, he stated, form a ventral pituitary body. In a longer paper this author ('08) gave the results of his studies on the development and evolution of the hypophysis in Torpedo. He described two main parts, the superior and inferior sacs. The superior sac is further divided into posterior and anterior parts. The hypophysis begins as an outpouching which in 45 mm. embryos shows a beginning of its division into the two main sacs. From the posterior part of the superior sac many cords grow dorsalward, these later becoming tubular. Gentes homologized the superior lobe with the 9,nterior lobe of mammals. The lateral lobes he believed persist through life.

Ziegler ('08) in a series of figures of an embryo of Chlamydoselachus, corresponding to Balfour's Stage L-M, showed the hypophysis as an upward anterior-extending evagination from the mouth. The connection with the mouth is still a wide canal.

Johnston ('09) described the hypophysis in Acanthias as consisting of a short anterior portion which grows toward the optic chiasma, and a longer posterior lobe directed toward the vascular sac.


396 E. A. BAUMGARTNER

Scammon ('11) in the normal plate series has figured the hypophysis in several of the younger stages. He described a distinct outpouching in 7.5 mm. embryos. In 18 mm. embryos a constriction of the anterior part of the hypophysis from the mouth has begun. In 24 mm. embryos shallow furrows separate two lateral portions from a median portion in the posterior part. A slight lateral constriction separates the anterior and posterior lobes.

In 1912 Sterzi described the development of the hypophysis in Acanthias. He noted that the front wall of the outpouching becomes dorsal in later embryos. A rostral lobe develops which is in part anterior to the stalk connecting the hypophysis to the mouth. Two lateral outpouchings arise which later form the endocranial portion. The dorsal lobe develops at the superior end of the early outpouching. An early differentiation takes place in the cells of the dorsal lobe. Buds grow out from this thickened wall and form epithelial cords between which are blood vessels and nerve fibers. Differences in the affinity for stains distinguish the rostral (and endocranial) lobes from the superior which is the chromophobic lobe in adults.

2. Anatomy and histology

Von Michlucho-Maclay ('68) described in Acanthias and in Scymnus a persistent connection between the mouth and the hypophysis. In his later work ('70) this investigator failed to find such a connection and believed his former observations to be incorrect.

Miiller ('71) found in later embryos and adults of Acanthias that the cells forming the glandular part are columnar while those toward the periphery of the cords and tubules are spindleshaped, with a finely granular cytoplasm. He also noted the large capillaries and the connective tissue between the tubules.

Viault ('76) briefly described the hypophysis in Raia. The hypophysis is surrounded by a connective tissue sheath which sends strands into the organ carrying large capillaries between the convoluted tubules. These tubules are 0.015 to 0.007 mm.


DEVELOPMENT OF THE HYPOPHYSIS 397

in diameter and usually have a small lumen. They are composed of cylindrical cells. The tongue-like anterior end, he stated, was of similar structure. He was of the opinion that the glandular portion arose from the mouth.

Rohon ('79) described an hypophysis composed of tubules in selachians. It is a triangular shaped organ with a long tonguelike projection which extends forward almost to the optic chiasma. He found no cavity within it. In Acanthias and Mustelus the hypophysis is larger than in Torpedo and Scyllium.

Sanders ('86) studied the central nervous system of Scyllium and Acanthias. He stated that the hypophysis is attached to the infundibulum by a glandular tube which lies between the inferior lobes. In a drawing of the brain of Acanthias (lateral view) he figured the hypophysis as lying caudal to the inferior lobes of the mesencephalon.

In 1892 Edinger described the mid-brain region. In Torpedo and Scyllium he found the hypophysis composed of tubules and cords of epithelium, among which are many blood vessels. He noted nerve fibers extending from the infundibular region ventralward into the hypophysis.

Haller ('96) described in detail the structure of the hypophysis of a 20 cm. Mustelus. He stated that in the adult the glands of the head (superior) portion are tubular. Secretion and cell detritus are found in the lumina. He also described an opening in the base of the anterior lobe. This is found in the thin portion of the floor in the position of the original connection with the mouth, and connects the cavity of the hypophysis with the subdural space.

Sterzi ('04) described the hypophysis in several selachians. He found the anterior part flattened dorso-ventrally, extending almost to the optic chiasma and containing a large cavity. The posterior part is separated from the remainder by connective tissue. A slender canal connects this to the anterior portion. The superior part is formed of anastomosing cords interlacing with sinusoids. The cords are formed of a peripheral layer of columnar cells and an inner mass of polyhedral ones. The nuclei of the outer zone are rich in chromatin. The


398 E. A. BAUMGARTNER

polyhedral cells contain small spherical nuclei. The cytoplasm is granular and stains deeply.

Pettit ('06) made a study of the hypophysis in Centroscymnus. He mentioned posterior, median and anterior parts, all communicating. He found ramifying cords or tubules surrounded by sinusoids.

Burckhardt ('07, '11) in his work on the central nervous system of selachians described, incidentally, the hypophysis in Scymnus. He recognized a terminal, a median and a posterior lobe. Apparently he included under his division of median lobe, the caudal end of the anterior lobe of Sterzi, or it may be that the hypophysis in Scymnus is quite different from that found in other forms.

Joris ('08, '09) described the dorsal lobe in Spinax and Mustelus as formed in part from the hypophyseal evagination and in part from the infundibulum. The cell cords arise from the hypophysis while neuroglia and nerve fibres from the infundibular region grow in between these.

In 1909 Sterzi in his comprehensive work on the central nervous system of selachians, gave a clear description of the hypophysis. According to this description the hypophj^sis is composed of perimeningeal and endocranial parts. The perimeningeal portion is further subdivided into a dorsal and a rostral lobe. The dorsal lobe has many columns from its dorsal surface. Among these are numerous capillaries. The rostral lobe is a long flattened part extending almost to the optic chiasma. The endocranial part is composed of two sacs connected medially to one canal which leads to the anterior or rostral lobe. The cords and tubules of the dorsal lobe anastomose forming a network in which is a rich vascular supply. These are primarily attached by means of connective tissue to the base of the brain or vascular sac. The structure of these cords, Sterzi had described before ('04) . The dorsal lobe, because it stained so lightly he termed the chromophobic portion. The rostral lobe has tubules extending from its ventral walls. The ventral wall has a distinct median ventral furrow and the tubules of either side


DEVELOPMENT OF THE HYPOPHYSIS 399

remain separated. The distal ends of the tubules are soUd and by means of branches anastomose with the cords of the other tubules. The arrangement of the cells here is the same as that in the dorsal lobe. This portion stains more deeply, the capillaries are smaller and less numerous. The endocranial portion in embryos is a sac with folded walls. In the adult, many tubules and cords have developed. In the main this resembles the rostral portion of the perimeningeal part.

Tilney ('11) in his studies on the comparative histology of the hypophysis, stated that in Acanthias there is a distal epithelial portion made up of parallel cell columns. The cells are deeply acidophilic with only a few faintly-staining acidophilic ones present. In the juxta-neural part the cells are larger, usually irregularly disposed, but forming some acini. However, they take the basic stains very markedly. He found this portion less vascular than the distal part. Tilney has homologized the different portions of the hypophysis in the various vertebrate groups. The distal epithelial portion of selachians corresponds to the intermediate lobe of mammals.

In 1913 Stendell made a comparative study of the hypophysis. He described intermediate and main lobes in Heptanchus. In the main lobe he described the peripheral cells of the tubules as of an acidophihc nature, while those toward the center are basophilic or neutral. Stendell has homologized the parts found in the hypophysis of selachians with those in the higher forms of vertebrates. His homology agrees with that of Tilney, i.e., the intermediate lobe of Heptanchus is homologous with the same portion in the higher vertebrates, being in Heptanchus very large and becoming smaller in the higher forms. The ventral sac of the main lobe is not found in higher forms.

There have been many terms used in the descriptions of the different parts of the elasmobranch hypophysis. A comparison with figure 1, which is a drawing of a model of the hypophysis of a pup, and a table of the terms employed may serve to explain the terms used in reviewing the literature.


400


E. A. BAUMGARTNER


TABLE 1

Tenns used by the various investigators for the different parts of the elasmobranch

hypophysis



MATERI.^L


REGIONS OF THE HYPOPHYSIS









Anterior


Inferior


Superior


Special portions


Muller (71)


Scymnus. Acauthias


Anterfor part ^secondary)


Posterior


main part)



Haller ('96)


Mustelus


Superior sac


Inferior sac


Head



Rossi ('02)


Torpedo


Median lobe;


Antero-Iateral


Terminal lobe


Lateral




antero-median


diverticulum



lobes




diverticulum





Sterzi ('04)


Mustelus, Acanthias


Anterior portion


Posterior portion


Superior portion



Pettit C06)


Centro-scymnus


Anterior


Median


Posterior



Burckhardt ('07)..


Scymnus


Terminal lobe


Posterior lobe


Median lobe



Gentes ('07)


Torpedo


Superior sac; anterior twothirds


Inferior sac


Superior sac; posterior onethird



Joris COS)


Mustelus



Posterior


Dorsal



Sterzi ('09)


Acanthias


Perimeningeal


Endocranial por

Perimeningeal





portion ; ros

tion


portion ; dor




tral lobe



sal lobe



Tilney ('11)


Acanthias


Juxta-neural part


Distal part



Stendell ('13)


Heptanchus


Main lobe


Ventral sac of main lobe


Intermediate lobe



Sterzi, Gentes, et







al









portion



MORPHOLOGY AND MORPHOGENESIS 1 . Description of the hypophysis in the pup stage

A description of the hypophysis in the pup stage may make clear the different parts of this organ. The formation of tubules has begun and the main portions or lobes are well formed at this time.

The anterior lobe is a tongue-like process with two somewhat wider extremities; these are connected by a more slender middle part which is less than one-third of the entire length. There is a deep sulcus in the median ventral wall which extends from about the middle of the posterior extremity to near the end of the anterior extremity (fig. 1). Several other more or less regular furrows occur in the ventral surface of the anterior extremity. Two lateral constrictions separate the middle portion from the some


DEVELOPMENT OF THE HYPOPHYSIS


401



402 E, A. BAUMGARTNER

what more dorsal posterior extremity. These extend as furrows posteriorly from the lateral dorsal side of the caudal end of the middle part (fig. 25). As just stated, the posterior extremity is somewhat dorsal to the other parts of the anterior lobe and is directly ventral to the superior lobe. Two deep horizontal furrows constrict the connection between the posterior extremity and the superior lobe.

The superior lobe is just below the saccus vasculosus to which it is closely attached, and is almost three times as wide as the posterior extremity of the anterior lobe (fig. 25). Its median part is about as long as the posterior extremity of the anterior lobe, but projects beyond it caudally and laterally. The caudal end extends somewhat dorsally. The lateral parts, or wings, of the superior lobe project somewhat upward and forward.

The inferior sacs extend laterally from a constricted median connection. They are much smaller than the wings of the superior lobe, but, viewed from above, give the appearance of two lesser caudal wings. From their median connection a short slender canal extends in a slightly oblique direction upward and forward to connect with the anterior lobe (fig. 25).

Fig. 2 Sagittal section of the hypophysis of an 8 mm. embryo. X 40 (H. E.G. 210). H, hypophysis; /, infundibulum; Mo, epithelium of mouth; Mm, median mass connecting the premandibular somites; N, notochord; Po, post optic groove.

Fig. 3 Sagittal section of the hypophysis of a 15 mm. embryo. X 40 (H.E.C. 228). For abbreviations, see figure 2.

Fig. 4 Sagittal section of the hypophysis of a 19 mm. embryo. X 40 (H.E.C. 138). For abbreviations, see figure 2.

Fig. 5 Sagittal section of the hypophysis of a 22 mm. ernbryo. X 40 (H.E.C. 231). AL, anterior lobe; Med, median connection of inferior lobes; SL, superior lobe; other abbreviations as in figure 2.

Fig. 6 Sagittal section of the hypophysis of a 28 mm. embryo. X 40 (H.E.C. 234). St, hypophyseal stalk; other abbreviations as in figure 5.

Fig. 7 Sagittal section of the hypophysis of a 34 mm. embryo. X 40 (H.E.C. 362). For abbreviations see figure 6.

Fig. 8 Sagittal section of the hypophysis of a 40 mm. embryo. X 40 (H.E.C. 370). P, parachordal plate; T, trabeculae; other abbreviations as in figure 5.

Fig. 9 Sagittal section of the hypophysis of a 50 mm. embryo. X 40 (H.E.C. 444). C, interhypophyseal canal; for other abbreviations, see figure 8.

Fig. 10 Sagittal section of the hypophysis of a pup, (reconstructed from transverse sections). X 40. For abbreviations, see figure 8.


DEVELOPMENT OF THE HYPOPHYSIS 407

The position of the adult hypophysis has been described by Sterzi ('09) and others. The long tongue-like anterior lobe lies on the median ventral wall of the inferior lobes of the brain. Its anterior end extends forward almost to the optic chiasma. The superior part is placed posterior to this and in a more dorsal plane. It extends as far caudally as the saccus vasculosus. The inferior sacs of Squalus do not extend as far ventrally as has been described for other selachians. But they are ventral to the superior lobe and extend farther caudalward (fig. 10) . From their middle connection a slender canal joins them to the ventral side of the caudal end of the anterior lobe.

2. Early development of the hypophysis

Recent work on the development of the hypophysis in elasmobranchs shows that it arises at an earlier period than was formerly believed. Hoffmann ('96) stated that the position of the future hypophysis is well marked in Acanthias embryos of 15 somites but that there is no indication of an evagination even in 8 mm. (50 somites) and 10 mm. embryos. Haller ('96) began his description of the hypophysis in Mustelus in embryos 22 mm. long. At that time, the hypophysis is already a distinct outpouching. More recently Johnston ('09) briefly described the earliest formation of the hypophysis in Acanthias. In an embryo of 24 somites the ectoderm from which the hypophysis develops is readily recognized. He stated that a short anterior lobe, developing later, extends toward the optic chiasma. Also, that the posterior part crowds between the brain and the median mass connecting the premandibular somites. Scammon ('11) mentioned a thickened hypophyseal plate in a 5.2 mm. embryo and a beginning evagination in a 6.2 mm. embryo (50-51 somites).

A median sagittal section of an Acanthias embryo 8 mm. in length is shown in figure 2. The anterior superior end is partially insinuated between the brain and the median mass connecting the premandibular somites, as had been noted by Johnston ('09) in about the same stage. During the time that the

3 In the description of the figures 'H. E. C has reference to the embryos of the Harvard Embryological Collection used for this study.

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 3


408 E. A. BAUMGARTNER

anterior part of the hypophyseal evagination is growing between the brain and the median mass connecting the premandibular somites, the posterior portion forms a more or less prominent ridge caudal to the connecting mass of the somites. A model of such an embryo shows as an evagination, the end of which is grooved transversely by the median mass connecting the premandibular somites (fig. 2). This groove may be quite prominent even in 11 to 12 mm. embryos and evidences of it are usually to be found at that time.

In 8 mm. embryos as well as in younger ones the anterior arm of the hypophyseal outpouching shows differentiation as far forward as the postoptic recess (fig. 2). Scammon ('11) stated that the notochord comes into contact with the early hypophyseal outpouching, and Sterzi ('12) has shown such a contact in Mustelus of 8 mm. (fig. 449). No such contact has been observed in this study of Acanthias.

A mid-sagittal section of a 15 mm. embryo shows that the superior end of the hypophysis has now grown well between the brain and the median mass connecting the premandibular somites. The evagination is more marked and the anterior arm is longer than in younger stages (fig. 3) .

A model of the hypophysis of an Acanthias embryo 19 mm. in length is shown in figure 11; this view is taken from the left lateral side. The hypophysis at this stage is an anteriorly and dorsally directed outpouching, concave on its ventral surface where it lies in close relation to the diencephalon (fig. 18). Its superior anterior end extends to the infundibular recess. The opening from the pharynx into the hypophysis is small. A sagittal section at about the median line (fig. 4) shows that the thickened anterior (ventral) wall of the hypophysis extends forward as far as the postoptic groove as Johnston ('09) has figured it.

Fig. 11 Left lateral view of a reconstruction of the hypophysis of a 19 mm. embryo. X 130. Mo, lining of mouth; H, hypophysis.

Fig. 12 Left lateral view of a reconstruction of the hypophysis of a 21 mm. embryo. X 100. a, anlage of anterior end of hypophysis; H, hypophysis; IL, anlage of the inferior lobes; Mo, lining of mouth.



409


410 E. A. BAUMGARTNER

Several changes have taken place in the hypophysis of a 21 mm. embryo. It is still concave, both laterally and dorso-ventrally in its ventro-anterior surface (fig. 12). The thickened anterior wall of the hypophysis, reaching almost to the preoptic groove, is now distinctly evaginated. Scammon ('11) mentioned this closing off of the anterior part in a 20.6 mm. embryo and Sterzi ('12) described the formation of this 'rostral diverticulum' in 20 to 24 mm. embryos. The lateral side of the anterior outpouching is sharply demarcated by the formation of the stalk connecting the hypophysis to the mouth (fig. 19). The anterior end at this stage is almost half as wide as the posterior, from which most of the hypophysis is developed. The opening from the mouth into the early anlage is located as before, but is smaller now. A view of a model from the oral side shows that the constriction of the front and lateral sides of the anterior end of the anterior lobe has begun.

In a 22 mm. embryo the hypophysis (fig. 13) is not as concave as in younger stages. The anterior part shows laterally more marked constriction from the stalk which connects it to the buccal cavity. The anterior end is also markedly constricted. The opening into the hypophysis extends now from this anterior constriction to the posterior (caudal) margin of the opening into the first outpouching. The opening into the first evagination is very small and connects the pouch with the stalk (fig. 5). The posterior end is wider transversely than before. On its dorso-lateral surfaces are small ridges (fig. 13), the anlagen of the inferior sacs. On the ventral surface of the posterior part are two slight lateral furrows which are beginning to separate the inferior sacs from Rathke's pouch (fig. 20). These furrows are present in a 20.6 mm. embryo, as Scammon ('11) stated. The lateral pouches or inferior sacs appear as dilated cavities at either side. The grooves or furrows are as yet shallow and indistinct. In an 18 mm. embryo, Scammon ('11) noted the beginning of the division, by slight furrows, of the posterior portion into a median and two lateral parts. Some embryos do indicate the beginning division of the inferior sacs about that time but the furrows are not as prominent as the dilated cavities.


DEVELOPMENT OF THE HYPOPHYSIS


411



Fig. 13 Left lateral view of a reconstruction of the hypophysis of a 22 mm. embryo. X 100. For abbreviations, see figure 12.


In a model of a 28 mm. embryo (fig. 14) the entire anterior portion is well closed off. It extends anteriorly almost to the postoptic recess. From its caudal surface a slender stalk connects the hypophysis with the mouth (fig. 6). The ventral end of the anterior lobe is somewhat wider than the mid part which connects it with the very much wider posterior portion or Rathke's pouch (fig. 21), Distinct furrows on the anterior (ventral) side partially separate the lateral anlagen of the inferior sacs from the original Rathke's pouch. A slight ridge on the caudal (superior) surface joins the two inferior sacs (fig. 14). In a median sagittal section (fig. 6) a shallow groove marks the connection between them. This connection is dorsal (caudal) to the stalk joining the hypophysis to the mouth. On the lateral dorsal surface at the posterior end of Rathke's pouch two very slight outpouchings indicate the position of the developing superior lobe (fig. 14).


412


E. A. BAUMGARTNER



Fig. 14 Left lateral view of a reconstruction of tlie liypophysis of a 28 mm. embryo. X 100. o, 7;, anterior and posterior extremities of anterior lobe; IL, inferior lobe;??i, middle part of anterior lobe; Mo, tv-all of mouth;. Si, hypophyseal stalk; SL, anlage of superior lobe.


3. Later development of the hypophysis

All the main outpouchings of the hypophysis are present in the 28 mm. embryo. The inferior lobes are quite prominently marked off, the anterior lobe shows two widened extremities and the superior lobe has just begun to evaginate. The anterior end of the hypophysis in a 33 mm. embryo is curved slightly forward. The anterior lobe is not so markedl}^ concave anteroposteriorly as in earlier stages (fig. 7). The ventral (anterior) furrows separating the inferior lobes from the posterior medial portion, are now quite deep (fig. 15). On the caudal side a distinct ridge extending between the inferior lobes indicates their


DEVELOPMENT OF THE HYPOPHYSIS


413



Fig. 15 Left lateral view of reconstruction of the hypophysis of a 33 mm. embryo. X 100. For abbreviations, see figure 14.


early connection with each other. On the posterior tip of the hypophysis two lateral grooves mark the beginning constriction of the superior lobe (fig. 22), the outpouching of which was seen in the previous stage. The stalk connecting the hypophysis to the pharynx is smaller than in earlier stages.

The anterior lobe of the hypophysis, particularly its anterior part, has increased in length in a 40 mm. embryo. The stalk joining the hypophysis with the mouth has disappeared, with


414


E. A. BAUMGARTNER



Fig. 16 Left lateral view of a reconstruction of the hypophysis of a 48 mm. embryo. X 100. For abbreviations, see figure 14.


the exception of a cone-shaped mass of cells connected with the oral epithelium, and an irregular area in the floor of the hypophysis which represents the remains of the former attachment (fig. 8). The furrow uniting the two inferior lobes across the middle line is more marked, and the superior lobe also is prominent. The anterior end of the hypophysis now extends forward and downward. The straightening out of the. head bend in the development of the embryo has probably helped to bring about this change.

More marked changes have taken place in a 48 mm. embryo (fig. 16). The anterior end of the anterior lobe is wider than in earlier stages., A short and narrow middle part connects this portion with the caudal extremity which is considerably- wider (fig. 23). The inferior lobes are attached to the, now, ventral (caudal) side of this part. The ridge connecting the two inferior lobes has become veiy pronounced but still opens widely into


DEVELOPMENT OF THE HYPOPHYSIS 415

the anterior lobe (fig. 16). The furrows separating the inferior lobes from the anterior are much deeper and wider. The inferior lobes have enlarged in their dorso-ventral and in their transverse diameters, and they extend laterally beyond the posterior extremity of the anterior lobe. Marked development has taken place in the superior lobe. The lateral furrows separating it from the anterior one are deeper, and the cranio-caudal length of the lobe has increased so that there is a projection caudally beyond the anterior lobe. The antero-lateral ends of the superior lobe have grown forward.

The median connection of the inferior lobes is constricted from the posterior (ventral) part of the anterior lobe in a 50 mm. embryo. The inferior lobes are directly ventral to the superior lobe. There remains a short slender tube in the mid-hne connecting the inferior lobes to the anterior (fig. 9). The duct connecting them to the anterior lobe extends almost straight anteriorly.

A median sagittal section of an 86 mm. embryo (fig. 27, G) shows an increase in the length of the hypophysis. The inferior lobes lie more caudally and the duct joining them to the anterior lobe is longer.

In a 95 mm. embryo the anterior lobe has increased greatly in length (fig. 17). A median ventral sulcus has appeared and the anterior third of this lobe is quite wide. A middle narrow portion, almost circular in cross section, connects the anterior extremity to a wider posterior end (fig. 24) . The caudal extremity is connected dorsally with the superior lobe. The inferior lobes are continuous across the median line. The connection between the inferior lobes and the anterior one is a small tube which extends almost straight forward to join the inferior surface of the caudal end of the anterior lobe just below where this opens into the superior one (fig. 10) . The inferior lobes have enlarged in their cranio-caudal axis. The lateral parts of the superior lobe have increased in their cranio-caudal diameters and extend forward beyond the median part. The latter has grown caudal ward and lies just dorsal to the tube joining the inferior and anterior lobes.


416


E. A. BAUMGARTNER



a.


o


X


S

a

a


n.


o ^


1-5 !/;


DEVELOPMENT OF THE HYPOPHYSIS 417

^ . The hypophysis of the adult

A detailed description of the pup stage has been given (p. 400) as typical of the morphology of the adult condition (fig. 1). A drawing of a dissection of the hypophysis of an adult will show the position and relation of some of the parts more clearly (fig. 26). The anterior end extends almost to the optic chiasma, as noted by Sterzi and others. From a study of the models of the 'pup' stage and of sections of adults it appears that the middle part of the anterior lobe is more than a mere constriction separating a larger rostral from a smaller caudal part as Sterzi ('09) described. In the pup this middle part is only a little shorter than either extremity, while in the adults it is much shorter. It is, however, distinctly marked. In some cases there are cystic outgrowths from the floor of this part; in others the walls and floor are quite regular and therefore are distinctly different from either extremity. The middle part is smaller than either extremity in its dorso-ventral and lateral diameters in pups (figs. 24, 25) and in adults, but the changes in diameter from either end to the middle part in adults takes place gradually and the parts are not so definitely marked, except in those cases in which no glandular outgrowths occur from the middle region which is much more prominent in sections and in wax reconstructions than is shown in dissections. The superior lobe projects some distance laterally on the ventral side of the vascular sac. It is difficult to make out the lateral limits of this part in the dissected specimen, as it seems to be continuous with the ventral surface of the vascular sac (fig. 26). In transverse sections, however, the lateral extent of the wings of the superior lobe is clear. The superior lobe is convex dorsally and closely attached to the saccus vasculosus. The latter dips down on either side of the caudal end of the anterior lobe and so partially separates the lateral wings of the superior from the anterior lobe.

The inferior lobes no longer resemble the sac-like structures with cystic outpouchings of the pup stage. Both superior and inferior walls have developed a mass of tubular-like glands. None of these was observed extending cranialward, as described by



Figs. 18-20 Antero-vpntral views of the reconstructions shown in figures 11 to 13. Figure 18, X 50; figures 19, 20, X 40.

Figs. 21-23 Anterior views of the reconstruction shown in figures 14 to 16. X 40.

Figs. 24-25 Ventral views of the reconstructions shown in figures 17 and 1. Figure 24, X 40; figure 25, X 25. For abbreviations, see figure 1.

418


DEVELOPMENT OF THE HYPOPHYSIS


419



Fig. 26 Drawing of a dissection of the hypophysis of an adult Acanthias from ventral side. X 20. AL, anterior lobe; B, inferior lobes of the brain; C, interhypophyseal canal; IL, inferior lobes of the hypophysis; SL, superior lobe of the hypophysis; VS, vascular sac.


Haller in Mustelus. From their median connection a long slender tube extends to the caudal end of the floor of the anterior lobe. As seen in figure 26, the median connecting portion of the inferior lobes may be large. In some specimens this part, as well as the lobes, shows a mass of tubules. The caudal ends of the inferior lobes are surrounded by cartilage.

The shifting of the hypophysis with reference to its position in the body and the development of different parts is brought out very clearly in figure 27. Sewertzoff ('99), in his studies


420 E. A. BAUMGARTNER

on the development of the skull, made use of such a figure to show the interrelation of development of the brain and skull. He made an outline drawing of the skull and brain of an embryo, choosing an arbitrary magnification. He then made drawings of different sized embryos of such a magnification that points, corresponding to two arbitrarily chosen in the first drawing, would coincide. In figure 27 of this paper, the same scheme was adopted. The magnification of the first drawing was such as to avoid as much as possible the confusion of lines. The points chosen were the extreme anterior end of the notochord and the axis of the notochord at the level of the first spino-occipital nerve. All the other drawings were then made so that these two points — the extreme anterior end of the notochord and the axis of the notochord at the level of the first spino-occipital nerve — should coincide with those of the first drawing. The outlines of the hypophysis were then drawn, using a line between the two points as a base.

The objections to such a drawing are readily apparent. There are, of course, individual variations in the embryos. Also, these points are probably continually changing during developr ment. For a comparative study, however, the variations can be no great objection and the points chosen are probably as reliable as any. A series of embryos from 11.5 mm. in length to the pup were drawn in this manner (fig. 27). One can see at a glance in all these stages the relative position of the hypophysis with reference to the anterior end of the notochord. Also, as was pointed out in the description of the different embryos, what is first the dorsal wall becomes in later stages the ventral, while the ventral or anterior becomes dorsal. The early superior end of the evagination shifts more and more caudalward with reference to the rest of the hypophysis, until, in the pup, the superior lobe, which develops from the superior dorsal end, is caudal in position. The inferior lobes, which develop from the sides of the superior end and are on the same horizontal plane as the superior position, take a position ventral to the superior lobe in the late embryo and adult. The furrows separating these inferior lobes, described as appearing in the 20 to 22 mm. embryos


DEVELOPMENT OF THE HYPOPHYSIS


421



422 E. A. BAUMGARTNER

on the ventral (anterior) side of the hypophysis, are later (50 mm. embryos) on the dorsal side. The anterior lobe, first directed almost vertically, grows ventrally and later extends more and more anteriorly mitil it is directed horizontally (craniocaudally) .

The comparative growth of the different portions is also made clear. The anterior lobe comprises all of the original outpouching and also the anterior tongue which evaginates later. The increase in length of the anterior lobe, particularly its anterior extremity, is marked. The inferior lobes, developing from the sides of the posterior portion of the early evagination, become continuous across the posterior side (34 mm. embryos) and finally constrict entirely except for a short duct connected with the anterior lobe. The inferior lobes increase greatly in size, but, in the adult, are largest transversely. The superior lobe, developed from the superior dorsal part, spreads far out transversely and later enlarges in its dorso-ventral axis.

The posterior extremity of the anterior lobe is then developed from the first outpouching. A little later (21 mm.) the part anterior or ventral to the original outgrowth evaginates, forming largely the ixiiddle narrower portion of the anterior lobe. The anterior extremity of the anterior lobe develops at the extreme anterior (ventral) end of this. The stalk connecting the hypophysis with the mouth is attached to the middle narrower portion. The connection between the inferior lobes develops caudal (dorsal) to this but arises from a part which later becomes the floor of the posterior extremity of the anterior lobe. From this description it is seen that the inferior lobes, developing from the posterior end of the hypophyseal anlage, and the superior lobe, from its extreme dorsal (anterior) end, are derived from a part of the anterior lobe. Such an explanation of the development of the parts is well borne out by a study of the models as well as of the various sections of embryos.

In comparing figures 6, 7 and 8 it is seen that the hypophyseal stalk in a 40 mm. embryo is attached nearer the caudal end of the hypophysis than it is in younger ones. This does not agree with what Haller has described for Mustelus when he found the


DEVELOPMENT OF THE HYPOPHYSIS


423


place of attachment of the hypophyseal stalk near the anterior end. In a 90 mm. Mustelus this place of attachment can be recognized by the very thin floor, and in the adult, by the opening into the subdural space (Haller '96, figs. 12 and 40).

The cavity in the hypophysis of elasmobranchs has been variously described as barely distinguishable, slit-like and large. It is large in some adult specimens of Acanthias. In the anterior lobe there is a distinct increase in the size of the cavity during its development. Table 2 will show the actual increase in the depth of the cavity. The measurements here given were taken in the caudal part of the anterior extremity of the anterior lobe.


TABLE 2 Showing depth of the hypophyseal cavity


Embryo 34 mm 50 mm 86 mm

Pup

Adult


THICKNESS

OF ROOF IN

MICRA


31 31 35 56

56


DEPTH

OF CAVITY IN

MICRA


37 13

12 90

470


THICKNESS OF FLOOR IN MICRA


25 18 22 50 including glandular

outgrowths 480 including glandular

outgrowths


The increase in size of the lumen from the pup to the adult is thus seen to be considerable. From the table and from comparison with figures 2 to 10 it is seen that the increase in size of the lumen is not gradual through all the stages. For example, in the 50 mm. embryo the lumen is actually smaller than in a 34 mm. embryo. It is only from the early pup stage on that the lumen increases to any considerable degree. The increase in size of the lumen of the inferior lobes is even more marked. The cavity in the superior lobe is never large. It is very small in a 48 mm. embryo where the lateral wings are first prominent. In a 95 mm. embryo this cavity is small, but is still distinct. The lumen of the middle portion of the superior lobe extends transversely and forward in the lateral wings of the superior


JOURNAL OF MORPHOLOGY, VOL. 26, NO. 3


424


E. A. BAUMGARTNER


lobe in pups. There is no evidence at any time of an extension of the lumen into any of the glandular columns of this part. In the adult all trace of a central cavity has disappeared, unless the small secretion spaces, to be described later, are remains of the original lumen. Occasionally there is a more or less prominent median dorsal extension of the cavity of the anterior into the superior lobe. In a very few cases this dorsally-extending cavity is continuous with a secretion space lying laterally.

Table 3 shows the increase in size of the hypophysis; also, the length of the hypophysis at the median line, the greatest


TABLE 3 Showing increase in size of the hypophysis



SIZE OF SPECIMEN


LENGTH OF HYPOPHYSIS IN MM.


GRE.\TEST WIDTH

OF SUPERIOR LOBE

IN MM.


GRE.^TEST WIDTH OF

.\NTERIOR EXTREMITY

IN MM.


Embryo 22 mm

28 mm

33 mm

48 mm

95 mm

Pup


0.69 0.81 1.04 1.00 1.34 3.54 6.00


0.40 0.96 1.07 1.30 1.91 4.00


0.23 0.22 0.30 0.35 0.40 0.53


Adult.


1.00


width of the anterior extremity of the anterior lobe, and the greatest width of the superior lobe. The table shows there has been a constant increase in length which is rather more rapid in earlier stages. The increase in width of the superior lobe is a gradual one in the beginning. The anterior lobe, however, grows very little at first. This part doubles in width in its growth between the 22 mm. and the pup stages. It almost doubles again in its later growth.

From a brief study of a few Torpedo embryos, I am inclined to believe, with Sterzi, that the lateral lobes, described in this form by Gehtes ('08) and others, are comparable with the inferior lobes of Acanthias. The embryos examined show no prehypophyseal body such as Chiarugi ('98) described in this genus, nor is there anything comparable to the prehypophyseal body found in the embryos or adults of Acanthias.


DEVELOPMENT OF THE HYPOPHYSIS 425

. HISTOLOGY AND HISTOGENESIS

1 . Histology of the adult hypophysis

a. Anterior lohe. The deep median sulcus in the floor of the anterior lobe has been noted above. It has been observed by Sterzi and others in various selachians. The furrows noted in the floor of the anterior extremity in the pup are evidence of the beginning formation of tubules. Haller observed the tubules on the ventral wall of the anterior part in Mustelus, Gentes observed them in Torpedo, and Sterzi in Acanthias. Tilney ('11) stated that there are many vesicles in the upper and lower walls of the juxta-neural (anterior) part in Acanthias. Haller figures cyst-like glands in the roof of the anterior lobe of Mustelus. Some adult specimens of Acanthias show glandular outgrowths in the superior wall of the anterior lobe, especially in the dorsolateral parts of the anterior extremity. As Sterzi ('09) observed, glands probably develop from the anterior lobe throughout life and, finally, even from the dorso-lateral walls and roof, and glands project ventrally from the floor of the anterior lobe. A model of some of the latter shows that branches are given off at all angles from the first large tubule extending ventrally and that secondary and tertiary outpouchings occur from the branches. The tubules anastomose among each other and with those from other glands both cranially and caudally,but not across the median sulcus with the glands of the other side. The lumina may be continuous through the anastomosing tubules. A cast of the lumina shows frequent enlarged cavities from which small openings lead to the cyst-like cavities found in some of the secondary and tertiary tubules. No such glands were observed in any other part of the anterior lobe. If any glands be present in the roof of the anterior end they are simple and cystic or acinar in character. In some specimens, probably older animals, there are large tubular glands in the floor and lateral walls and even in the roof of the caudal extremity. The floor of the middle part occasionally shows cystic glands. The walls of the tubules are two or three cells thick and the columnar cells forming them are at right angles to the surface (fig. 28) . There is a periph


426


E. A BAUMGARTNER



Fig. 28 Transverse section of the floor and a gland of the anterior lobe of an adult. X 400. s, sinusoid.


eral zone of cytoplasm which is very finely granular and contrary to the statement of Tilney, it is acidophilic. Haller observed that the peripheral zone of cytoplasm in Mustelus stained intensively with borax carmine and Stendell stated that the peripheral layer of cells is acidophilic. Sterzi ('09) found that the cytoplasm of the cells in the floor of the caudal part of the rostral lobe is not granular. However, there are glandular outgrowths here in some adults and these walls have the same character as the other glandular parts, except in the mid- ventral line where no outgrowths are found, and here the wall is composed of low epithelial cells. The nuclei are oval and crowded nearer the inner free surface. They have a finely granular chromatic network. The roof of the anterior lobe is also composed of several layers of cells. If no glands are present, the nuclei here are very irregularly placed, some of them lying parallel to the surface along the inner free side, others being placed at various angles to the surfaces. The cytoplasm is very scant. The roof comes into close relation with the overlying brain, but a thin connective tissue layer containing small blood vessels and capillaries separates them. One cannot speak of a fusion of the roof of this lobe with the brain tissue above, as noted by Tilney.


DEVELOPMENT OF THE HYPOPHYSIS


427



Fig. 29 Transverse section of a gland of the inferior lobe of an adult. X 400.


b. Inferior lobes. The inferior lobes are large glandular structures, from the walls of which are many tubular outgrowths. These tubules are especially numerous on the ventral surfaces of the inferior lobes although there are some on the roof also. Two or three layers of columnar cells form the walls. In this case a wider cytoplasmic zone lies along the inner free surface (fig. 29). The cytoplasm is clear and the cell membranes stand out distinctly. The cells are very faintly acidophilie. The nuclei here, as in the anterior lobe, are oval in outline, and the long axis is always at right angles to the free surfaces. The chromatin here also is in a fine network with a more or less definite layer along the nuclear membrane.

c. Superior lobe. As has been observed in all selachians, there is a great glandular outgrowth from the roof of the superior lobe. As Sterzi has noted, these glands are not tubular but solid. The floor of this portion shows no glandular outgrowth. It is made up of several layers of columnar cells. The roof is several cell-layers deep and from it many thick columns extend upward.


428


E. A. BAUM GARTNER



Fig. 30 Sagittal section of the glandular cords of the superior lobe of an adult. X400.


The nuclei of the columns are spherical in shape, crowded close together in the center, and contain a light chromatin network and a nucleolus. Between these columns are numerous sinusoids. Along the periphery of the columns is a thick layer of granular cytoplasm, the granules being small and closely crowded (fig. 30) . The cytoplasm frequently takes a distinctly acid stain. In some eosin-methylene blue preparations the cytoplasm of the superior lobe along the periphery of the columns is stained blue. With iron hematoxylin this same zone sometimes retains the hematoxylin longer than does the chromatin. The granules of this part often stain an intense blue with Mallory's phosphotungstic acid stain. On either side of a granular area which stains very deeply there may be a clear area where the secretion possibly may be forming or has just been given up. I cannot, therefore, agree with Tilney ('11) who finds only eosinophilic cells in the superior lobe and basophilic in the anterior lobe. Besides, as has been noted, in many cases the cells of the anterior lobe take eosin quite as readily. Stendell ('13) thought that the division of chromophobic and chromophilic portions as described by Sterzi and others might not be true in all cases. In the adult Acanthias, it seems to me this distinction cannot be sharply drawn, at least not with all stains. The cytoplasm of the supe


DEVELOPMENT OF THE HYPOPHYSIS 429

rior lobe stains readily, and indeed, sometimes more deeply than that of the anterior lobe. The nuclei, on the other hand stain less deeply than those of the anterior lobe. Those of the inferior lobe frequently stain very lightly. Sterzi has stated that the cytoplasm of the superior lobe stains with difficulty.

d. Connective tissues, blood vessels and nerves. In very small embryos there are only occasional connective tissue cells lying between the hypophysis and the brain and vascular sac above, and these cells are along the dorso-lateral walls. Beginning in 33 mm. embryos, however, there is a thin layer of mesenchyma between the dorsal (posterior) end of the hypophysis and the anlage of the vascular sac. In 50 mm. embryos small blood vessels are found between the superior lobe and the saccus vasculosus (fig. 37). At this time, also, a thin layer of mesenchyma separates the hypophysis from the brain floor. In the superior lobe of the adult occasional small strands of connective tissue are found in the center of the core of nuclei of the columns.

They anastomose with the connective tissue between the columns but have no cytoplasmic zone bordering them as do the larger, more vascular connective tissue strands between the columns.

In the pup, there are small capillaries in the connective tissue over the anterior lobe of the hypophysis. On the ventral side there is considerable connective tissue between the developing tubules. The few capillaries here are not large. The capillaries between the columns of the dorsal lobe are large and numerous. These capillaries or sinusoids are to be found in the interstices between the cell columns and also between the ends of the columns and the overlying vascular sac. In the adult the capillaries over the anterior lobe are somewhat larger. There has been some increase in the size and number of the capillaries between the tubules of the anterior lobe and the same is true in the superior lobe between the cell columns.

Nerves have been described in the hypophysis by Edinger ('92), Sterzi ('09) and others. Sterzi described the floor of the brain in the hypophyseal area (above the superior lobe of the hy


430 E. A. BAUMGARTNER

pophysis) as composed of three layers, of which the middle was made up of nerve fibers coming from the caudal end of the inferior lobes of the brain. When these fibers reach the area above the superior lobe, numerous bundles of them go ventrally between the columns. These bundles are large and composed of large nerve fibers. According to Stendell ('12) a distinct lumen, continuous with the lumen of the vascular sac, extends into these bundles in Heptanchus. As Sterzi ('09) showed, these bundles are solid in Acanthias. Sterzi was not able to see any of the fibers ending in the cell columns. In some material stained with Mallory's phosphotungstic acid hematoxylin these fibers are well shown. I can not affirm that they do end in the cell columns between the cells as Sterzi was inclined to believe.

e. Secretions. Haller ('96) stated that the lumina of the superior lobe may contain cell detritus or a secretion. Tilney ('11) observed a colloid-like substance in the vascular sac above the superior lobe. Stendell ('13) described deep acidophilic secretion granules in the cytoplasm of the cells of the 'Zwischenlappen.' These colloid-like secretions were found in the cells lying towards the blood vessels. The adult Acanthias studied show no colloid-like secretion granules, such as Stendell found in the cytoplasm near the sinusoids in Mustelus and Scyllium. Considerable colloid-like secretion, however, is found in the tubules of the anterior lobe, also some in the main lumen of this part. The tubule drawn in figure 28 contains secretion. Some secretion was found in the tubular glands and in the main lumen of the inferior lobes. There are also many spaces in the superior lobe which are partially filled with secretion! The spaces are cylindrical in shape, sometimes as much as 20 fx in diameter and 50 to 200m long. They have no special walls, the cells and nuclei lining them appear to have been crowded aside. Frequently the inner layer of nuclei lies flat along the wall. These spaces never come in contact with the sinusoids, but are always found in the middle of the columns and are surrounded by nuclei which are crowded close together. Aresu (14) has described similar cyst-like spaces in Chimaera, containing a substance which stains lightly with basic stains. That the secretion does not fill the


DEVELOPMENT OF THE HYPOPHYSIS 431

spaces may be due to shrinkage, as has been suggested in the case of the colloid in the thyreoid follicles. In all respects it is similar to that found in the anterior lobe. There is no direct outlet by means of which a secretion can reach the cavity of the vascular sac and thus gain entrance to the ventricles of the brain. The absence of any secretion in the vascular sac also argues against there being a pathway for the secretion to enter it. It would seem probable that another secretion, distinct from this, is formed in the lumen of the tubules. As stated in the description of the histology of the adult hypophysis, the granular cytoplasm is always found on the peripheral side of the cell cords or tubules and never on the side toward the lumen. It is probable that this secretion finds its way into the numerous capillaries along the periphery. In the formation of two secretions, therefore, the hypophysis resembles the thyreoid in some forms, and also the hypophysis in some mammals.

2. Histogenesis of the hypophysis

In 7.5 mm. embryos the walls are formed of a single layer of cells of a large cuboidal type. The cytoplasm is slightly granular and somewhat acidophilic. The nuclei are large, somewhat oval, placed near the basement membrane, and contain considerable chromatin network. Usually several nucleoli, although sometimes only one, are found near the nuclear membrane (fig. 31). Very soon the walls of the hypophysis are composed of several layers of cells. In a 13 mm. embryo the large nucleoli are no longer so prominent and, as a rule, only smaller pseudonucleoli, as observed by Sterzi, are to be seen.

a. Anterior lobe. In a 21 mm. embryo, very little of the floor of the anterior lobe is as yet present. The walls are composed of two layers of columnar cells which have a thin outer and a thick inner, slightly granular cytoplasmic zone. The large and elongately oval nuclei contain a dense chromatin network, especially in the anterior end (fig. 32). Sterzi has observed that there are some elongated nuclei in the roof of the hypophysis in which the long axes are at right angles to the surfaces, and


432


E. A. BAUMGAETNER



Fig. 31 Transverse section of a portion of the hypophyseal anlage of a 7.5 mm. embryo. (H.E.C. 1503). X 500. B, region of brain; P, premandibular somite.



Fig. 32 Sagittal section of the roof of the anterior lobe in a 21 mm. embryo. (H.E.C. 1493). X 450. R, roof of anterior lobe; Med, medial connection of inferior lobes; P, pigment granules.


DEVELOPMENT OF THE HYPOPHYSIS

4


433



Fig. 33 Sagittal section of the floor of the anterior end and of one of the tubules of the anterior lobe of a pup. X 400.

between these are spherical shaped nuclei. He has described these in the roof of the superior lobe. These become more evident in 40 and 50 mm. embryos. In an 86 mm. embryo, the nuclei of the roof are to some extent irregular in arrangement. The cytoplasm takes orange G quite readily and hence is somewhat acidophilic. In the pup, the floor is well formed and is now the thicker wall of the anterior lobe. As shown in figure 33, it is composed of four or five layers of nuclei. The oval nuclei are crowded close to the inner free surface. There is, however, a narrow outer cytoplasmic zone which is very granular. The character of the cells of the glandular outgrowths is shown in figure 33. Here, too, there is a narrower outer rim of granular cytoplasm. A wider inner zone of non-granular cytoplasm bounds the lumina of the tubules. The nuclei, as in the floor, are oval. Usually two or three layers form the walls. The nuclei have a distinct chromatin network with occasional denserstaining chromatin masses resembling nucleoli.


434


E. A. BAUMGARTNER


b. Inferior lobes. The development of the inferior lobes as constrictions of the lateral walls of the early hypophyseal outpouching has been described above. The point where these lobes will grow together across the median line is indicated in figures 5 to 10. The character of the cells forming this part differs from the rest of the hypophyseal outpouching as early as the 21 mm. stage (fig. 32). The cytoplasm here stains less densely than that of the rest of the hypophysis. The nuclei are distinctly spherical in shape and have a very scant chromatin network. A part of this floor, immediately posterior to the hypophyseal stalk, contains a considerable amount of a granular yellowish pigment. Both Miiller ('71) and Hoffmann ('96) have noted the



Fig. 34 Sagittal section of the floor of the hypophysis near the median plane, showing the part Avhieh connects the inferior lobes. (H.E.C. 362). X 400.


pigment in the stalk of the hypophysis. The nuclei become more oval in older embryos. This is well shown in figure 34 which is a mid-sagittal section of the region which later forms the connection between the inferior sacs. The nuclei here are very irregularly placed. Extending caudally from the stalk are several nuclei flattened along the inner free surface. The cytoplasm stains very lightly. The pigment masses are numerous. Caudally there is a sudden transition to columnar cells of the kind found in the wall of the anterior lobe. The floor in this region is as just described until the 48 to 50 mm. stages when the inferior sacs are completely constricted from the anterior one. The pigment and the flattened nuclei are found only near the median line. Both are still present in a 41 mm. embryo.

In the inferior sacs proper, however, which are formed at the lateral sides, the cells are similar to those of the floor of the anterior lobe. The outer, narrower zone of cytoplasm, as well


DEVELOPMENT OF THE HYPOPHYSIS

o


435



Fig. 35 Sagittal section of a portion of the inferior lobe of the hypophysis of a pup. X 400.


as the inner, wider one, stains lightly in a 50 mm. embryo. In an 86 mm. embryo the outer zone has fine granules. The cells are slightly acidophilic. The nuclei are elongately oval in outline as in the anterior lobe. In the pup many outpouchings indicate the beginning glandular development. The upper wall is three or four cell-layers thick (fig. 35). There is a wide, inner, clear-staining cytoplasmic zone. The outer narrower rim is slightly granular. The nuclei are oval in outline, as in the anterior lobe. Numerous densely staining chromatin masses are to be seen. The lower wall or floor is much thinner. It is composed of only one or two layers of cells and has a narrow, inner, cytoplasmic rim. This zone, as in the roof, stains very lightly and is non-granular. The outer rim is, however, quite granular. The nuclei are like those in the roof but contain a somewhat denser chromatin network.

c. Superior lohe. In a 21 mm. embryo the wall of the superior end of the hypophyseal anlage is thickest where the superior lobe later develops (fig. 36). The nuclei are large and oval and have a light chromatin network. In a 50 mm. embryo (fig. 37) the wall in this region is considerably thicker than in the 21 mm. embryo because of an increase in the number of cell layers. There


436


E. A BAUMGARTNER



Fig. 36 Sagittal section of the superior end of the hypophysis of a 21 mm. embryo. (H.E.C. 1493). X 450.

Fig. 37 Sagittal section of the superior lobe of a 50 mm. embryo. (H.E.C. Series 444). X 450.

is an outer cytoplasmic zone which is non-granular and stains lightly. The nuclei are smaller than in the 21 mm. embryo and contain less chromatin. As Sterzi has stated, there are some spherical nuclei and some more slender oval nuclei, but no regular arrangement of these, such as Sterzi described, was observed. In a 95 mm. embryo, the roof of the superior lobe has increased in thickness (fig. 38). The nuclei are oval and contain a denser network of chromatin than is found in 50 mm. embryos. Along the periphery many of the nuclei are spherical. There is an inner zone of cytoplasm as in the 50 and 21 mm. stages, which is


DEVELOPMENT OF THE HYPOPHYSIS


437



Fig. 38 Transverse section of the superior lobe of a 95 mm. embryo. (H.E.C. Series 1882). X 450.

Fig. 39 Sagittal section of the superior lobe of the hypophysis of a pup showing one of the cell columns and a portion of the roof. X 400.


non-granular. Fewer mitotic figures are to be found at this time. In the pup the roof is a Httle thicker than in the preceding stage. Above the roof there are many cell columns which are outgrowths from the roof proper (fig. 39). The relations* of cell columns and roof to each other are shown in figure 39. The roof has a narrow outer zone of granular cytoplasm. Its nuclei are oval and have a light chromatin network. The cell columns come into close contact with the overlying floor of the vascular sac (fig. 39). Numerous capillaries and a loose connective tissue fill the spaces between the cell columns and between the roof of the hypophysis and the floor of the vascular sac. The columns have an outer, granular cytoplasmic zone. The cells are acidophilic. The nuclei are spherical in outline, have a light chromatin network and usually one or two larger chromatin masses or nucleoli.

Only a brief statement can be given at this time concerning the development of the glandular columns of the superior lobe. In the region of the superior lobe in all embryos up to the 50 mm. stage, the nuclei are elongate-oval in outline and are arranged in two or three layers (fig. 36). In a 50 mm. embryo some of the nuclei at the periphery are spherical (fig. 37). It is possible


438 E. A. BAUMGARTNER

that the elongate-oval nuclei in this region in the younger embryos are changed into spherical ones. In the pup there are numerous columns extending dorsally, which consist of a central group of spherical, light-staining nuclei and a peripheral zone of cytoplasm. Until some material between the 95 mm. embryos and the pup stage is studied, it must remain a question how these cell columns are formed. . It is possible that the cells with spherical nuclei arrange themselves in groups and these groups then evaginate from the roof. The scarcity of these groups in the 95 mm. embryos and in all late embryonic stages argues against such a possibility, although Sterzi's observations on the presence of groups of spherical nuclei lying between masses of cells with oval nuclei should be taken into consideration. Numerous cyst-like outpouchings are present in some adults in the anterior part of the superior lobe, or, rather, between this and the roof of the caudal extremity of the anterior lobe. Some of these outpouchings show areas of cells similar to those forming the columns of the superior lobe, interspersed with areas of cells like those of the anterior lobe. The areas of cells resembling those of the superior lobe may form the entire wall or may lie on a basement of cells resembling those forming the anterior lobe which line the cavity. This would indicate that the regions of the anterior and superior lobes are not sharply separated, or, that the cells of this region which still resemble the embryonic condition change into cell columns of the superior lobe. This need not imply, however, that the cells of the anterior and inferior lobes are of a more embryonic type, although they may be more primitive phylogenetically.

3. Development of the interhypophyseal canal

The formation of the ridge connecting the inferior lobes on the dorsal (posterior) side of the hypophyseal anlage had been described. The character of the epithelium in this region in a 21 mm. embryo, as stated above, differs from that of other parts. A ridge is prominent in a 34 mm. embryo. In a 40 mm. embryo the groove on the inside of this ridge is marked (fig. 8). In a


DEVELOPMENT OF THE HYPOPHYSIS 439

48 mm. embryo the ridge is very distinct and the connection of the lumen of this part with the lumen of the anterior lobe has become constricted. The constriction forms the narrowed connection of the, inferior sacs to the anterior lobe. As previously stated, the growth of the furrows separating the inferior sacs from the lateral sides of the anterior lobe is well marked at this time. A sagittal section of the hypophysis of a 50 mm. embryo shows a short interhypophyseal canal (fig. 9). In a 95 mm. enjbryo the canal has lengthened considerably. The walls are composed of one or two laj^ers of low columnar cells. In the pup the canal is longer than in the embryos but the diameter is about the same as in younger specimens. In the adult the canal has increased in length and diameter and is attached in the floor of the anterior

T.\BLE 4 Showing the size of the interhypophyseal canal


SIZE OF" SPECIMEN


LENGTH OP CANAL IN MM.


DIAMETER OF CANAL IN MM.


Embryo of 50 mm

95 mm

Pup


0.13

0.36

0.44 ^

0.68


0.042

0.048 048


Adult


080




lobe at or near its caudal end. Its other attachment is near the dorsal side of the connection between the inferior lobes. There are no tubular outgrowths from it such as Haller found in Mustelus. Table 4 shows the size of the canals at different stages. It will be seen that there is a continual increase in the length of the canal. The diameter in the older specimen is somewhat greater than in the 50 mm. embryo, although there is no great change. A distinct lumen is present. In the median line there is a well defined layer of connective tissue extending from the tip of the parachordal plate (fig. 9) forward to the floor of the anterior lobe of the hypophysis. This layer develops rapidly after the canal is formed and surrounds it. It is well defined in a 50 mm. embryo, but becomes thicker in the adult. This layer separates the inferior lobes from the rest of the hypophysis and

JOUHNAL OF MORPHOLOGY, VOL. 26, NO. 3


440


E. A. BAUMGARTNER


makes their dissection difficult. The cells and fibers in it are arranged concentrically around the canal. In the adult, the layer of connective tissue is still prominent and extends the entire length of the canal.





■i3^


Fig. 40 Transverse section of the hypophyseal stalk of a 28 mm. embryo. (H.E.C. Series 1357). X 350.

Fig. 41 Transverse section of the hypophyseal stalk of a 33 mm. embryo. (H.E.C. Series 307). X 350.


4. Development of the hypophyseal stalk

The stalk connecting the hypophysis with the mouth is formed in 22 to 24 mm. embryos. It is present in one 21 mm. embryo which shows the anterior end constricting from the mouth. In these stages it is oval in cross-section, the lumen is very large, and its walls are formed of a layer of low columnar cells, the nuclei of which are somewhat elongated and contain considerable chromatin. In the posterior (superior) margin are found many yellow pigment granules, as has been described by Hoffmann ('86). These granules are found within the cell, sometimes apparently in the nucleus (fig. 32). As Hoffmann stated, they are first seen in the bucco-pharyngeal membrane, but later occur also in the wall of the stalk. Miiller (71) had described them in the stalk in Acanthias embryos of 30 mm. length.

In 28 mm. embryos the lumen in the stalk is small. The wall consists of a double layer of epithelial cells, the outer of which


DEVELOPMENT OF THE HYPOPHYSIS 441






Q^ Q)




43


Fig. 42 Transverse section of the hypophyseal stalk of a 37 mm. embryo. (H.E.C. Series 363). X 350.

Fig. 43 Transverse section of the hypophyseal stalk of a 41.5 mm. embryo. (H.E.C. Series 369). X 350.

is low columnar in type (fig. 40) ; the inner is irregular and the nuclei are oval in outline but not regularly placed. Figure 40 is of a section through approximately the middle of the stalk. The lumen and stalk are larger where it connects with the hypophysis and with the mouth, both ends being funnel-shaped and joined by a narrower middle part. The connective tissue around the stalk is mesenchymal in character. Immediately around the stalk the cells are concentrically arranged. This character is more pronounced in later stages.

Ia a 33 mm. embryo the two ends of the stalk are funnelshaped, as before, and contain a lumen, while the middle part of the stalk is made up of a mass of cells in which no lumen is present (fig. 41). The outer layer of cells, though still definite at the ends, is no longer so in the middle part. The nuclei are more spherical in shape and contain a denser chromatin network. The arrangement of the mesenchymal cells around the stalk is concentric.

In a 37 mm. embryo the stalk is greatly reduced in size. The nuclei are massed in the center and are surrounded by a densely staining cytoplasm (fig. 42) . Some pigment is scattered throughout the stalk, as in all of the specimens. The concentric arrangement of connective tissue cells is more marked.

In some embryos 40 mm. in length no remnant of the stalk is found. In a 41.5 mm. embryo a small elongated densely stain


442 E A. BAUMGARTNER

ing stalk is present (fig. 43). What apparently is the remains of the nuclear mass is surrounded by a narrow densely-staining cytoplasmic rim. The chromatin network has disappeared, but larger masses of densely staining chromatin are to be seen. The concentric arrangement of the connective tissue cells is apparent, but not so marked as in the younger stages. In another specimen, 40 mm. in length, a strand of connective tissue, extending from a funnel-shaped mass of epithelial cells — continuous with the epithelial lining of the mouth — to the base of the hypophysis, indicates the position of the degenerated stalk (fig. 8) . The hypophyseal attachment of the stalk is anterior to the groove connecting the inferior lobes. Soon after this time the cartilages at the base of the brain become continuous across the mid line.

I wish to thank Dr. R. E. Scammon for his many helpful suggestions throughout this work. Thanks are also due to Dr. R. J. Terry for his kindly interest during its completion.

SUMMARY

1. The terms 'anterior lobe,' 'inferior lobes' and 'superior lobe' have been used for the several parts of the hypophysis of Acanthias.

2. Rathke's pouch forms the posterior part of the anterior lobe. The later evagination of the ectoderm, anterior to this, forms the middle portion and the anterior extremity of the anterior lobe.

3. The inferior lobes develop from the lateral sides of the posterior extremity of the anterior lobe, i.e., from the lateral sides of Rathke's pouch.

4. The superior lobe develops from the caudal (superior) end of the hypophyseal anlage.

5. In the course of development the hypophysis shifts in position about 145 degrees, so that the upper wall becomes the floor and the ventral (anterior) surface the roof.


DEVELOPMENT OF THE HYPOPHYSIS 443

6. There is glandular growth from the roof of the superior lobe and the inferior lobes, as has been described by Sterzi and others, and from the floor of only the anterior and posterior extremity of the anterior lobe in all adults.

7. The cells of both anterior and inferior lobes are acidophilic in character.

8. The cell columns of the superior lobe are solid as Sterzi described them.

9. Frequently the anterior and inferior lobes stain more densely than does the superior lobe. In these cases, it is the nuclei which take the darker stain. In general the anterior and inferior lobes may be considered the chromophilic ones.

10. Spaces containing some colloid-like secretion are present in the superior lobe. A similar secretion is present in the lumina of the tubules of the anterior and inferior lobes and in the large main lumen.


444 E A. BAUMGARTNER

ADDENDUM

After the completion of the present work a paper by M. W. Woerdeman: Vergleichende Ontogenie der Hypophysis" appeared (Arch. f. mikr. Anat., Bd. 86). This investigator figured Rathke's pouch in an 8 mm. Torpedo embryo. In somewhat older embryos (12-15 mm.) the region where Rathke's pouch opens into the mouth evaginates and in still later embryos a region anterior to this is constricted from the mouth. The hypophysis then consists of a small Rathke's pouch somewhat constricted from an anterior (ventral) 'Mittelraum' and anterior to the latter, the 'Vorraum.' The middle division, in 20 mm. embryos, divides by a circular constriction into a dorsal and a ventral part. In this way the ventral sacs are formed. The hypophyseal stalk now opens into the ventral sacs, in which observation Woerdeman agrees with that of Gentes and Stendell. According to Woerdemann's comparison of the parts of the hypophysis with those described by Stendell, Rathke's pouch is homologous with the superior lobe (table 1, p. 400) and the 'Mittelraum' and 'Vorraum' are homologous with the anterior lobe. The ventral sacs and lateral lobuli which he described are probably homologous with the inferior lobes. In Squalus I have described Rathke's pouch, or the early anlage of the hypophysis as giving rise to the caudal extremity of the anterior lobe. A later evagination ventral to this gives rise to the middle portion and anterior extremity of the anterior lobe. The secondary evagination is early recognized, as the epithelium here is thickened (page 408). The opening from the mouth to the early hypophyseal anlage, or Rathke's pouch, secondarily comes to open in the later evagination (page 410) of which there is only one in Squalus. The inferior lobes and the superior lobe develop from the early hypophyseal anlage in Squalus, as has been described (pp. 410-11). The hypophyseal stalk is not constricted from the anterior lobe with the developing ventral lobes but remains connected with the caudal wall of the middle portion of the anterior lobe until it disappears.


DEVELOPMENT OF THE HYPOPHYSIS 445

BIBLIOGRAPHY

Aresu, M. 1914 L'ipofisi in Chimaera monstrosa L. Anat. Anz. Bd. 47. Balfour, F. M. 1874 A preliminary account of the development of the elasmo branch fishes. Quart. Jour. Micr. Sc, vol. 14.

1878 A monograph on the development of elasmobranch fishes.

London. BuRCKHARDT, R. 1907 Das Zentral'-nervensystem der Selachier als Grund lage fi'ir eine Phylogenie des Vertebratenhirns. I Teil: Einleitung

und Sc>Tnnus lichia. Abh. d. Kais. Leop -Carol. Deutschen Akad. d.

Naturf., Bd. 73.

1911 Das Zentral-nervensystem der Selachier. II Teil. Die i'lbriger

Palaoselachier. Nova Acta. Abh. des Kais. Leop. -Carol. Deutschen

Akad. d. Naturf., Bd. 94. Chiarugi, G. 1898 Di un organo epitheliale situato al dinanzi della ipofisi

e di altri punti relativi alio sviluppo della regione ipofisaria in embrioni

di Torpedo ocellata. Monit. Zool. Ital., vol. 9. Edinger, L. 1888 Untersuchungen liber die vergleichende Anatomie des

Gehirns. I Das Vordenhirn. Frankfort a. M.

1892 Untersuchungen liber die vergleichende Anatomie des Gehirns.

II Das Zwischenhirn. Frankfort a M.; also, Anat. Anz., Bd. 7. Gentes, L. 1906 Signification choroidienne du sac vasculaire. Compt. rend.

Soc. Biol., T. 60; also Reun. Biol., Bordeaux.

1907 Recherches sur I'hypophyse et la sac vasculaire des vertebres. Travaux des Laborat. Bordeaux. Soc. Sci. d'Arcachon Sta. biol!, Pt. 1.

1908 a Developpement comparee de la glande infundibulaire et des plexus choroides dorsaux chez la torpille. Compt. rend. Soc. Biol. Paris, T. 64.

1908 b Sur le developpement des lobes infcrieurs chez les Selaciens. Compt. rend. Soc. Biol. Paris, T. 64.

1908 c Les lobes lateraux de I'hypophyse de Torpedo marmorata Risso; developpement du sac inferieur de cette hypophyse. Compt. rend. Soc. Biol. Paris, T. 64.

1908 d Developpement et evolution de I'hypencephale et de I'hypophyse de Torpedo marmorata Risso. Trav. Soc. Sci. Arcachon, Sta. Zool., T. 11.

Haller, B. 1896 LTntersuchungen liber die Hj-pophysis und die Infundibular organe. Morph. Jahrb., Bd. 25. Herring, P. T. 1908 A contribution to the comparative physiology of the

pituitary body. Quart. Jour. Expt. Phys., vol. 1. Hoffmann, C. K. 1896 Beitrage zur Entwicklungsgeschichte der Selachii.

Morph. Jahrb., Bd. 24. Johnston, J. B. 1909 The morphology of the forebrain vesicle in vertebrates.

Jour. Comp. Neur., vol. 19, no. 5. JoRis, H. 1908 Le lobe posterieur de la glande pituitaire. Mem. d. I'Acad.

de Med. d. Belgique, T. 19.

1909 Le glande neuro-hypophysaire. Compt. rend. d. I'Assoc. des Anat., 11 Reun.


446 E. A. BAUMGARTNER

VON KuPFFER, C. 1894 Die Deutung des Hirnanhangs. Sitzungsber. d. Ges.

f. Morph. u. Phys. in Munchen. VON Miklucho-Maclay, N. 1868 Beitrag zur vergleichenden Anatomie des

Gehirnes. Jena. Zeitsch., Bd. 4.

1870 Beitrage zur vergleichenden Neurologie der Wirbethiere. Das

Gehirn der Selachier. Leipzig. MtJLLER, W. 1871 Ueber Entwickelung und Ban der Hypophysis und der

Processus infundibuli cerebri. Jena. Zeitsch. f. Med. u. Naturwiss.,

Bd. 6. NisHiKAWA, T. 1899 Notes on some embryos of Chlamydoselachus anguineus

Garm. Annotationes zool. japonenses, vol. 2. Pettit, a. 1906 Sur I'hypophyse de Centroscjonnus caelolepis Boc. et.

Cap. Compt. Rend, de la Soc. de Biol., T. 61. Rabl-Rxjckhard, H. 1880 Das gegenseitige Verhaltnis der Chorda, Hypophysis

und des mittleren Schiidelbalkens bei Haifisch embryonen, nebst

Bemerkungen iiber die Deutung der einzelnen Theile des Fischgehirns.

Morph. Jahrb., Bd. 6. Reichert, H. 1877 Ueber das vordere Ende der Chorda dorsalis bei friihzeiti gen Haifisch Embryonen (Acanthias vulgaris). Abh. d. kongl. Akad.

Wiss. Berlin. RoHON, J. V. 1879 Das Zentralorgane des Nervensystems der Selachier.

Denkschr. k. Akad. Wiss. Wien., math, naturw. Kl. Rossi, U. 1902 Soprailobilateralidellaipofisi. Arch. Ital.Anat.Embr., vol. 1. Sanders, A. 1886 Contribution to the anatomy of the central nervous system

in vertebrate animals. Phil. Trans. Roy. Soc. London, vol. 177. Sasse, H. F. a. 1886 Bydrage tot de kennis von de ontwikkelung en beteekenis

der Hypophysis cerebri. Diss. Utrecht. ScAMMON, R. E. 1911 Normal plate of the development of Squalus acanthias.

Normt. z. Entwicklungsgeschichte d. Wirbeltiere, H. 12. Sedgwick, A. 1892 Notes on elasmobranch development. Quart. Jour. Micr.

Soc, vol. 33. Sewertzoff, a. N. 1899 Die Entwicklung des Selachier SchJidels. Festsch.

zum 70. Geburtstag von. C. v. Kupffer, Jena. Stendell, W. 1913 Zur vergleichenden Anatomie und Histologic der Hypophysis cerebri. Arch. f. mikr. Anat., Bd. 82. . Sterzi, G. 1904 Intorno alia struttura dell' ipofisi nei Vertebrati. Atti dell'

Acad. sc. veneto- trentino- istriano, vol. 1.

1909 II sistema nervosa centrale dei Vertebrati, vol. 2, libro 1, P. 1;

Padova.

1912 II sistema nervosa centrale dei Vertebrati, vol. 2, libro 1, P. 2;

Padova. Tilney, F. 1911 Contribution to the study of the hypophysis cerebri with

especial reference to its comparative histology. Memoirs of The

Wistar Institute of Anatomy and Biology, no. 2. ViAULT, F. 1876 Recherches histologiques sur la structure des centres nerveaux

des Plagiostomes. Arch. d. Zool. Experimentale, T. 5. ZiEGLER, H. E. 1908 Ein Embryo von Chlamydoselachus anguineus (Garm).

Anat. Anz., Bd. 33.


THE ANATOMY OF HETERODONTUS FRANCISCI

II. THE ENDOSKELETON^ J. FRANK DANIEL

From the Department of Zoology, University of California

THIRTY-ONE FIGURES (EIGHT PLATES)

CONTENTS

a. Introduction 447

b. The endoskeleton 448

I . Axial skeleton 449

1. The skull 449

a. The cranium 449

b. The visceral skeleton 455

2. The spinal column 466

II. Appendicular skeleton 470

1. The skeleton of the fin girdles 470

a. The pectoral girdle 470

b. The pelvic girdle 471

2. The skeleton of the fins 471

a. The paired fins 471

1 . The pectoral fin 471

2. The pelvic fin of female 472

3. The pelvic fin of male 473

b. The unpaired fins 473

1 . The first dorsal fin 473

2. The second dorsal fin 474

3. The caudal fin 474

4. The anal fin ' 475

Literature cited 475

INTRODUCTION

The skeleton of the elasmobranch .fishes has served for numerous investigations which have contributed much to our knowledge. Principal among these researches may be mentioned ' Das Kopfskelet' of Gegenbaur ('72) dealing with the head; studies on the

^ Part I, The exoskeleton, published in L^niv. Calif. Pub., Zool., 1914, vol. 13, p. 147.

447


448 J. FRANK DANIEL

column by Hasse ('79); and on the anatomy and development of the fins by Thacher (77); by Mivart (79), by Gegenbaur ('65), and by Balfour ('81). From exhaustive studies of the first group we have corrected our understanding of the nature of the skull, while from those of the third we have gained illuminating evidence as to the origin of paired limbs.

While considerable attention has been given to parts of the skeleton of the heterodont sharks, yet, so far as I am aware, no approximately complete study of any member of this group has previously been made. This may be due, at least in part, to the fact that the heterodont sharks are located in widely separated regions, and, furthermore, that they are not abundant in the localities where they occur. Heterodontus is more or less difficult to obtain on the California coast, yet through the efforts of the Scripps Institution of the University of California I have been provided with numerous specimens of various ages. I wish here to re-express my thanks to that Institution for its assistance.

While the head region of Heterodontus is specialized, as Gegenbaur maintained, yet it is by no means without generalization. In fact marked simplicity may be found side by side with specialization. Such a case is seen in the generalized type of quadrato-mandibular joint, accompanied by a highly specialized mode of ligamentous articulation.

It would be generally conceded that the fin skeleton also is specialized, if one agree with Mivart's conception of specialization meaning concrescence. However this may be, it is certain that, had Mivart known of the unpaired fins of Heterodontus francisci, his argument for similarity of plan between paired and unpaired fins would have been even more convincing.

THE ENDOSKELETON

In the following paper I shall discuss at some length the endoskeleton of Heterodontus francisci. In such a consideration the skeleton naturally is divided into: (1) The axial part, including the skull and spinal column; and (2) the appendicular skeleton, embracing fins and fin-girdles.


ANATOMY OF HETERODONTUS : ENDOSKELETON 449

I. AXIAL SKELETON

1. The skull

The skull, like that of elasmobranchs in general, is composed of: (a) A cranium or brain case to which the sense capsules are fused in the adult; and (b) a series of cartilaginous visceral arches which support the buccal and pharyngeal regions.

a. The cranium or brain case in dorsal view (fig. 1) is roughly quadrilateral in shape, slightly bifurcated at the anterior and posterior margins, and constricted along the sides at the first and second thirds — the first of these indentations being much the more pronounced. The cranium is a closed box except at the antero-dorsal end, where there is a large opening, the anterior fontanelle (F.) and at the posterior end, where is located the foramen magnum (f.m.) through which the spinal cord joins the brain.

In the mid-dorsal line joining these two openings several structures appear. These are, passing forward, a ridge, the occipital crest (o.cr.) which runs to the parietal fossa (p.f.); from the bottom of the latter the endolymphatic ducts lead to the ears. In front of this. pit is a slight elevation which sinks immediately into a long groove — ^the parieto-frontal groove (p-f.g.) which, in turn, broadens out into the anterior fontanelle.

On each side of and running parallel to the parieto-frontal groove there is a row of foramina extending posteriorly to the level of the parietal fossa. Anteriorly the first two of these on each side are the ophthalmics, through the first of which passes the ophthalmicus profundus nerve (/.o.p.'), through the second, the ophthalmic division of the seventh nerve (f.o. VIT') . Through the numerous and smaller perforations which follow pass branches of nerves, and through the succeeding large foramina, blood vessels. These openings terminate posteriorly at an elevation produced by the anterior oblique semicircular canal {a.o.s.) which with a similar elevation from the opposite side roughly forms a broad V enclosing at its apex the parietal fossa. Below this there is a large inverted lower V, the arms of which enclose the foramen magnum (f.m.), and the apex of which abuts


450 J. FRANK DANIEL

against that of the V above described so that the two V's roughly form an hour-glass.

Viewed from the ventral side (fig. 2) the cranium is roughly flat-iron-shaped, with the apex projecting between the olfactory capsules (oLc). At the most posterior part of the cranium is a niche, the sides of which are produced by the occipital condyles (o.cd.). In the mid- ventral line, one-fourth the distance from the posterior border to the tip of the nose, is a foramen (or a pair of foramina) through which the internal (posterior) carotid arteries reach the brain (f.i.c); laterad of these on each side are similar perforations (J. ex.) through which the external (anterior) carotids pass on their way to the orbital region. A line through the internal carotid foramina, and at right angles to the long axis of the cranium, divides the ventral cartilaginous mass into two regions, the anterior of which is the embryonic trabecular region, the posterior region that of the parachordal cartilages.

Along the lateral margin, in ventral view, from behind forward are the post-articular processes (po.hm.) bounding posteriorly the deep fossa into which the hyomandibular cartilage fits. Anterior to the fossa is a pre-articular process [pr.hm.). In front of the latter is a constriction, anterior to which is a wide projection — the basal plate (b.p.) ; considerably in front of the basal plate, at the sides, is the palatal fossa (pl.f.) into which a projection of the palatoquadrate cartilage fits. At the anterior tip of the cranium the basitrabecular cartilage (b.tr.) arches upward to be met by two dorsolateral rostral pieces coming down from the dorsal part of the cranium. At the sides of the basitrabecular piece are the external openings for the olfactory capsules {ol.c).

In this view may be described the olfactory capsules and the nasal cartilages at their margins. The capsules are thin, cartilaginous structures which are formed as the skeletogenous protection for the olfactory organ. Dorsally the capsules are continuous with the cranium (see also fig. 1); ventrally they thin out to delicate lamellae of cartilage which surround the nasal aperture, excepting in the postero-medial part, where the


ANATOMY OF HETERODONTUS : ENDOSKELETON 451

wall is membranous (fig. 2). The olfactory cup or inside of the capsule communicates internally with the cranial cavity by the olfactory foramen, through which the first cranial nerve passes.

At the free margin of the capsule there is a scroll-like nasal cartilage {n.c.l , fig. 2), which runs on the outer margin around the aperture of the capsule. Both the anterior and posterior ends of the cartilage recurve upon themselves medially so as to form a narrow ellipse, across which, from its antero-lateral third, a projection extends backward and inward, forming of the ellipse a figure 8.

A second nodule of cartilage {n.c.2; not figured by Gegenbaur '72 for H. philippi, pi. 16, fig. 2) is loosely attached to the anterior end of the first nasal cartilage. The attachment is made at its broader anterior end and its free tip extends backward to be connected by tissue with the deeper recurved anterior end of the first cartilage.

In side view (fig. 3) the olfactory capsules occupy a position remote from the main part of the cranium. Projecting from the postero-lateral part of the cranium are the thick-walled auditory capsules (ax.) which give protection to the organs of hearing. Between the auditory and nasal capsules is the large socket or orbit in which is located the eye. Overhanging the orbit is the broad supraorbital crest (s.o.) from the anterior part of which arises the preorbital process {pr.o.) and from the posterior, the post-orbital process (po.o.). The floor of the orbit extends outward as the basal plate. Anterior to this plate and running between the orbit and the olfactory capsule is the elongated palatal fossa (pl.f.) previously noted in ventral view.

Perforating the brain case in the orbit are numerous foramina through which nerves or blood vessels course between the brain on the one hand and the structures of the eye and the facial region on the other. Ventrally and a little in front of the middle of the orbit is a large opening, the optic foramen (/.//) , through which the optic nerve reaches the brain. Above and slightl}^ anterior to the optic is a small trochlear foramen (f.IV) through which the fourth cranial nerve passes to the superior oblique muscle of the eye. Behind the optic foramen, and in the lower


452 J. FRANK DANIEL

posterior angle of the orbit, is the large facial foramen (f.VII), through which branches of the seventh or facial nerve pass. Almost in the same foramen but slightly ventralward and forward is a small opening for the entrance of the external carotid artery to the orbital region {f.e.c.'). Between the facial and optic foramina is a small perforation for the entrance of an artery, the ramus anastomoticus of Hyrtl (f.r-a.). This, in Gegenbaur's ('72) plate 2, figure 1, has been marked incorrectly the 'Querer Basalcanal.' Above the facial is the large orbital fissure (o./.) (trigeminal opening) through which pass the fifth, sixth and the first part of the seventh cranial nerves. Slightly above the middle part of a line connecting the orbital fissure and the optic foramen is the oculomotor {f.III) for the exit of the third cranial nerve from the brain to muscles of the eye. Between the orbital fissure and the foramen for the ramus anastomoticus artery is the interorbital canal (i.o.) by means of which the orbital sinuses of the two sockets communicate. In the anterodorsal angle of the socket are two foramina, the larger and upper of which is the ophthalmic (f.o.VII) for the superficial branch of the seventh nerve; the smaller and more ventrally placed is for the deep ophthalmicus profundus (f.o.p.). In the last mentioned opening is a second smaller foramen for the anterior cerebral vein. This leaves the cranial cavity in the region above the olfactory lobe. Below this, in the anteroventral angle of the socket is the posterior entrance to the orbito-nasal canal (o-n.) through which a vein passes from the olfactory region. (For the anterior end of this canal see o-n.', fig. 2.)

A median sagittal section through the cranium (fig. 5) shows the cavity for the brain. Surrounding this are the walls of the brain box through which the foramina lead. Dorsally the cranial roof or tegmen cranii varies considerably in thickness. Posteriorly and above the foramen magnum {f.m.) is a thick portion through the occipital crest (o.cr.). Anterior to this the wall pits sharply downward forming the parietal fossa. From this fossa the roof again arches upward and then, as the parietofrontal groove, passes forward to the anterior fontanelle.


ANATOMY OF HETERODONTUS: ENDOSKELETON 453

From the fontanelle anteriorly the walls are extended by the rostral and basitrabecular cartilages.

Along the ventral margin the floor or basis cranii also shows extremes in thickness. Directly under the anterior fontanelle it is relatively thick. It then becomes thinner and thinner posteriorly until it reaches the foramen for the internal carotid artery {J. i.e.). As we have said (p. 450) this foramen divides the basis cranii into two parts, the anterior of which is the embryonic trabecular, and the posterior the parachordal region. The parachordal or the part accompanying the notochord is greatly thickened. It extends to the posterior part of the cranium as a somewhat spool-shaped segment. Inside of this is the cranial notochord (c/i.), and surrounding it posteriorly are calcified tissues.

Posterior to the end of the cranial notochord but not in the middle line appears the occipital condyle (o.cd.). Other structures are seen below the basis cranii and in the background. These in front of the occipital condyles are the post- and prehyomandibular processes; under the socket is the down-curving basal plate ; and under the anterior fontanelle, the margin of the palatal fossa.

The foramina perforating the cranium are here seen to advantage. The most anterior of these is the large opening through which the olfactory tract passes (/./). Midway between this and the occipital condyle is the optic foramen (/.//) ; above and anteriorly is the anterior cerebral foramen (f.a.c). Almost directly above the optic is the trochlear (J. IV). SHghtly posterior to the optic are two foramina, the upper for the oculomotor nerve {J. Ill) , the lower for the ramus anastomoticus artery (f.r-a.) . Above the entrance to the internal carotid artery is the interorbital canal (i.o.). Above the anterior tip of the cranial notochord are two foramina, the upper of which is the large orbital fissure (p.f.); the lower of the two is a double foramen, the anterior division of which is for a part of the facial nerve; the posterior gives the acustic or eighth cranial nerve access to the ear (f.VIII). Posterior to this is the smaller foramen for t'he glossopharyngeal nerve, behind which is the larger


454 J. FRANK DANIEL

foramen for the vagus (f.X) . Below the vagus are three smaller foramina (two in fig. 5) through which trhe spino-occipital nerves of the ventral root type pass. Finally between the posterior end of the cranial notochord and the ventral margin of the occipital crest is the large foramen magnum {f.m.).

At the posterior end of the cranium (fig. 4) ventral to the foramen magnum is the cranial notochord, at the sides of which are the occipital condyles (oxd.) by which the spinal column is joined to the cranium. At the side of and slightly above each condyle is the large foramen for the vagus nerve (f.X) ; above this are two smaller foramina through which a spino-muscularis artery (f.s-m.) perforates the cartilage without entering the brain case. These, I take it, are the foramina which Haswell ('84, p. 93) describes for Crossorhinus as "a pair of small apertures of unknown function." Still further laterally is the opening for the glossopharyngeal or ninth cranial nerve {J. IX). At the lower angles of the posterior part of the cranium are the post- and superior articular processes {po.hm. and s.hm.) by which the hyomandibular suspensorium is fixed to the cranium.

A view of the rostral region (fig. 1 and text fig. A) explains the structures there involved. It is noted that the pointed anterior end bifurcates into dorso-lateral halves which, near the middle line, bend downward and fuse with the median ventral trabecular piece. These, in Heterodontus francisci, were they compared with a form in which the rostral cartilages compose a wellmarked framework, as for example Pseudotriacis microdon (Jaquet '05), would show but slightly their rostral nature. A comparison with another type is instructive in this respect. In Crossorhinus (Orectolobis) Haswell ('84) figures a cranium (h of text fig. A) in which he describes paired pieces {rst.) as being prolongations of the ventral floor. From the condition present in the heterodont sharks it would seem not improbable that they are in fact projections from the dorsal and the lateral walls rather than from the floor. If such be true the likeness between Crossorhinus and Heterodontus in this regard is striking, for a union of the median rostral piece (rst.) with the olfactory wing {ol.ivg.) above and of the olfactory wing and the basitra


ANATOMY OF HETERODONTUS : ENDOSKELETON


455


becular cartilage (ba.tr.) below would give to Crossorhinus a type of rostrum much like that of Heterodontus.

b. The visceral skeleton is composed of a series of right and left cartilaginous arches which more or less completely surround the buccal cavity and the pharynx. These, in Heterodontus, if viewed, say, from the left side, are like those of other pentanchid sharks, seven in number; they may be divided into two groups. The' first group comprises the first and second arches,



t.


Text-fig. A Nasal region of Heterodontus francisci and Crossorhinus barbatus ; ba.tr., basi-trabecular cartilage; f.op.pr.', ophthalmic foramen for profundus nerve; f.op.Vir, ophthalmic foramen for facial nerve; Fo., anterior fontanelle; ol.wg., olfactory wing; rst., rostral cartilage.

the first of which, the mandibular, is composed of the upper and the lower jaw; the second, the hyoid arch, is similarly made up of two segments. The second group consists of five branchial arches which support the pharynx. In structure the branchial arches are essentially similar to the first two arches excepting that in these there are typically four segments to an arch. These differ among themselves, however, in minor details.

The mandibular, or first arch (fig. 7), has become the most highly specialized of all the visceral arches. Its upper segment, representing the palatal and quadrate regions, is called the


456 J. FRANK DANIEL

palato-quadrate (p-g.) ; the lower segment is the mandibular or Meckel's cartilage {md.). In Heterodontus this arch is closely attached to the cranium in the preorbital region by a capsular ligament which keeps the upper and anterior margin (a. p.) of the palato-quadrate in the palatal fossa of the cranium. A slip from the capsular ligament {c.l.' fig. 7), arising along the ventral margin of the cranium under the fossa (*fig. 2), extends backward and downward to join the quadrate on the ventral part of the transverse median ridge {tr.m.r.). Just under the orbit both the upper and the lower segments of the mandibular arch flare outward in Heterodontus francisci so that the distance to the spiracular cartilage or to Huxley's so-called otic process is, I take it, greater than that described by Huxley for Heterodontus philippi. Posteriorly, the arch has no direct attachment to the cranium but is held in position by ligaments soon to be described.

As a cartilage the palato-quadrate {p-q., figs. 6 and 7) is longer than the mandible. Its upper margin is irregular, due principally, to a dorsal indenture in the anterior third. The anterior wall of this indenture comes in contact with the ethmoidal region, while the posterior wall of the indenture, as seen in figure 7 {a. p.) serves as a process of the palato-quadrate which fits into the palatal fossa of the cranium. Medially from this articular surface a sharp ridge {tr.m.r.) runs, to the lower part of which is attached the slip from the capsular ligament above described. Externally, at the beginning of the posterior third of the quadrate tl^ere is a strong lateral transverse ridge which passes almost across the cartilage -(ir.Lr., fig. 6). To this ridge are attached tendinous fibers of the adductor mandibularis muscle. Posteriorly and dorsally the palato-quadrate is provided with a lateral flattened enlargement, the hyal process (hl.p.), also for muscular attachment. The hyal process is continuous with a similar process on the mandible.

The mandible {md., figs. 6 and 7) is an unusually heavy cartilage. The angular part of this is high, considerably elevating the quadrato-mandibular joint. If seen from below, the mandible would appear as a strongly crescentic cartilage, the poste


ANATOMY OF HETERODONTUS : ENDOSKELETON 457

rior tip of which extends considerably laterad of the anterior. Inside of and below the teeth, there is a long ridge {md.r., fig. 7) from which a tendinous bridge passes to a similar ridge on the other side; to the lower sides of this ridge the strong coracomandibularis muscle is attached; near the quadrato-mandibularis joint and mediad there is present a prominent mandibular knob (kb.) against which the second arch abuts.

The joint between the palato-quadrate and the mandible, like that in Chlamydoselachus (Goodey '10, pp. 544-545) and Heptanchus (Gadow '88, pp. 452-453) forms a double ball and socket. The anterior articulation is formed by a ball of the mandible fitting into a socket of the quadrate. The posterior articulation consists of a large socket in the outer angular part of the mandible, into which a ball from the hyal process of the quadrate fits. Between the two articulations in Heterodontus is a space, somewhat like that described by Gadow ('88) for Heptanchus.

A description of the articulations of the first arch, further than the attachment of the palato-quadrate to the cranium as above described (p. 456), may be deferred until a study is made of the second or hyoid arch.

The hyoid arch (fig. 6) as we have seen, is also composed of two segments. The upper division, the epihyoid, becomes in Heterodontus an important suspensorium for the mandibular or first arch; it is called the hyomandibula (hm.). The lower segment of the arch is the ceratohyoid or hyoid proper (c-h.). Connecting the two ceratohyoids of opposite sides is a median unpaired piece, the basihyal cartilage {h.h., fig. 11).

Both of the segments of the hyoid arch are heavy cartilages. The hyomandibula is thickened both proximally, where it fits into the deep fossa under the auditory capsule, and distally, where it joins the ceratohyoid and touches the mandible near the quadrato-mandibular joint. The ceratohyoid is considerably longer than the hyomandibular segment, and extends forward and inward to meet the basihyal.

Articulations of the hyoid arch. The hyomandibula is bound by a strong capsular ligament to the hyomandibular fossa in the


458 J. FRANK DANIEL

cranium. This, in figure 6, has b^en removed so as to show the proximal end of the hyomandibula. The superior post-spiracular Hgament (s.p-s.l.) of Ridewood '96; (see also W. K. Parker 79), arising in the postero-ventral angle of the socket and anterior to the auditory capsule, attaches itself to the distal third of the hyomandibula. Further, the hyomandibula is bound to the ceratohyoid by a hyomandibulo-hyoid ligament {l.h7n-h, fig. 6) which arises on the side of the distal end of the hyomandibula and passes over to the anterior and inner face of the ceratohyal segment, extending thence to its distal third.

A series of ligaments may next be described which are effective in swinging the first or mandibular arch, all but one of which connect this arch directly to the second. That one, however, indirectly and in part, attaches the first arch to the cranium. Those binding the first arch to the second directlj^ and appearing externally are three in number. The first of these is a dorsal ligament {l.hm-q., fig. 6) which passes from the upper part of the hyal surface of the quadrate posteriorly to the medial and anterior part of the hyomandibula. This ligament is doubtless that part of the superior post-spiracular ligament which Ridewood ('96, p. 427) described for Scyllium as attaching on the quadrate. In Heterodontus francisci, however, its attachment is on the hyomandibula, few of its fibers being continuous with the superior post-spiracular ligament. I have therefore called it by a separate name, the ligamentum hyomandibulo-quadratum {l.hm-q. ).

At the joint there is a complex median ligament {l.m., figs. 6 and 7) which passes from the inner side of the quadratomandibular joint externally, principally, to the cerato-hyoid cartilage. The quadrate part of this ligament {l.m., fig. 7), however, arising under the large ligament which joins the mandible to the quadrate {l.q-m.i.), runs upward and posteriorly to attach to the hyomandibula, mediad of and slightly distal to the attachment of the ligamentum hyomandibulo-quadratum. x-ill of those fibers of the median ligament which arise from the joint and from the mandible {l.?n., fig. 7) are attached to the ceratohyoid.


ANATOMY OF HETERODONTUS : ENDOSKELETON 459

From the ventral angle of the mandible a third ligament, the ligamentum hyoideo-mandibulare (l.h-m., fig. 6) (the ligamentum hyoideo-mandibulare externum of Goodey '10) extends posteriorly to be attached to the inner margin of the ceratohyoid segment. Near the mandibular attachment this ligament is perforated by an artery.

A most complex suspension is made by a ligament which passes from the medial side of the mandible to the ventral side of the cranium. For want of a better name I shall call it the ^ligamentum complexum' (Ixp.). Only a part of its course can be shown in figures 6 and 7. This arises as a double band (l.cp.., fig. 7), ventral to the mandibular knob, and passes outward over the ceratohyoid to which some of its deeper fibers are attached {l.cp., fig. 6); it then gives a bundle of fibers to the hyomandibula and, with fibers from the hyoid, runs upward and mediad of the hyomandibula to be attached vent rally to the base of the cranium slightly anterior to the external carotid foramen (see fig. 2). It seems probable that the upper part of this ligament at least is comparable to the inferior post-spiracular ligament of Ridewood ('96).

It is thus seen that, excepting the suspension rendered by this complex ligament just described and by a few fibers from the ligamentum hyomandibulo-quadratum, the first arch in the posterior region is suspended entirely by the second. The attachment of the first by the second is so complex that it would be hard to agree with Huxley ('76, p. 43) that "The 'epibranchial' (hyomandibula) of the hyoidean arch of Cestracion (Heterodontus) is just beginning to take on a new function, that of suspending the palato-quadrate cartilage and mandible to the skull."

It may be added that the union of the quadrate and mandibular cartilages is made principally bj^ a large ligament, the ligamentum quadrato-mandibulare internum (l.q-m.i., fig. 7) already mentioned. This attaches to the upper internal border of the quadrate above and extends along the posterior border of the transverse median ridge of the palato-quadrate {tr.m.r.) and over the anterior articulation of the quadrato-mandibular

JOURNAL OP MORPHOLOGY, VOL. 26, NO. 3


460 J. FRANK DANIEL

joint to be attached to the lower ventral margin of the mandible. Further, it may be said that a slip from this ligament, the ligamentum hyomandibulo-mandibulare (l.hm-m.), passes upward to be attached on the hyomandibula, just proximal to the quadrate slip of the median ligament and directly under the attachment of the hyomandibulo-quadratum. It thus results that a puncture through the fibers of the superior post-spiracular ligament and through the attaching fibers of the ligamentum hyomandibulo-quadratum would perforate the attaching fibers of this ligament.

Finally other ligaments may here be mentioned. Strong fibrous bands run lengthwise of the lateral or concave surface of the mandible and the palato-quadrate to the quadratomandibular joint. The one on the mandible sends a slip upward to attach on the quadrate just mediad of the first (anterior) articulation. A similar slip from the quadrate attaches on the mandible just mediad of the second (posterior) articulation. These two attaching ligaments form a curious type of scissor ligament.

The cartilaginous gill-rays. There are present on the hyomandibula (epihyal) and on the ceratohyal segments of the second arch, cartilaginous rays which project outward and backward as supports for the gill septa. These show considerable variation in different specimens. In one of the large males the first six of these on the hyomandibula fuse at their proximal ends into two masses. The sixth is followed by nine single rays which meet and fuse at their proximal ends into a half arch. Eight single rays of the ceratohyoid, similarly fusing at their base, complete the arch. This arch then encircles the articulation between the two segments. Following these upper ceratohyal rays there are six pairs of rays fused at the bases into three pieces, and following these there are two or three stout rays.

The first branchial arch (fig. 10) consists of four segments, three of which are shown in figure 10. These, counted from the dorsal to the ventral side, are: (1) the pharyngobranchial (p-h.), (2) the epibranchial (e-h.), (3) the ceratobranchial (c-h.), and (4) the hypobranchial {h-h., see fig. 11). From below, the


ANATOMY OF HETERODONTUS: ENDOSKELETON 461

arch slants obliquely backward so that the pharyngobranchial is considerably behind the outer segments of the arch.

The upper or pharyngobranchial segment is a triangularshaped cartilage, the apex of which points ventro-laterally, and the broad base of which forms its dorso-median margin. It is not bound by pronounced ligament to either the pharyngobranchial of the opposite side or to the spinal column, but it is held in place dorsally by connective tissue. A ligament is attached to the neck of the pharyngobranchial just above its union with the epibranchial segment. To a further consideration of this ligament we shall return. Here it may be said simply that it passes posteriorly to the head of the following epibranchial segment.

The epi- and ceratobranchials are stout cartilages, the latter being considerably longer than the former. Near their joint both cartilages are hollowed out (not seen in fig. 10) so as to increase the angle between the two. The joint between them is simple, the articulating surfaces being held together by a connective tissue capsule.

The epi- and ceratobranchial segments are of great importance to the area since they alone possess cartilaginous branchial rays for the support of the gill tissues (b.r., fig. 10). On the first arch fourteen such rays are usually present, five on the epiand nine on the ceratobranchial segment; the first on the ceratobranchial is in all cases the longest of the series.

The hypobranchial segment of the first arch {h-b.l , fig. 11) is much smaller than any of the other segments. It is not connected with the hypobranchial of the other side or with a median basibranchial cartilage, but remains as a rudimentary cartilage connecting the ventral ends of the first ceratobranchial with the cerato- and basihyal cartilages.

Dorsally the segments of the second, third and fourth arches are essentially like the first, except that the pharyngobranchial segment of the fourth has fused with that of the fifth arch (fig. 14) . Ventraliy, these arches differ from the first principally in that their hypobranchial segments are well developed {h-b.2-4, fig. 11). The hypobranchial segment in these is so arranged as to


462 J. FRANK DANIEL

be attached by pads of tissue to the end of its own ceratobranehial and also to that of the ceratobranchial just in front. These hypobranchials then run posteriorly and medially to join unpaired cartilages soon to be described.

The fifth branchial arch is greatly modified (fig. 14). Its pharjmgobranchial segment has been so distorted as almost completely to change the appearance of the upper part of the arch. Its epi- and ceratobranchial segments (e.b.5 and c.h.6) are stout cartilages, devoid of branchial rays. The hypobranchial segment is absent, and its ceratobranchial is attached directly to a median unpaired cartilage — -the basibranchial (fig. 11) .

The median unpaired pieces and their relation to the segments of the arches are shown in figure 1 1 . The most anterior of these is the basihyal cartilage, to which reference above has already been made and which may be described as a more or less star-shaped cartilage joining the right and left halves of the ceratohyoids in the mid-ventral line. It has an anterior triangular glossal projection (g.p.) which bends upward in the floor of the mouth to form a support for the so-called tongue.

A first basibranchial cartilage has been mentioned by White ('92, p. 299) as characteristic of Heterodontus (Cestracion). (See also Gegenbaur '72, pi, 19, fig. 3). This, according to Gegenbaur, is located in H. philippi as a free nodule of cartilage in the middle line, posterior to the basihyal cartilage. For the same form Karl Fiirbringer, ('03) describes paired basal cartilages. In Heterodontus francisci I have not found either the azygos cartilage of Gegenbaur or the paired cartilages described by Fiirbringer. I have found, however, cartilages in this region which I have described as extra-hyoid cartilages. These are identical in shape and direction with those given by Fiirbringer ; but I am convinced that they are not first hypobranchial cartilages since they lie superficial to the afferent artery. The second basibranchial forms a large median piece (b.h.) to which the hypobranchials of the second branchial arch are united. Posteriorly this median piece joins an enlarged backwardly-directed, arrow-shaped piece (m.p.) to which the third and fourth pairs of


ANATOMY OF HETERODONTUS : ENDOSKELETON 463

hypobranchials and the fifth ceratobranchials are attached. This piece bears a posterior segment.

The discovery of a vestigial sixth branchial arch in the Heterodontidae (Hawkes '05) has been of considerable interest. This arch was found in the young both of Heterodontus francisci (Gyropleurodus francisci) and of H. philippi. Since this discovery, however, an additional rudimentary arch has been described in several other elasmobranchs.



Cr:"B<6


Text-fig. B Rudimentary sixth branchial arch of Heterodantus francisci; Cr.Br.5., fifth ceratobranchial cartilage; Cr.Br.6., sixth or rudimentary cerato branchial cartilage; Ep.Br.5., fifth epibranchial cartilage; Ep.Br.6., sixth or rudimentary epibranchial cartilage; Lg., ligament passing under pit; pt., pit in neck of pharyngobranchial cartilage.

The rudiments of the arch found by Hawkes in Heterodontus francisci consist of a pair of pieces located back of the fifth arch. The upper piece is attached by a ligament to the epibranchial segment of the fifth arch and is joined below to the second rudimentary segment. These pieces are interpreted by Hawkes either as the cerato- and hypobranchial, or as the epi- and ceratobranchial segments which have become closely joined to the fifth arch.

In the adult specimen these cartilages {ep.hr.6 and cr.br.6, text-fig. B, and fig. 14) are much like those described for the


464 J. FRANK DANIEL

young. The upper segment {ep.hr.6) is a slender cartilage, (loosely) attached dorsally to the fifth pharyngobranchial, but ventrally, as is seen from the figure, it is fused solidly to the fifth epibranchial segment. If viewed from the median side, however, it is seen to be continuous with the lower segment. .

I have already described for the first branchial arch a ligament which extends from the neck of the pharyngobranchial backward and downward to be attached to the epibranchial following. To this I shall now return with a hope that it may be of service in interpreting the dorsal piece in question. It so happens that this ligament forms the floor of a pit, the anterior wall and the roof of which are formed by the neck of the first pharyngobranchial and the posterior wall by the upper part of the following epibranchial segment."^ Similar pits follow the second, third and fourth {Ig., text fig. B) arches. Now, following the fifth pharyngobranchial there is also a pit {pt., text-fig. B) which, in its surroundings, is identical with the first. From the neck of the fifth pharyngobranchial the ligament passes under the pit, giving some of its fibers to the posterior wall; other fibers from the fifth pit continue across with the ligament from the fourth pit to join the pectoral girdle. The piece forming the posterior wall {ep.hr.6) I therefore interpret as the sixth epibranchial segment.

The lower or second segment, the ceratobranchial, is displaced forward so as to lie laterad of the joint of the fifth arch. This displacement in Heterodontus is due to the enlargement and crowding forward of the massive pectoral girdle.

There is present in the anterior wall of the spiracle a thin spiracular cartilage {sp.c, pi. 3, and fig. 12) which, like branchial rays, supports a septum for gill filaments. This cartilage is (generally) interpreted as a fused series of cartilaginous rays which originally belonged to the palato-quadrate segment. Because of the outward flaring of the quadrate the spiracular cartilage comes to be widely separated from the quadrate.

^This ligament probably represents the median interarcuales muscles.


ANATOMY OF HETERODONTUS: ENDOSKELETON 465

Extra-visceral cartilages. The visceral arches are provided with superficial pieces, the extra-visceral cartilages. These for convenience may be separated into the labial cartilages, the extra-hyoid and the extra-branchial cartilages. The labials are located at the sides of the mouth and consist of three pieces of cartilage on each side, two dorsal and one ventral {d.l.1-2 and V.I., fig. 6). The posterior dorsal labial is about twice the length of the anterior dorsal labial cartilage; it articulates with the ventral labial at its distal end so that the two serve to reduce the gape of the mouth.

An extra-hyoid cartilage, so far as I have been able to make out, is lacking dorsally, and the one which appears ventrally is small. This, in the adult, is generally a nodule of cartilage less than half the length of the one shown in figure 9. In all cases the extra-hyoid cartilage was located where the termini of the ventral aorta bifurcate to form the first and second afferent arteries, the body of it lying superficial to the base of the second afferent. In no case did I find it further out over the first gill pocket as is shown for Heterodontus philippi (Max Flirbringer '97, pi. 6, fig. 5). In one of the cases examined the extrahyoid on the right side was elongated as is shown in figure 9, while on the left it was a nodule very much like the enlarged end of the cartilage seen in figure 9. Usually both of the cartilages were almost identical in shape with that figured by Gegenbaur and Karl Flirbringer as the first basibranchial. I am at a loss to know whether what Gegenbaur described as a single piece and Flirbringer described as paired cartilages are not in fact what I have regarded as the extra-hyoids. If these be the cartilages described by them I am convinced that they are not basibranchials since they lie entirely superficial to the second afferent artery.

Extra-branchial cartilages are located over all of the (internal) branchial arches in Heterodontus excepting the fifth. The extra-branchials of the first branchial arch are usually large and like succeeding arches overlap terminally {ex.h., fig. 10). The first three cartilages are hook-shaped at their attached ends, the dorsal ones only slightly and the ventral pieces in a very pro


466 J. FRANK DANIEL

nounced fashion. In both dorsals and ventrals the body tapers toward the free end. The fourth extra branchial (not added in fig. 14) is smaller and simpler in form both above and below, than the preceding.

The four dorsal extra-branchial segments are united in such a way as to make the upper attachment in a continuous line above the gill pockets. Each of these cartilages curves around the tip of the branchial rays at the margin of a gill septum. The attachment of the lower or ventral extra branchials is less concentrated. The first three of these are joined to the connective tissue ventrally at the base of their respective gill-pockets, but the fourth is shorter and tends to migrate upward so as to rise from the side of its ceratobranchial.

2. The spinal column

The spinal column in Heterodontus, although somewhat variable in the number of its segments, consists of about one hundred and ten clearly marked vertebrae. Of these the first thirty-one have ribs growing from their basiventrals (h.v.) or transverse processes; the five or six following these have their basiventrals bent downward, and the sixth or seventh (thirtyseventh or thirty-eighth of the column) usually has them meeting below to form the first hemal arch. Thirty-four similar arches follow back from the first haemal arch to the beginning of the ventral rays of the tail (on the seventy-first vertebra). There are next thirty-nine or forty caudal vertebrae, behind which in the adult is a mass but slightly differentiated, tapering gradually to a point (see also T. J. Parker '87, p. 31, pi: 8, fig. 28).

The first vertebra in the column (fig. 15) is obscured by an incomplete anterior segment having a short centrum and bearing enlarged transverse processes for articulation with the occipital condyles of the cranium. The second complete vertebra (vt.^, fig. 15), appearing behind the incomplete segment, may be described as provided with a strong centrum upon which rests a basidorsal (basal) piece (h.d.^), above and posterior to which is a iarge interdorsal (intercalary) plate (i.d.^). Both of these plates are perforated, the former giving passage to the ventral root


ANATOMY OF HETERODONTUS ! ENDOSKELETON 467

nerve (f.v.), the latter to the dorsal root (f.d.) of the first spinal nerve. Capping the column between the first and second interdorsal plates on each side is a smaller plate — the suprabasidorsal (s.h.d.) or neural spine. The centrum itself has extending from its side a basiventral (b.v.) from which the rib (r.) projects.

In an end view of the fifth vertebra the structures there concerned are seen to advantage (fig. 18). Resting upon the centrum is the neural arch (n.a.), the sides and the top of which are made up entirely of the cut edge of the interdorsals {i.d.), but the basidorsal plates (b.d.) of the vertebra appear in this end view. At the ventro-lateral angles of the centrum are the ribs (r.) which stand out almost at right angles.

A similar view taken of a vertebra toward the tail region, say the fifty-third, in addition to a neural arch above, shows a haemal arch below the centrum (h.a., fig. 21). The neural arch in this case shows the interdorsal plate with a basidorsal (b.d.) seen in end view. The haemal arch is formed by the downward bending of the basiventrals and by the adjoined haemal spine; the basiventrals make up the larger part of the wall and all of the floor of the canal. Contiguous to the centrum, however, are seen the interventral plates of the vertebra. In such a view it will be observed that the haemal canal is divided by a partition into a dorsal and a ventral part, the dorsal being for the caudal aorta and the ventral for the caudal vein.

If a series of transverse sections be taken through the two above vertebrae, the ends of which are figured (figs. 18 and 21), the composition of the arches as well as the finer structure of the vertebrae may be made out. The neural arch in the second section (fig. 19) shows a great extent of the basidorsal cut and a decrease in the amount of the interdorsal. In the third section (fig. 20) the suprabasidorsal or neural spine (s.b.d.) also appears, so that all three of the parts making up the arch are seen here. In the caudal vertebrae the neural arch is similar to that just described. In figure 22 a bit of the suprabasidorsal is cut, and in both figures 22 and 23 the haemal arch shows the basiventral, that part of the interventral which appears in end view (fig. 21) not being touched.


468 J. FRANK DANIEL

A study of the finer structure of these vertebrae is of interest. At the end of the vertebra the calcified tissue appears as a whitish ring like the walls of a funnel (cl.r., figs. 18 and 21). In this funnel is to be found primitive gelatinous notochordal substance which at the ends also forms an intervertebral pad. As the sections pass farther toward the middle of the centrum this calcified ring becomes smaller and smaller, and there pass off from it numerous radiating calcified plates (d.p.). As the middle of the centrum is reached (figs. 20 and 23) the apex of the funnel appears as a tiny circlet of calcified tissue containing the constricted part of the notochord (ch.) . The calcified ring {cl.r.) may here be likened to a hub from which the calcified plates radiate like the cogs of a wheel. It will be noticed that the calcified ring diminishes in thickness as well as in size and that the cogs decrease in number the nearer we approach the middle of the centrum (compare figs. 19-20, also 22-23), showing that some of these do not extend the entire length of the centrum. It is also to be seen that the cogs are not so numerous in the region of the fifty- third vertebra as in the region of the fifth.

Other calcifications of less importance also appear in the vertebrae. These are confined principally to the lining of the neural canal, to the outside circle around the body of the centrum (not shown in the figures), and to the roof and sides of the haemal canal.

Considerable interest attaches to the region of the spinal column between rib-bearing and the haemal arch segments. This area, which may be designated as a transition between the body and the caudal regions, may be represented by a section of the column extending from the thirtieth vertebra — the last but one to bear ribs — to the thirty-eighth, usually the second to form a complete haemal arch (fig. 16). In such an area there is a sudden change in length of centrum, the centra of the nonrib-bearing vertebrae being markedly shorter than those of a ribbearing nature. This change begins usually with the thirtysecond vertebra but in one case I found that the thirty-second was still fused with the succeeding vertebra dorsally, although the two were separate ventrally. On the opposite side, however,


ANATOMY OF HETERODONTUS : ENDOSKELETON 469

the two plates were fully separate both above and below. In Heterodontus francisci only six or seven vertebrae form the transition between rib-bearing and haemal vertebrae (see also Gegenbaur '67, pi. 9, fig. 19, and Secerov '11, pi. 1, fig. 5). Upon these centra and those following, the plates of the neural arches are also modified, being so arranged that to each myomere two basidorsal and two interdorsal plates occur. Such vertebrae are called diplospondylous.

In the transitional vertebrae alternate basidorsals, which are odd in number, are perforated by the ventral roots of the spinal nerve if. v.), and alternate interdorsals are usually separated by the foramina of the dorsal nerves (f.d.). On the thirty-seventh vertebra (fig. 16) the foramen for the dorsal root, however, migrates forward so that it perforates the interdorsal (intercalary) plate. In some other cases the dorsal root foramina pass between the interdorsal and the basidorsal, producing a more or less pronounced niche in the anterior part of the basidorsal plate. In this transitional area, the suprabasidorsal (s.b.d.) above imperforate basidorsals is single where the foramen completely separates these. Above each perforate basidorsal it is usually doubled. These occur in regular fashion, capping the interspaces between basidorsals and the interdorsals so that in the case of figure 16. they comprise three (pairs of) nodules to each myomere.

It will be observed, further, that haemal arches corresponding to the imperforate and perforate segments may be determined by their shape without reference to the basidorsals in question. This will be made especially clear by reference to the fortieth and forty-first basiventrals. The ventral termini of these point in opposite directions, the fortieth pointing anteriorly, and hence corresponding to the imperforate basidorsal, and the forty-first posteriorly, and hence belonging to the same segment as the perforate basidorsal. In a later study of the caudal region we shall have reason to refer to the foramen formed between the haemal arch of the imperforate segment (anterior) and the haemal arch of the perforate segment (posterior). It is through this that the main segmental artery leaves the caudal aorta.


470 J. FRANK DANIEL

The question has been raised: Do the diplospondylous vertebrae return to the monospondylous type as the tip of the tail is approached. This is stated to occur in Acanthias by Ridewood '99), who says, The change from diplospondylous to the monospondylous condition occurs at about the twenty-fourth centrum from the end." In Heterodontus fran.cisci such is not the case (fig. 17). It is difficult, as Ridewood says, to delimit a myomere in the posterior region because of the thinness of the muscle, yet in an injected specimen the segmental arteries arising from the caudal aorta are clearly definitive of boundaries, only two to a myomere, one in front of it, the other bounding it posteriorly. Since two vertebrae occur between each two arteries, at least as far back as the ninety-eighth segment, the diplospondylous nature is retained. Some of the segments posterior to this retain their regularity although in these the arteries themselves are not sufficiently regular to be definitive.

II. THE APPENDICULAR SKELETON

The part of the skeleton known as appendicular consists of pectoral and pelvic girdles and the frame-work for the fins attached thereto.

1 . The skeleton of the fin girdles

(I. The pectoral girdle in Heterodontus (fig. 8) is a strong arch, open dorsally, to which the frame-work of the pectoral fin is attached. It is composed of a right and a left cartilaginous part solidly fused in the middle line below. The part of the girdle which extends dorsal to the attachment of the fin is the scapular portion (sc). That part which meets a similar part from the opposite side below is the coracoid portion {co.). At the middle of the postero-lateral portion of each half of the girdle is a projection or articular surface (a.pt.) which fits into the fossa of the pectoral fin skeleton. In front of this articular surface is a strong antero-ventral projection (a.pr.), like that in Crossorhinus (see Haswell '84), to which is attached strong musculature. A foramen through which nerves and blood vessels pass


ANATOMY OF HETERODONTUS : ENDOSKELETON 471

perforates the girdle between these two projections. Above the articular process {a. ft.) and on the scapula is the postscapular projection to which the heavy lateral musculature attaches. Between the anterior projection and the mid-line there is a deep concavity on the coracoid from which the arcuales communis muscles arise.

h. The pelvic girdle (fig. 13) consists of a flattened bar — slightly cupped up in the middle and expanded at the ends. The right and left halves of this are firmly fused together. Three foramina perforate the pelvic girdle near each end (f.pL); through the median one the iliac artery passes, and through the other two, nerves. At the termini of the girdle are the articular processes for the right and left fins. Each articular surface consists of two protuberances (a.pl.) which fit into depressions of the fin skeleton proper.

« 2. The skeleton of the fins

a. The paired fins. 1. The skeleton of the pectorals (fig. 26) is made up of a group of large basal cartilages from which radiate numerous rows of radialia. The basal cartilages in the pectoral of Heterodontus francisci are unlike those previously described for the adult of Heterodontus philippi. In the former there are three piieces, pro- meso- and metapterygium, the first (the propterygium) being absent in the adult of the latter species. (Mivart 79, p. 449; Huxley 76, p. 50; and Gegenbaur '65, II, pi. 9, fig. 3; see also Howes '87, pi. 3.)

This propterygial basal (pr-p.) in Heterodontus francisci is a clearly marked cartilage, quadrilateral in shape and somewhat elongated. It is followed by a series of four radialia, the first of which is large and plate-like. Contiguous to this plate-like radial is a hexagonal plate, a part of which evidently belongs to the first radial of the mesopterygium.

The. mesopterygium (ms-p.) is a stout cartilage, from the enlarged distal end of which five rows of rounder radials radiate. The most proximal segment of the first row of radialia joins distally the hexagonal plate of the propterygium just described.


472 J. FRANK DANIEL

In the right fin of my dissection this plate was separated into two parts so as to be included in the first line of the mesopterygial radials. The second row of the mesopterygium consists of six segments. The first segment of the third row of radials is followed directly by a similar more flattened segment which, in turn, abuts against a double row, the anterior of which is composed of four or five flattened plates, the posterior row having but four. The proximal segment in the fourth line of mesopterygial radials, like the third, is followed by a slightly shorter segment which abuts distally against two rows, the posterior of which consists of four radials. The anterior plates have just been described. The proximal segment of the fifth row of radials is the longest of the series. It is continued distally by a pentagonal piece which fits against a double row of radials, the anterior proximal line of which was previously described, and the posterior row has but three segments capped distally by small cartilages.

The metapterygium (mt.p.) is a spatulate cartilage, the wider part of which points distally. From this ten or eleven rows of radialia diverge. The second two segments in the first two rows of these abut against double rows of radialia, each row usually consisting of two segments capped by smaller pieces; the third, fourth and fifth are similar except that the double rows are usually uncapped. The sixth to the eighth rows are of two segments each, between which terminally single terminal cartilages abut. The proximal segments of the ninth and tenth radials of the metapterygium are followed by segments equal in length to themselves. Each one of these segments is capped by a single piece. In an older specimen the ninth and tenth radials were fused proximally and cleft distally. The eleventh radial gives evidence of being a fusion of two. The accessory cartilages accompany it; one appears at its apex, and another along its edge.

2. The frame-work of the pelvic fin (fig. 29) in the female consists of a long posteriorly projecting basal cartilage, the basipterygium, to which radialia are joined. The basipterygium iha.p.) in Heterodontus is separated into five sections, the proxi


ANATOMY OF HETERODONTUS : ENDOSKELETON 473

mal of which is long, the distal represented by a tiny cartilaginous tip. Anteriorly a much enlarged fused first radial strikes the basal plate almost at right angles. In the end of this, as well as in the end of the basal piece, is a fossa, by means of which articulation with the pelvic girdle is effected. From this first radial cartilage three rows of radialia project. In the first are one or two small cartilages; in the second, two or three; and in the third there are four cartilages similar to the segments in the first radialia of the basal piece. From the basipterygium thirteen rows of radialia are given off. These are terminated by smaller radial cartilages in the more anterior rows but in the three posterior rows each one consists of two segments.

3. The skeleton of the pelvic fin of the male is, with slight modifications, like that of the female. It consists of the basipterygium and its radials. In the male there are fourteen radials which meet the basipterygium at an angle of forty-five degrees. The most anterior of the radials represents the fusion of three cartilages which, like those following, are segmented into two or three pieces. The most posterior radials, unlike those in the female, are unsegmented. The inner lobe of the pelvic fin in the male (fig. 27) is modified as a framework for the claspers or copulatory organ.

The basipterygium in the male is continued by the basal piece (ba.) to which it is connected by two short segments (b^~^). At the angle between the basipterygium and the basal piece there arises from the former the so-called 'beta' (jS) cartilage. The basal piece is generally round, but terminally it is flattened and possesses a groove which passes obliquely across to the dorsal side. Several cartilaginous pieces appear near the terminal part of the groove. These are two dorsal terminals {d.tr.1-2), a dorsal marginal (d.mg.), and one ventral terminal cartilage (v.tr.). Proximal to the ventral terminal there is an accessory terminal (tr.3).

b. The skeleton of the unpaired fins. 1. First dorsal fin (fig. 24). Extending from the vertebral column one-half the length of the long anterior fin-spine, or to the point externally where the spine emerges from the skin, is a thin basal cartilage, the base of


474 J. FRANK DANIEL

which extends over two and one-half vertebrae (17-18§) in the female; (15-18) in the male. From the top of this plate arise two other cartilages, the anterior of which is twice the width of the posterior. From the former there appear four rows of radiating pieces, each row of which contains three cartilages. Capping the second and third rows is an extra cartilage and over the fourth is a similar cap of a double piece.

2. Second dorsal fin (fig. 25). The second dorsal fin, like the first, is provided with a basal piece (6.c.) one-half the height of the spine; it extends over three (46-48) diplospondylous vertebrae in the female; (44-48) in male. From it arise three radials which point in a more posterior direction. The most anterior of these is the smallest. Passing from it is a double row of two segments capped with a broader piece. The second radial, though a single piece proximally, is bifid distally. From the distal end arise two rows of three cartilages each. The third radial is a truncate cone which rests on its apex. Upon its base it supports in an irregular fashion si^ cartilaginous pieces.

3. The caudal fin (fig. 17). The ventral rays of the caudal fin or tail, as we have seen are an integral part of the axial skeleton, being the prolonged haemal spines. These consist of a series of forty rays, each of which corresponds to a vertebra. Beyond the tip of these, in the adult is an undifferentiated (fused) mass. In this region interdorsal pieces are present as far back as the ninety-fourth to the ninety-seventh vertebrae, while interventral pieces extend back to the ninety-ninth vertebra. The dorsal lobe of the fin is supported by rays which unlike those supporting the ventral lobe, are not equal in number to the centra, forty-six being present in one specimen examined. The first of the dorsal rays is small and the second arises as a broad clear piece of cartilage over the seventieth or seventy-first vertebra. Back' of this the rays are numerous and are more or less regular to the ninety-fifth vertebra. At the tip of the tail they fuse into a common mass (fig. 17).

In the caudal region alternate basidorsals are perforate back to the ninety-fifth vertebra, with the exception of the eightyninth and ninety-third. The eighty-ninth although perforate


ANATOMY OF HETERODONTUS I ENDOSKELETON 475

on the right side was imperforate on the left. No suprabasidorsals are found just anterior to the dorsal rays, and the interdorsals usually cease at about the ninety-fifth, although they may sometimes extend a few segments further back. It is difficult to make out the exact perforations in the plates posterior to the eightieth vertebra, for they may cut the edges of the plate and hence the nerve may emerge between the plates.

4. The anal fin. The base of the anal fin in the specimen before me abuts against the fifty-eighth centrum of the spinal column. ' But this position varies between the fifty-eighth and sixty-third vertebrae. From the basal piece, which is remarkably like that of the dorsals, proceed four rows of radials, the first composed of two segments; the second has two rows arising from it, in the first of which are five cartilages, in the second four; the third radial is elongate and is continued by three successively shorter pieces; and the fourth, more elongate still, is followed by a broader radial. Upon this are three rounded pieces, over the first and a part of the second of which is a broader cap.


Berkeley, California March, 1915


LITERATURE CITED


Balfour, F. M. 1881 On the development of the skeleton of the paired fins of Illasmobranchii, considered in relation to its bearings on the nature of the limbs of the Vertebrata. Proc. Zool. Soc. London.

FtJRBRiNGER, Karl 1903 Beitrage zur Kenntnis des Visceralskelets der Selachier. Morph. Jahrb., Bd. 31.

FtJRBRiNGER, Max 1897 Ucber die Spino-occipitalen Nerven der Selachier und Holocephalen und ihre vergleichende Morphologie. Festschrift flir Gegenbaur, TIL

Gadow, Hans 1888 On the modifications of the first and second visceral arches, with especial reference to the homologies of the auditory ossicles. Phil. Trans. Roy. Soc, B, vol. 179.

Gegenbaur, C. 1865 Untersuchungen zur vergleichenden Anatomie der Wirbelthiere. IL Brustflossen der Fische.

1867 Ueber die Entwickelung der Wirbelsaule des Lepidosteus, mit vergleichend-anatomischen Bemerkungen. Jena. Zeitsch., Bd. 3.

1872 Untersuchungen zur vergleichenden Anatomie der Wirbelthiere. III. Das Kopfskelet der Selachier, ein Beitrag zur Erkenntniss der Genese des Kopfskeletes der Wirbelthiere. Leipzig.


476 J. FRANK DANIEL

GooDEY, T. 1910 A contribution to the skeletal anatomy of the frilled shark — Chlamydoselachus anguineus Garman. Proc. Zool. Soc. London.

Hasse, C. 1879 Das natlirliche System der Elasmobranchier auf Grundlage des Baues und der Entwicklung ihrer Wirbelsaule. Jena.

Haswell, W. a. 1884 Studies on the elasmobranch skeleton. Proc. Linn. Soc. N. S. Wales, vol. 9.

Hawkes, O. a. M. 1905 The presence of a vestigial sixth branchial arch in the Heterodontidae. Journ. Anat. Physiol., vol. 40.

Howes, G. B. 1887 On the skeleton and affinities of the paired fins of Ceratodus, with observations upon those of the Elasmobranchii. Proc. Zool. Soc. London.

Huxley, T. H. 1876 Contributions to morphology, Ichthyopsida — No. 1. On Ceratodus fosteri, with observations on the classification of fishes. Proc. Zool. Soc. London.

Jaquet, M. 1905 Description de quelques parties du squellette du Pseudotriacis micridon Capello. Bull. Mus Ocean. Monaco, no. 36.

Mivart, St. Geo.. 1879 Notes on the fins of elasmobranchs, with considerations on the nature and homologues of vertebrate limbs. Trans. Zool. Soc. London, vol. 10.

Parker, T. J. 1887 Notes on Carcharodon rondeletti. Proc. Zool. Soc. London.

Parker, W. K. 1879 On the structure and development of the skull in sharks and skates. Trans. Zool. Soc. London, vol. 10.

RiDEWooD, W. G. 1896 On the spiracle and associated structures in elasmobranch fishes. Anat. Anz., Bd. 11.

1899 Some observations on the caudal diplospondyly of sharks. Jour. Linn. Soc. Zool., vol. 27.

Secerov, Slavko 1911 liber die Entstehung der Diplospondjdie der Selachier. Arb. Zool. Inst. Wien, Bd. 19.

Thacher, Jas. K. 1877 Median and paired fins, a contribution to the history of vertebrate limbs. Trans. Conn. Acad., vol. 3.

White, Phil, J. 1892 The skull and visceral skeleton of the Greenland shark, Laemargus microcephalus. Trans. Roy. Soc. Edinburgh, vol. 37, (2).


PLATES


JOURNAL OF MOHPHOLOGY, VOL. 26, NO. 3





^


^■o



)











"3

t/3







d



r^



a







rt




en




>





'm







a

3 o


-2



3


Ji



o p

3d



3 -5


_c3








o


73


CQ



3


o3


"3

3


c3


3 03

S


3

c3


o3




'S





3


cu


"— •


c3


CQ


o

o .2


CQ




CO

«  O


3

a '8

IB


a

3


s

3

'2


■ c! 3


O

o '3

o

o


5

"ci

'o o o


03 03

3 O

o


o 3


CQ

o


03 O

"3

P.


o >>

-3

03

o


s

o

_3 03

p. a


'o

-4-=

3

03

s

-3 o c3


w


[X.

o



o


c3




c

i


^f


-^


s



"p.



-S


o3


H




0)














< h-1


2

O




"+3









^



03



f— t


-t^


^4-H


O









•-^



A



Ph


><


CI

o o


o

'>

"^

o -a


-! C

>









03 >

s

3

CQ

-5



£ p

o





3


< <









3



>

s





bO


r— 1


(M









O



•"















t-i







S




S3








m



>



.£2 O


o3 03


^ T3 -a


(D ^ C 3^ "^

3 3^^


<» — -3 O


tH ^ S S3


o3


- .- ^ S


c5 03


J5 fc. >-; •




=: 03

!= ^ 2 O e

c3 O ^ ^ 3

O . :: C "

- ^ • 3 i-H ^-,

r 55. 2 t^ tr^


PLATE 2


ANATOMY OF HETERODONTUS: ENDOSKELETON

J. FRANK DANIEL n S.O.

•fill




f.S-ni





r,3.4



DUNCAN DUNNING, del.


PLATE 2


EXPLANATION OF FIGURES

All figures of Heterodontus francisci (natural size).

3 A side view of the cranium.

4 Posterior view of the cranium.

5 A median sagittal section through the cranium, seen from the inside, nasal capsule has been removed.


The


a.c, auditory capsule

ch.. notochord

/.a.c, foramen of anterior cerebral vein

f.e.c'., external carotid foramen (entrance to socket)

J. i.e., internal carotid foramen

/.?/?., foramen magnum

f.o.p., foramen of ophthalmicus profundus nerve (leaving the socket)

f.o.VII, ophthalmic foramen of Vllth nerve (leaving the socket)

f.r-a., foramen for ramus anastomoticus artery

f.s-m., foramen for spino-muscularis artery

/./, foramen through which the olfactory nerve leaves the cranium

/.//, foramen for the second cranial or optic nerve

f.III, foramen for third cranial or oculomotor nerve


f.IV, foramen for fourth cranial or trochlear nerve

J. VII, foramen for seventh cranial or facial nerve

J. VIII, foramen for eighth cranial or auditory nerve

f.IX, foramen for ninth cranial or glossopharyngeal nerve

f.X, foramen for tenth or vagus nerve

i.o., interorbital canal

o.cd., occipital condyle

o.cr., occipital crest

O.J'., orbital fissure

o-n., orbito-nasal canal (entrance to socket)

pl.f., palatal fossa

po.hm., posterior hyomandibuiar process

po.o., postorbital process

pr.o., preorbital process

s.hni., superior hyomandibuiar process

S.O., supraorbital crest


48]


PLATE 3





ANATOMY OF HETERODONTUS: ENDOSKELETON

J. FRANK DANIEL



i^'r


I


A


-Icp


v.l.


-h.


riQ.o


c.l' /' ,P-q


l.hni>ni


DUNCAN DUNNING, del.


7,3.


482


PLATE 3


EXPLANATION OF FIGURES

Figures of Heterodontus francisci (natural size).

6 Lateral view of the cranium with articulation of first and second visceral arches.

7 Median view of mandibular arch showing articulation.


a.p., articular process of palato-quad rate c-h., ceratohyoid c.l'., slip from palato-cranial capsular

ligament (l.l. 1-2, first and second dorsal labial

cartilages hl.p., hyal process hm., hyomandibula kb., mandibular knob l.cp., ligamentum complexum l.h-m., ligamentum hyoideo-mandibu lare l.hm-h., ligamentum hyomandibulo hyoideum l.hm-m., ligamentum hyomandibulo mandibulare


l.h-nt.q., ligamentum hyomandibuloquadratum

I. III., ligamentum mediale

l.q-iii.i., ligamentum quadrato-mandibulare internum

md., mandible

md.r., mandibular ridge

o.p., optic pedicle

p-q., palato-quadrate cartilage

sp.c, spiracular cartilage

s.p-s.l., superior postspiracular ligament

Ir.l.r., transverse lateral ridge

Ir.m.r., transverse median ridge

V.I., ventral labial cartilage


483


PLATE 4


ANATOMY OF HETERODONTUS: ENDOSKELETON

J. FRANK DANIEL



ex.b.


-f.pt


7,0-11


7io,.14


DUNCAN DUNNING, del.


484


PLATE 4


EXPLANATION OF FIGURES

All figures of Heterodontus francisci.

8 Antero-lateral view of left side of pectoral girdle (one-half natural size) .

9 Lateral view of extrahyoid cartilage ( X 4) .

10 Antero-lateral view of first branchial arch; (one-half natural size).

11 Ventral view of median basibranchial cartilages (natural size).

12 Posterior view of the spiracular cartilage (X 1\) .

13 Dorsal view of right half of pelvic girdle (natural size) .

14 Antero-lateral view of the fourth and fifth branchial arches, with attached rudimentary sixth arch (one-half natural size) .


a.pl., articular process pelvic fin a.pr., anterior projection of pectoral

arch a.pt., articular process pectoral fin b.b., basibranchial 5./i.,- basihyal cartilage b.r., branchial ray

c-b. 1-5, first to fifth ceratobranchials CO., coracoid cartilage e-b. 1-5, first to fifth epibranchials


e.v.b., extrabranchial

J'.pl., foramen through pelvic girdle

J'.pt., foramen through pectoral girdle

g.p., glossal process

h-b. 1-4, first to fourth hypobran chial m.p., posterior median piece p-b. 1-5, first to fifth pharyngobrancli ials sc, scapula


485


^ ? p.




S '"




<!


»— 1



QJ



J



CJ


-^



PLi

X


X





fn


.^


<1?


^




o



^^




_ai


b


rt ->J





a;


r^ fc.





>


1 >




rt


!-i





a:



13





4)


.2




e*-.


^


03 03


3


-1-=

ai



03

03



>




'>


■>


73


'3






'P



e+-(


'S


^


'^



+=



P!



c3


OT



^

S-i


^


73


c



C


1


,




CD


(D


S


QJJ


1


<D



1:3


o3



c3


o3





_>


B

u


s


a


s


3


i-:i


1-5


h^



c3


tn


o3


c3



c3


bO






-Q


"S




^


y3


lO



l^


r



q;








•>



o Q 2: w .. J

O M

9 « 

W

a o

o

H <5 IS

<;






^:


PLATE 6


EXPLANATION OF FIGURES

All figures of Heterodontus francisci (X 2). 18 End view of fifth vertebra. 19-20 Cross-section of fifth vertebra. 21 End view of fifty-third vertebra. 22-23 Cross-sections of fifty-third vertebra.


b.d., basidorsal plate b.v., basiventral plate c, centrum ch., notochord cl.p., calcified plate cl.r., calcified ring


h.a., haemal arch

i.d., interdorsal plate

i.v., interventral plate

n.a., neural arch

r., rib

s.b.d., suprabasidorsal plate


488


ANATOMY OF HETERODONTUS: ENDOSKELETON

J. FRANK DANIEL


PLATE 6



♦- 1 b d


-dr.



YioAS


Fl£ 21



-s.b.cT.



F.3.19


"Fia. 2.0


'-^^N :-^^-^


\. ^


Y\Q.2Z


DUNCAN DUNNING, del.


4S9


c\.r.



"FiQ.23


PLATE 7


EXPLANATION OF FIGURES


All figures of Heterodontus francisci (three-fourths natural size).

24 Lateral view of left side of first dorsal fin (female) .

25 Lateral view of left side of second dorsal fin (female) .

27 Dorsal view of left pelvic fin of male, showing skeleton of clasper.


6. 1-2, first and second segments following the basipterygium

b.c, basal cartilage

ba., basal piece

ba.p., basipterygium

j3., beta cartilage

d.nig., dorsal marginal cartilage of clasper


(J.lr. 1-2, first and second dorsal terminal cartilage of clasper

pi., pelvic girdle

ra., radial cartilage

Ir. S, accessor}^ terminal cartilage of clasper

v.tr., ventral terminal cartilage of clasper


490


ANATOMY OF HETERODONTUS: ENDOSKELETON

J. FRANK DANIEL


I'LATI':


/

I


^^b.c


Fia. 2,5




"FiQ. VI


Fio,£4


DUNCAN DUNNING, del.


491


"to s


1^ o


._, y2 "" O

.25 75 S =

S f^ p y=;


a >


o o




cj o Is "5 k e


go

bib U3 <£>




.2 g^-a



0 d


en



da



en


STUDIES ON GERM CELLS

IV. PEOTOPLASMIC DIFFERENTIATION IN THE OOCYTES OF CERTAIN

HYMENOPTERA

ROBERT W. HEGNER

From the Zoological Laboratory of the University of Michigan, Ann Arbor , Michigan, U. S. A.

NINETY-EIGHT FIGURES (THIRTEEN PLATEs)

CONTENTS

I. The differentiation of the oocytes and nurse cells in the ovaries of the

honey-bee, Apis mellifica 495

II . The bacteria-like rods and secondary nuclei in the oocytes of Camponotus

herculeanus var. pennsylvanica DeG 506

III. The history of the nuclei and germ-line determinants in the oocytes of

certain parasitic Hymenoptera and Hymenopterous gall-flies 521

1 . Copidosoma gelechiae 621

2. Apanteles glomeratus 526

3. Hymenopterous gall-flies 520

Literature cited 933

I. THE DIFFERENTIATION OF THE OOCYTES AND NURSE CELLS IN THE OVARIES OF THE HONEY-BEE, APIS MELLIFICA

As the writer has recently pointed out (Hegner '14 c), there are in many animals two definite periods in the germ-cell cycle during which germ cells and somatic cells arise from the same mother cells. One period occurs during embryonic development when the primordial germ cells are segregated. This segregation takes place at different stages of development in different species. For example, in the midge, Chironomus, one of the first four cleavage cells gives rise to all of the germ cells (Hasper '11); in the paedogenetic fly, Miastor, the primordial germ cell is differentiated at the eight-cell stage (Kahle '08; Hegner '14 a) but in most cases where a very early segregation has been observed, one cell at the thirty-two-cell stage is the primordial germ cell, as in Ascaris (Boveri '92), in Cyclops (Haecker '97;

495

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 3


496 ROBEKT W. HEGNER

Amma '11), and in Sagitta (Elpatiewsky '09, '10; Buchner, 10 a, '10 b; Stevens '10). The other period is that of the differentiation of the oocytes and nurse cells in the female and, at least in man, of the differentiation of the spermatocytes and Sertoli cells in the male (Montgomery '11; Winiwarter '12). These two periods seem rather distantly removed from each other, since we ordinarily begin our ontogenetic studies after the eggs are laid, but in reality they are very close together in the germ-cell cycle since the oocytes and nurse cells often become differentiated shortly before the deposition of the eggs, and the primordial germ cells are segregated shortly after cleavage begins.

This contribution deals entirely with the second period described above and the data have been derived from a study of the cellular elements in the ovaries of the queen honey-bee, Apis mellifica. Bees of three ages were employed: (1) those still within their pupal cells, (2) virgin queens three days old, and (3) virgin queens shortly before 'the deposition of eggs. The ovaries were dissected out in Ringer's solution and fixed in five different fluids: (1) Towers', (2) Carnoy's, (3) Bouin's, (4) Altmann's, and (5) Meves' modification of Flemming's solution.^

^ The formulae are as follows:

(1) Tower's solution.

Saturated sol. HgCl2 in 35 per cent alcohol 95 vols.

Glacial acetic acid 2 vols.

Nitric acid, c.p 3 vols.

(2) Carnoy's solution.

Absolute alcohol 1 vol.

Glacial acetic acid 1 vol.

Chloroform 1 vol.

HgCl2 to saturation

(3) Bouin's solution

Picric acid, sat. aqueous sol : 75 vols.

Formol 25 vols.

Glacial acetic acid 5 vols.

(4) Altmann's solution

Bichromate of potash, 5 per cent 1 vol.

Osmic acid, 2 per cent 1 vol.

(5) Meves' solution

Osmic acid, 2 per cent 100 cc.

Chromic acid 0.5 cc.

NaCl 1 gram.

Glacial acetic (just before using) 30 drops


STUDIES ON GERM CELLS 497

The general structure of the ovary and ovarioles was obtained frorn in toto preparations and from thick sections. Detailed studies were made of sections 4jLt thick and stained in the following ways: (1) Heidenhain's iron hematoxyhn; (2) Rubaschkin's mitochondrial method ;2 (3) Altmann's acid fuchsin; (4) Benda's crystal violet; and (5) Delafield's hematoxylin. These methods of fixation and of staining were selected in order that both cytoplasmic and nuclear bodies could be studied.

The ovaries of insects consist of a number of tubes, the ovarioles, which are attached at the anterior end by means of terminal threads and open at the posterior end into the oviduct. The variations in the structures of the ovarioles are due principally to the presence or absence of nurse cells and the distribution of these when present. Certain ovaries, for example, those of the Orthoptera and Aptera, are not provided with nurse cells. In others the nurse cells may remain within a terminal chamber and supply the growing oocytes through a nutritive strand, as in certain Hemiptera and Coleoptera; or a rather definite number of nurse cells may become separated from the terminal chamber and accompany each oocyte, as in the Neuroptera, Hymenoptera, Diptera, and Lepidoptera. The result of the last named method of nutrition is the formation of ovarioles which resemble rows of beads.

In the bee each oocyte is accompanied by a group of nurse cells. An outline of a single 'ovariole is shown in figure 1. The terminal filament (t) consists of a row of long slender cells which extend entirely across the filament. Following this is a region occupying about half of the entire ovariole which is characterized by rosettes of cells (r). These cells are apparently all alike

- Rubaschkin fixes tissues in Meves' modification of Flemming's solution for one or two days. Sections are treated as follows :

Potassium permanganate, I per cent 1 minute

Wash in water •

Oxalic acid and potassium sulphate, i per cent 1 minute

Wash in running water 15 minutes

Ferric alum, 4 per cent 24 hours

Weigert's hematoxylin 2-3 days

Differentiate in ferric alum, 2 per cent


498 ROBERT W. HEGNER

and those in a single rosette have descended from a single mother cell which may be called the oogonial mother cell. The actual differentiation of the oocytes and nurse cells occurs in a much shorter part of the ovariole (d) . After the oocytes are definitely established, they move down the ovariole, become arranged in a single row (o) and are graduallj^ separated from each other by groups of nurse cells (n) which lie in nurse chambers just above them. The elements within ovarioles of different ages differ, of course, both in their stage of development and in their distribution.

The three kinds of cells within the ovaries of insects are the oocytes, nurse cells, and epithelial cells. These three kinds of cells arise differently in different groups of insects. Thus the nurse cells and epithelial cells in the paedogenetic fly, Miastor (Kahle '08; Hegner '14 a) are of mesodermal origin and the germ cells give rise only to oocytes. In the Hymenoptera on the other hand Korschelt ('86) in Bombus, Paulcke ('01) in Apis, and Marshall ('07) in Polistes agree that the three cellular elements within the ovaries arise from one sort of cells, the germ cells. I have been unable to determine the origin of the epithelial cells in the ovarioles of the bee because of the lack of young ovaries, but that nurse cells and oocytes arise from oogonia there can be no doubt.

Part of the rosette region of an ovariole is shown in figure 2. Two kinds of cells are present, (1) Those that make up the rosettes (r) ; and (2) the epithelial cells (e) among the rosettes. The ground substance within the ovariole in this region appears to be a loose cytoplasmic reticulum containing a few scattered nuclei. These nuclei are rather irregular in shape, and contain a clear matrix in which may be seen one or two large chromatin masses and a very delicate reticulum. No epithelial cell boundaries could be observed in this part of the ovariole and it seems probable that the rosettes are imbedded in a syncytium. There seems to be no regular arrangement of the rosettes; they do not crowd one another, but the cells in each are closely united, hence it is a very simple matter to distinguish the separate rosettes in an ovariole even with low magnification. It seems strange


STUDIES ON GERM CELLS 499

because of this perfect distinctness that Paulcke ('01) failed to observe these rosettes.

The evidence for the statement that all of the cells in a single rosette have descended from a single mother cell is irrefutable. In figure 2 the cells of the rosette to which the guide line(r) extends are grouped about a branching strand which stains black in iron hematoxylin. A similar rosette is shown enlarged in figure 3. One branch of the black strand extends into the cytoplasm of each cell. These strands consist of the spindle fibers remaining after previous mitotic divisions, and, as will be pointed out later, such strands are not uncommon in either the ovaries or the testes of insects. A section through one end of a rosette at right angles to that shown in figure 3 is illustrated in figure 4. The spindle remains form a sort of axis about which the strands from the most recent divisions are radially arranged. The entire rosette is therefore oblong and may be sectioned longitudinally or transversely. The number of cells in each of the rosettes figured is sixteen, indicating that four divisions had occurred since the oogonial mother cell was established. No evidence was obtained which indicated the presence of amitotis in these ovarioles, and very few mitotic division figures were observed. Those that were found were invariably restricted to the cells in single rosettes (fig. 5), thus indicating that the cells in a rosette divide synchronously.

A Critical examination of both the cytoplasm and nuclei of the cells in the rosettes failed to reveal any constant differences among the cells of any particular rosette. Giardina COl) discovered a difference in the nuclei of certain rosette cells in the ovarioles of Dytiscus, and Kern ('12) has reported a difference in the cytoplasm of similar cells in Carabus, but no such distinguishing marks were found in the bee. This indicates that all of the cells at this stage in the oogenesis of the bee are probably potentially alike. At any rate no visible differences were discovered in material fixed and stained so as to bring out to the best possible advantage both nuclear and cytoplasmic bodies.

The rosette zone in the ovariole is followed by the zone of differentiation (fig. 1, d). Certain of the cells increase in size


500 ROBERT W. HEGNER

and are recognizable as oocytes (fig. 6, o). This is brought about by an increase in the amount of cytoplasm and by the enlargement of the nucleus. The arrangement of the chromatin within the nucleus changes during this differentiation; that of the nurse cells (fig. 6, n) retains the condition characteristic of the rosette stage (fig. 2), whereas in the newly formed oocytes the chromatin forms threads which are scattered about irregularly within the nucleus (fig. 6, o). The connecting strands, so noticeable in the rosettes (fig. 3), either disappear at this time or lose their staining capacity since they are apparently absent from this stage on. Nevertheless it is very easy to determine which cells have descended from a single mother cell since a dark double ring remains where the strands passed from one cell to another (fig. 7). These rings are quite conspicuous but were completely overlooked by Paulcke ('01).

The change from the rosette zone to the zone of differentiation in the ovariole of the bee is an abrupt one — a fact which makes a study of the differentiation of the oocyte difficult, since no intermediate stages can be studied unless material in just the proper condition is obtained. In several cases which will be described later, investigators have found that a single rosette gives rise to gne oocyte and a group of nurse cells. This is certainly not true in the bee, since the oocytes in the zone of differentiation are much too numerous, compared with the number of rosettes, and many instances were observed of two or *hiore oocytes which had been directly connected by spindle remains as indicated by the presence of double rings between them (fig. 7) .

If all of the cells in a single rosette are potentially aUke the question arises, what causes some of the cells to become oocytes and others nurse cells? Three explanations have occurred to me: (1) There may be differential changes during the mitotic divisions in rosette formation as in Dytiscus (Giardina '01) resulting in one or more cells (oocytes) which differ in content from the others (nurse cells). No Visible changes of this sort were observed. (2) The polarity of the rosette may influence the cells in such a way that those near the center of the ovariole and closest to the zone of differentiation tend to develop into


STUDIES ON GERM CELLS 501

oocytes. (3) Those cells of the rosette which reach the zone of differentiation first are stimulated to become oocytes and by their growth and differentiation prevent the other cells of the rosette from similar changes. It would be futile to argue on the basis of known facts in favor of any of these hypotheses.

The arrangement of the oocytes and nurse cells within the ovariole resulting in a linear series of oocytes which alternate with groups of nurse cells takes place a short distance back of the zone of differentiation (fig. 1, n). Paulcke ('01) has satisfactorily described and figured the formation of the epithehum around the oocytes and the structure of the nurse chamber, but, as stated above, he failed to see the intercellular rings which indicate the descent of the cells concerned. A group of nurse cells about an oocyte is shown in figure 8. This oocyte is connected with at least three nurse cells. One of the nurse cells (a) lies below the oocyte in the ovariole; since this is never true in later stages it is probable that such a cell would either degenerate or become separated from the oocyte and forced over to one side. This has evidently happened in the case of the oocyte illustrated in figure 9, since a ring is present here at the lower end (a), but it does not connect the oocyte with a nurse cell. The relation between the oocyte and its accompanying nurse cells is shown in figure 10. All of the nurse cells are not included, since this is a camera drawing of a section. It illustrates, however, the way in which the nurse cells form into rows converging toward the oocyte.

The descent of the cells within the zone of differentiation would be impossible to determine if it were not for the presence of the rings between them. These rings continue to connect the nurse cells with the oocyte, even in late stages in the growth of the latter (fig. 11) and many of them may also persist between the nurse cells after the nurse chamber is fully formed, as in the stage illustrated in figure 12. Kern ('12) also finds these rings connecting the nurse cells with the oocytes of Carabus, and claims that nutritive material passes through them during the growth of the egg.

As soon as the oocytes are differentiated, numerous granules of various sizes appear within their cytoplasm; in the earlier


502 ROBERT W. HEGNER

stages these lie mostly near the nucleus (figs. 7-8), but later (fig. 9) become scattered throughout the cytoplasm. These granules stain best in iron hematoxylin after fixation in Meves' modi' cation of Flemming's solution. No evidence was obtained that they are of nuclear origin, although their early position near the nucleus indicates that they may have arisen in this way; or if not directly from the nucleus, at least through its influence. On the other hand, their sudden appearance within the cytoplasm indicates that they are cytoplasmic bodies which have resulted either from the ag'gregation of smaller pre-existing bodies of a similar nature or from the synthesis of other substances under the stimulus of the metabolic processes set up at the inauguration of the growth period. Duesburg ('08) has recognized granules in the peripheral layer of cytoplasm in the full grown egg of the bee, especially near the nucleus in the thickened area which Petrunkewitsch ('01) has called the 'Richtungsplasma,' and considers them to be mitochondrial in nature. It seems probable that the bodies we have observed are the 'mitochondria' of Duesberg at an earlier stage. Paulcke ('01) failed to observe them.

Discussion. The differentiation of the cellular elements in the ovaries of insects and the relations of the oocytes to the nurse cells has interested students of histology and cytology for three quarters of a century. Mayer, as early as 1849, expressed the opinion that the nurse cells are abortive eggs. The connections between them and the oocytes were observed by Huxley ('58) in oviparous aphids, and were considered by him a nutritive canal for the conduction of food material from the nurse cells to the growing egg — a conclusion concurred in by Lubbock ('60) and Claus ('64). Balbiani ('70), however, proved this 'nutritive canal' to be a protoplasmic strand, but, as Wielowiejski ('85) has pointed out, he was in error when he stated that the terminal chambers of the ovarioles of aphids contain a large central cell which gives rise to both the oocytes and nurse cells (abortive eggs). He nevertheless established the fact of a protoplasmic cellular bridge between these two kinds of cells.


STUDIES ON GERM CELLS 503

Protoplasmic bridges between the cells of Metazoa are not uncommon and may exist in all tissues. As a rule, they are delicate strands which pass through pores in the cell walls. The cellular elements in a syncytium, such as occurs during the cleavage of the insect egg, must be even more closely united physiologically, since here the cytoplasm forms a continuous network. Cellular bridges similar to those described above in the queen bee, have been observed in the germ glands of a number of other animals, especially insects,, but mostly during spermatogenesis. Thus Platner ('86) found in Lepidoptera that often two neighboring spermatocytes, and sometimes three, were connected by intercellular ligaments which were attached to an intracytoplasmic body in each cell. The latter were considered 'Nebenkerne.' Similar conditions were discovered by Prenant ('88), Zimmerman ('91) and Lee ('95) in the male germ cells of Gastropoda. Lee, in his work on Helix, recognized the true origin of the intercellular bridges and their significance. They were found to be the remains of the spindle fibers following a mitotic division. The term *pont fusorial' was applied by Lee to the bridge itself and 'moignons fusoriaux' to the ramification of the fibers within the cytoplasm of the cells. Similar intercellular ligaments w^ere observed by Henneguy ('96) in the seminal cells of Caloptenus; by Erlanger ('96, '97) in both the testes and ovaries of the earthworm; by Wagner ('96) in the male germ cells of spiders; by Meves ('97) in both the testes and ovaries of the salamander; by Giardina ('01), Debaisieux ('09) and Giinthert ('10) in the ovaries of Dytiscus; by Marshall ('07) in the ovarioles of Polistes; by Kern ('12) in the ovarioles of Carabus; by Govaerts ('13) in the ovarioles of Carabus and Cicindela; by Maziarski ('13) in the ovarioles of Vespa; and by Hegner ('14 a) in the testes of Leptinotarsa.

By far the most interesting results are those obtained by Giardina and confirmed by Debaisieux ('09) and Giinthert ('10). Giardina proved conclusively that a single oogonium in the ovary of Dytiscus undergoes four divisions, thus producing sixteen cells, one of which is the oocyte and the remaining fifteen nurse cells. The processes of differentiation in this genus are partic


504 ROBEET W. HEGNER

ularly interesting, because they include a separation of the chromatin of the mother cell into two masses. One of these masses of chromatin forms an 'anello cromatico;' the other gives rise to forty chromosomes which divide equally, half of each passing to each daughter cell. The chromatic ring remains undivided and becomes situated entirely in one of the daughter cells. At each of the three succeeding divisions the chromatic ring is segregated entirely in one cell; this cell is the oocyte, whereas the other fifteen which have a common origin with it are nurse cells.

Since the publication of Giardina's observations many investigators have attempted to discover similar visible differentiations in the ovaries of other insects, but without much success. Thus Govaerts ('13) made detailed studies of beetles of the genera Carabus, Cicindela, and Trichisoma but was unable to find anything resembling the chromatic ring which occurs in Dytiscus. He found however that the spindle fibers ('residu fusorial') persist after the daughter cells are formed during the differential divisions, just as they do in Dytiscus, and that a definite polarity is marked by the position of these spindle remains. The conclusion is reached that something more fundamental than the unequal division of chromatic elements is responsible for the differential divisions and decided in favor of a 'polarite predifferentielle.' No explanation is offered, however, as to the origin of this polarization.

A brief account of the oogenesis in carabid beetles has also been published by Kern ('12), who finds that during the differential mitoses, the oocyte mother-cell may be distinguished by the presence of certain intracytoplasmic granules which he describes as follows:

Befinden sich die Zellen der Zellrosetten in Teilung, so findet man mitunter in einer Zelle neben der Teilungsfigur eine Anhaufung von farbbaren Kornchen, ahnlich denjenigen, die in spateren Stadien in der jungen Eizelle im Cytoplasma gefunden werden. Es liegt nahe, an einen Diminutionsvorgang , ahnlich demjenigen, welchen Giardina bei Dytiscus beschrieben hat, oder auch an einen Vergleich mit den Ectosomen bei Cyclops zu denken; doch gelang es mir bisher nicht, alle Einzelheiten festzustellen. Die Kornchen im Cytoplasma junger Eizellen werden nach und nach aufgelost.


STUDIES ON GERM CELLS 505

The origin of these granules was not determined, and although Kern is inclined to consider them similar to the chromatic-ring substance in Dytiscus, there is a possibility that they may be mitochondrial in nature or may consist of some other cytoplasmic material.

The presence of intercellular bridges is important, since it makes it possible to determine the relationship of the groups of cells in the ovarioles. But in the queen bee these bridges do not persist to any considerable extent after the zone of differentiation has been reached. Here, however, as shown in figures 7 to 12, there are well defined rings between the cells which indicate their relationship. It might be argued that these rings may arise where two cells happen to come into contact, if it were not for the fact that all stages between the fully developed bridges and the presence of clearly defined rings have been observed. These are no doubt the persisting mid-bodies or 'Zwischenkorper' which remain between the cells after division. They have been noted especially by Giardina ('01) in Dytiscus; by Marshall ('07) in Polistes; by Kern ('12) in Carabus; and by Maziarski ('13) in Vespa.

Summary of Part I. 1. Four rather definite regions may be recognized in the ovariole of the queen honey bee (fig. 1): (a) the terminal filament; (6) a rosette region; (c) a zone of differentiation; and {d) the posterior part in which the oocytes are arranged in a linear series and separated from each other by groups of nurse cells.

2. The rosette region is filled with rosette-like groups of cells, each group consisting of the descendants of a single mother oogonium. The cells of a rosette are united by strands which are the persisting spindle fibers from earlier mitoses (fig. 3). The cells in a rosette divide synchronously (fig. 5).

3. Oocytes and nurse cells are both derived from the oogonia. Their differentiation occurs in the zone of differentiation (fig. 1, d). One or more cells of each rosette enlarges and becomes an oocyte, whereas the others retain more of their earlier characteristics and become nurse cells. Although the strands which connected the cells in a rosette disappear, the descendants of a single oogonium


506 ROBERT W. HEGNER

may still be determined, because of the presence of deeply staining rings between the cells (figs. 7-12).

4. The causes of differentiation could not be definitely determined, but several hypotheses are mentioned (p. 5C0).

5. Granules appear near the nucleus of oocytes shortly after their differentiation. Later they become distributed throughout the egg cytoplasm. These granules appear to be mitochondrial in nature and to arise from, or under the influence of the nucleus.

II. THE BACTERIA-LIKE RODS AND SECONDARY NUCLEI IN THE

OOCYTES OF CAMPONOTUS HERCULEANUS VAR.

PENNSYLVANICA DEG.

The important contributions by Blochmann ('84, '86) upon the growth of the oocytes in ants seem to be the only reports that have ever been made on this subject. Blochmann discovered two very interesting facts regarding these oocytes: (1) the presence of rod-shaped bodies almost completely filling the growing egg which he considered symbiotic bacteria, and (2) the formation of nuclear-like bodies around the oocyte nucleus. Recently Tanquary ('13) has described, in the freshly laid eggs of the carpenter ant, a body which he calls a cleavage nucleus, but which resembles very closely bodies that have been discovered in the eggs of other animals and to which I have applied the term keimbahn- or germ-line determinants. The observations recorded in the following pages were made in order to trace the genesis of the eggs of ants with special reference to the origin, distribution, and fate of the bacteria-like bodies, nuclearlike bodies, and the germ-line determinants.

The material used for these studies consisted of the ovaries of the carpenter ant, Camponotus herculeanus var. pennsylvanica DeGeer. A large number of virgin queens were obtained from a dying apple tree on April 3, 1914, and some of them were kept alive until June 9, 1914. The ovaries were dissected out in Ringer's solution and immediately fixed in the same manner as were those of the honey bee (page 496). Ovaries were preserved at intervals of a few days during the period of two


STUDIES ON GERM CELLS 507

months. In this way oocytes in all stages of growth were obtained up to almost the period of deposition. Sections were cut and stained as in the queen bee.

The ovaries of the carpenter ant resemble those of the queen bee in general structure and the ovarioles are likewise similar. The youngest ovaries obtained had already passed the period when the oocytes and nurse cells are differentiated, so there was no opportunity to study the events that occur during this differentiation. Four regions may be distinguished in the ovarioles as shown in figure 13. There is a terminal filament (t) at the anterior end. This is followed by a region which we may call the terminal chamber (i.e.) containing oocytes, nurse cells, and epithelial cells without any special arrangement. The next part of the ovariole is short and contains oocytes which have grown considerably but have not yet taken a position in the axis of the tubule. This we may call the first zone of growth (g) . The rest of the ovariole consists of a linear series of oocytes (o) each with its accompanying group of nurse cells (n). Each oocyte is larger than the one anterior to it and the nurse cells gradually become grouppd into a definite nurse chamber (n.c). The bacteria-like bodies discovered by Blochmann are present only in the last described zone. The first signs of nuclear-like bodies around the oocyte nucleus also appear here. For the sake of convenience oocytes in the various stages which need to be referred to have been drawn in outline and to scale as shown in figures 14 and 15.

The posterior end of the terminal filament (t) and anterior end of the terminal chamber {t.c.) are shown in outline in figure 16. The cells of the terminal filament are long and slender and extend entirely across it. One is shown enlarged in figure 17. Within the terminal chamber are two kinds of cells, oocytes and nurse cells. The oocytes, as indicated in figure 18, are the youngest to be found in the ovarioles at this time and I have regarded them as Stage A (fig. 14). The cell walls of the nurse cells are not very distinct. Their nuclei (fig. 19) are much smaller than those of the oocytes and contain a single irregular mass of chromatin granules. The structure of the oocytes and nurse cells is similar throughout the entire terminal chamber.


508 ROBERT W. HEGNER

The terminal chamber is separated from the first zone of growth (fig. 20) by what appears to be a distinct membrane (m). The condition of all of the oocytes is similar throughout this zone (Stage B, fig. 14). The oocytes have grown considerably and their nuclei (fig. 21) contain a few clumps of chromatin granules lying near the nuclear membrane. Outside of the nucleus (fig. 21) is a layer of darkly staining substance which resembles chromatin in some respects and may represent chromatin which has passed through the nuclear membrane into the cytoplasm. The nurse cells now have definite cell walls (fig. 22) and are also characterized by a layer of darkly staining material lying around the nucleus. Among the oocytes and nurse cells are a few epithelial cells (fig. 23) ; these have no definite cell walls, and their nuclei are rather irregular in shape and contain a single mass of chromatin.

Whether or not the first zone of growth is definitely separated from the remaining part of the ovariole could not be determined with certainty, but its limit is conspicuously marked by the abrupt appearance of the bacteria-like bodies of Blochmann. This is indicated in figure 24, which shows. the posterior portion of the first zone of growth and the anterior part of the rest of the ovariole. In the upper part of this figure is a single oocyte in Stage B and a number of nurse cells. These are apparently embedded in a loose reticulum of cytoplasm. Further down the ovariole the spaces surrounding the nurse cells and epithelial cell nuclei are filled with more or less wavy rods w^hich Blochmann considered symbiotic bacteria. These rods extend throughout the ovariole in all directions, being represented by distinct spherical granules where cut across.

From this point on, the oocytes are arranged in a linear row in the central axis of the ovariole (figs. 13 and 25). The cytoplasm of the oocytes increases rapidly in amount, but the nuclei enlarge very little. The nurse cells (fig. 25, n) become arranged more or less definitely into rows which radiate toward the upper end of the oocyte. Those nurse cells closest to the oocyte increase more rapidly in size than do the others. Compare, for example, that lettered a in figure 25 with its companions, and


STUDIES ON GERM CELLS 509

those accompanying the upper oocyte with those of the lower oocyte. Surrounding the oocytes, nurse cells, and epithelial cell nuclei are the groups of bacteria-like bodies.

The transition of the oocyte from Stage C (fig. 14, C; fig. 25, C2) to Stage D (fig. 14, D, fig. 26) is accompanied by an invasion of the oocyte cytoplasm by the bacteria-like rods. Some of these rods form almost perfect circles, resulting in what at first sight appear to be vacuoles. Some of the epithelial-cell nuclei are in very close contact with the oocyte but these were not observed actually within the oocyte cytoplasm.

The principal difference between an oocyte in Stage D (fig. 26) and one in Stage E (fig. 14, E; fig. 27) is the sudden appearance of nuclear-like bodies around the nucleus, which I shall call secondary nuclei. The nucleus itself is about equal in size to that of the preceding stage (fig. 26). The chromatin, which in younger oocytes (figs. 24-26) has gradually migrated from the periphery toward the center of the nucleus where it formed an irregular clump, has again become scattered, being represented by a few smaller and widely separated masses. In the illustration (fig. 27) three secondary nuclei are shown lying below but in contact with the oocyte nucleus. These likewise contain a delicate reticulum and from one to three chromatin masses. No intermediate stages between the nucleus of Stage D (fig. 26) and that of Stage E (fig. 27) were discovered, and it was thus impossible to determine with certainty the origin of these secondary nuclei. If, however, the oocyte nucleus continued to increase in size at the same rate as indicated in Stage C (fig. 25) and in Stage D (fig. 26) it would be about the size of that in figure 27 after having given rise to the secondary nuclei by the method of budding or in some other way. This subject will be discussed more in detail later.

During the interval between Stage E (fig. 14, E; fig. 27) and Stage F (fig. 14, F, fig. 28) the oocyte enlarges until it extends almost across the ovariole, and the epithelial cell nuclei become arranged in a single layer around it, forming a follicle. At this time (fig. 28) the cytoplasm of the oocyte and that surrounding the nurse cells and epithelial-cell nuclei is crowded full of the


510 ROBERT W. HEGNER

bacteria-like rods. The secondary nuclei also increase in number around the oocyte nucleus; the. nucleus itself does not increase in size. Both the oocyte nucleus and the secondary nuclei are sometimes irregular in shape, a condition that may be due to the effects of fixation, or that may represent a stage in budding or in amitotic nuclear division (page 518).

The next phase of the growth period (Stage G, fig. 14, G, fig. 29) witnesses the lengthening of the oocyte and the further arrangement of the nurse cells to form a compact group, which becomes surrounded by epithelial cells, thus producing a definite nurse chamber. The bacteria-like bodies increase in number as the oocyte grows and continue to fill it completely with bundles of rods. The secondary nuclei near the oocyte nucleus also increase slightly in number.

Shortly after this condition is reached the oocyte is invaded just beneath the nurse chamber by an influx of cytoplasm elaborated by the nurse cells (fig. 30, c). This cytoplasm is free from the bacteria-like bodies and it seems very probable that it either forces these rods out of its path or else dissolves those which it encounters. There is evidence that, from this stage on, the number of bacteria-like rods does not increase, the rods gradually lose their compact grouping and become further separated from one another, the spaces between them probably being occupied by the cytoplasm added to the oocyte by the nurse cells. The oocyte nucleus by this time (fig. 30) is completely surrounded by secondary nuclei from which it differs in appearance. The secondary nuclei contain a rather dense reticulum and one or several large chromatin granules, whereas the oocyte nucleus is very irregular in shape and contains a delicate reticulum which causes it to appear clearer. The irregular shape of the oocyte nucleus is probably due to the pressure upon it of the secondary nuclei which surround it. Its decrease in size is also noticeable and one cannot but suspect that this decrease is directly related to the increase in the number of secondary nuclei. A transverse section through an oocyte near the nurse chamber is shown in figure 31.


STUDIES ON GERM CELLS 511

The nurse chamber is now completely formed (fig. 13, n.c). The nurse cells are still free from the bacteria-like rods and their nuclei, as pointed out by Blochmann ('86), possess very thick membranes (fig. 30, n) . Part of one of these nuclei greatly enlarged is shown in figure 32. The membrane contains, in a homogeneous matrix, a number of vacuoles and a great many granules of various sizes which appear in material fixed and stained by a number of different methods. Their reactions all indicate that they are chromatic in nature and their position suggests that they may have migrated into the membrane from inside of the nucleus and are on their way into the cytoplasm. It could not be definitely determined, however, whether this is a true case of chromatin emission or simply a condition due to the action of the fixing solutions used.

A further increase in the amount of cytoplasm within the oocyte is evident when Stage H (fig. 15, H; fig. 33) is reached. Here an opening (a) is present in the follicle connecting the oocyte directly with the nurse chamber. The small plug of cytoplasm filling this channel is no doubt homologous with the nutritive string present in the ovarioles of insects whose oocytes are not accompanied by a group of nurse cells, but are connected with the terminal chamber by a protoplasmic thread. In this stage the oocyte nucleus (o) is still closely pressed by the secondary nuclei (s) surrounding it and the entire group lies within the cytoplasmic zone. Such a group is shown enlarged in figure 34, in which the oocyte nucleus may be distinguished from the secondary nuclei by its irregular shape, central position, and clearness.

The succeeding stages in the growth of the oocyte (fig. 15, /, J, K, L; figs. 35-39) are characterized by a decrease in the number of bacteria-like rods, by the formation of yolk globules, and by the increase in number and the scattering of the secondary nuclei. Part of a section through an oocyte of Stage I (fig. 15, 1) is shown in figure 35 which represents a portion extending from a point midway between the two poles out to the middle of the oocyte. Just within the follicular epithelium (e) is the suggestion of a clear layer {k) which later becomes the 'Keimhautblastem.' The

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 3


512 ROBERT W. HEGNER

black spherical bodies are yolk globules (y) which appear to originate near the periphery and gradually to migrate into the central region. The bacteria-like rods are still present but they are widely scattered and faintly staining.

By the time the next stage is reached (fig. 15, J; fig. 36) the bacteria-like rods have completely disappeared everywhere except near the periphery, around the lower part of the oocyte. According to Blochmann ('86) they are still present in this region after the eggs are laid, and they are also mentioned by Tanquary ('13) in the freshly deposited eggs. The latest oocyte studied by the writer is Stage L (fig. 15, L), which is considerably younger than the fully grown egg. A few faintly staining rods still exist at this stage near the posterior end.

The compact group of secondary nuclei which surround the oocyte nucleus up to this stage now breaks up, and the individual nuclei become scattered throughout the entire egg near the periphery. Quite a number of them still appear near the anterior pole of an oocyte in Stage K (fig. 15, K; fig. 37) where they surround the opening into the nurse chamber. At some distance back of this pole the secondary nuclei are imbedded in the cytoplasm especially near the perpihery. They retain at this time (Stage K) the characteristics noted in early stages; i.e., they are more or less spherical, filled with a reticulum, and contain one or several large chromatin granules (fig. 38). Later (Stage L, fig. 15, L; fig. 39) they seem to be more numerous and a single egg must contain hundreds of them. In this, the last stage examined, these secondary nuclei have changed in appearance as indicated in figure 40. The chromatin granules have become aggregated into a few irregular strands, a condition which may be a phase of degeneration or, as Loyez ('08) believes, a stage in the formation of yolk globules. The fate of the secondary nuclei was not discovered and, so far as I know, none of the investigators who have described similar bodies has been able to determine with certainty what becomes of them.

The posterior ends of the older oocytes in my series were carefully examined with a view to tracing the origin of the body which Tanquary ('13) observed near the posterior pole of freshly


STUDIES ON GERM CELLS 513

laid eggs of Camponotus herculeanus var. ferrugineus Fabr. and called the cleavage nucleus (fig. 41, n). This body is obviously not a cleavage nucleus since it is not in the usual position occupied by this nucleus, and does not possess the characteristics of a cleavage nucleus. Furthermore, it persists during the early cleavage stages at the posterior end, whereas the cleavage cells (nuclei) are shown by Tanquary in their proper position near the anterior pole (fig. 42, cc). It seems probable therefore that this body belongs to the class of substances which have been found in the eggs of many different kinds of animals and which later become part of the material within the primordial germ cells — substances to which I have applied the term 'Keimbahn or germ-line determinants.' This seems all the more probable since it persists at least until a late cleavage stage (fig. 43, rn.) and later there is present a group of cells (fig. 44, kc.) which Tanquary describes as a "group of small cells applied to the posterior end of the inner peripheral protoplasm," and which further research will doubtless prove to be germ cells, No bodies were discovered in my material that could be recognized as an early stage in the formation of the 'cleavage nucleus' described and figured by Tanquary.

Discussion. No opportunity was afforded by the material in my possession for determining the differentiation of the oocytes from the nurse cells, since these two sorts of cells are established in ovarioles younger than those in the virgin queens collected in the spring. The problem of the separation of germ cells (oocytes) from somatic cells (nurse cells) therefore could not be solved. The most interesting phenomena exhibited by the ovarioles are (1) the presence of the bacteria-like rods and (2) the formation and distribution of the secondary nuclei.

The bacteria-like rods. Blochmann ('84, '86) was the first to observe the bacteria-like rods in the ovarioles of two species of ants, Camponotus ligniperda and Formica fusca. Recently Tanquary ('13) has observed these bodies in the cytoplasm at the posterior end of freshly laid eggs of Camponotus herculeanus var. ferrugineus. Blochmann found those in the eggs of Camponotus to be from 10 to 12ju in length, whereas those in Formica


514 ROBERT W. HEGNER

were only from 4 to 5^ long and were not so regularly arranged in bundles as in the former species He at first supposed these bacteria-like bodies to be cytoplasmic structures, but, after observing them in various stages of division, expressed the opinion that they are symbiotic bacteria.

Bodies of a similar kind have been observed in many other insects. Those that occur in the cockroaches most closely resemble the bacteria-like rods in the ovarioles of ants. These likewise were first discovered by Blochmanh ('87, '92) in Periplaneta orientalis. They occurred not only in the eggs but also among the blastoderm cells and in the spaces formed by the liquefaction of the yolk in the embyros. Later they were observed in the anlage of the fat body where they persist in the adult stage. Wheeler ('89) described them in the 'Keimhautblastem' of Blatta germanica as "minute rod-shaped bodies so numerous in the surface protoplasm as to make it appear reticulate. They look like bacillar micro-organisms and stain deeply."

Mercier ('07) has subjected these bacteria-like rods in Periplaneta orientalis to careful study. He agrees with Blochmann regarding their distribution and confirms Blochmann's statement that they multiply by division. Mercier was able to cultivate the rods and concludes that they are true bacteria and thinks them to be of a symbiotic nature although he was unable to suggest any advantage that the host receives because of their presence. They are given the name Bacillus cuenoti by Mercier.

Many other investigators have reported bacteria within the eggs or tissues of insects. Blochmann ('87) observed them in Pieris, Musca, and Vespa; Stuhlmann ('86) shows them in many of his figures, and Forbes ('91) found them in the caecal glands of various Heteroptera. The 'green or yellow granular mass' described by Leydig ('50) in the embryos of viviparous aphids 'and later called the 'pseudovitellus' by Huxley ('58) and the 'green body' by Witlaczil ('84) is considered now to be due to symbiotic organisms. Of particular importance are the contributions of Mercier ('07) on the cockroach and of Sulc ('06, '10), Pierantoni ('10), and Buchner ('12) on the Hemiptera. Buchner ('12) has given a full historical discussion of the subject


STUDIES ON GERM CELLS 515

besides adding considerable new material, and any one desiring a comprehensive review of ihe present state of our knowledge of these symbiotic organisms is referred to his paper. Thirtyfour species are described and figured by Buchner. Some of them are bacteria, such as those in the cockroach, but others are more like yeasts. The infection of the egg, which reminds one of the infection of the egg of the Texas fever tick by Piroplasma bigeminum, may be diffuse, as in the cockroach, or localized, as in the aphids. Buchner decides that these organisms are symbiotic, but, like Mercier, was unable to discover any advantage to the insect host from the relationship.

The secondary nuclei. One of the most interesting features of the growth of the oocyte in certain insects is the formation of small nuclear-like bodies around the oocyte nucleus. Bodies of this sort were first described by Blochmann ('84, '86) in Hymenoptera. Since then they have been observed in insects belonging to this order by Stuhlmann ('86) and Marshall ('07) and similar bodies were noted by Korschelt ('86) near the nuclei of both the oocyte and nurse cells of the fly, Musca vomitoria. Korschelt was unable to determine the origin, function, and fate of these 'helle Blaschen' but noted their resemblance to those discovered by Blochmann.

The two ants, Camponotus ligniperda and Formica fusca, and the wasp, Vespa vulgaris, were all found by Blochmann to be very much alike so far as the growth of their oocytes is concerned. The origin of the nuclear-like bodies is described in Camponotus as follows:

Bei etwas alteren Eiern beginnt nun an der Oberflache des Kernes ein Knofpungsprocess, der schliesslich zur Entstehung einer grossen Anzahl kleiner Kerne ftihrt. Man bemerkt als erste Andeutung dieses Processes kleine, helle, rundliche Gebilde, die dicht an der Oberflache des Kernes anliegen, und die ich in meiner vorlaufigen Mittheilung als 'knotchenformige Verdichtungen' bezeichnet hatte. Ich neige jetzt zu der Ansicht, dass es von vornherein kleine Vacuolen sind, da die Kernmembran sich meist etwas farbt und stets sehr scharf erscheint, wahrend ich an diesen kleinen Gebiklen bei ihrem ersten Auftreten keine derartige Membran unterscheiden konnte. Bald tritt in diesen Vacuolen ein kleines mit Pikrocarmin sich farbendes Kornchen auf diese Vacuolen oder, wie ich sie jetzt nach dem Auftreten des Chro


516 EGBERT W. HEGNER

matinkornchens nennen will, Nebenkerne, nehmen allmahlich an Grosse zu, wobei sie dami eine sehr deutliche Membran an ihrer Oberflache erkennen lassen, zugleich nimmt der Inhalt an festen, farbbaren Substanzen zu. Diese treten theils als kleine, rundliche Nucleolen, oder als feine, wenig sich farbende Fadchen auf.

Da nach und nach immer mehr solche Nebenkerne entstehen, finden wir bei etwas weiter in der Entwicklung fortgeschrittenen Eiern in der Region der Eirohre, wo bereits Eifacher und Nahrzellenfacher deutlich abgegrenzt sind, die Oberflache des Eikernes von einer ganzen Schicht solcher Nebenkerne von verschiedener Grosse bedeckt, die sich gegenseitig beriihren. So bleiben die Verhaltnisse auch in noch etwas alterne Eiern, (pp. 144-145).

These Nebenkerne, accordmg to Blochmann, after multiplying by self-division become scattered within the yolk where they degenerate, none being present in the ripe egg.

Among the other investigators who have observed similar bodies in the oocytes of insects are the following: Will ('84) and Ayers ('84) observed them in Hemiptera and considered them follicular epithelial cells which contributed to the formation of the yolk. Stuhlmann ('86) has described them in many insects, including Musca, Periplaneta, Locusta, Pieris, Aphrophora. Sphinx, and certain Coleoptera and Hymenoptera. They were called 'Reifungsballen' by him and were thought to be similar to the polar bodies which at that time had not yet been observed in insects. The 'Reifungsballen' appear at different stages of the growth period in different species and also have different fates; some of them fuse to form a large 'Dotterkern' which lies near the posterior end of the egg and resembles what I called ^Keimbahn-determinants,' and others become widely distributed and disappear in the yolk. The possible origin of the 'Ballen' from epithelial cells is suggested but not considered probable. Korschelt ('89) from a study of them in Bombus, concludes, as did Will and Ayers, that they are derived from epithelial cells.

In Blatta germanica and Leptinotarsa decemlineata, Wheeler ('89) has described as 'maturation spheres' a number of globular bodies which appear after the egg nucleus migrates to the periphery and prepares for maturation. In Blatta several of these spheres may be present. In Leptinotarsa a number of oval


STUDIES ON GERM CELLS 517

hyaline masses likewise occur which are considered the equivalents of the 'maturation spheres' inBlatta and homologous to the 'Reifungsballen' of Stuhlmann. No chromatin masses were observed in any of these spheres, but in Leptinotarsa the wandering of part of the chromatin into the yolk, where it disappears, is described. As Stuhlmann pointed out, these spheres -may appear in different species at different stages in the growth period and it seems therefore possible that the 'Nebenkerne' of Blochmann, the 'Reifungsballen' of Stuhlmann and the 'maturation spheres' of Wheeler may be homologous, although the first two contain chromatin whereas the 'maturation spheres' do not. Lameere ('90) was able to confirm Blochmann's account regarding the origin of the Nebenkerne in Camponotus and Henneguy ('04) found them in both the wasp and the honey-bee. In the former they appear around the germinal vesicle and disappear very early, but in the bee they seem to be derived from follicular epithelial cells and persist until a later developmental stage. None of these bodies could be found in the oocytes of the honeybee which I have studied.

Marshall ('07) made a careful study of the secondary nuclei in Polistes, but, like previous investigators, was unable to determine definitely regarding their origin and fate. He agrees with Blochmann that they probably arise from the germinal vesicle by budding, but was unable to find any stages in such a process. Concerning their function Marshall was likewise unable to come to a definite conclusion, but suggests that they may act upon the nurse-cell substance making it available for the oocyte.

As described on page 509, the secondary nuclei of Camponotus make their appearance at Stage E (fig. 14, E; fig. 27) in the growth of the oocyte. From this stage on the size of the primary nucleus does not increase but actually decreases and the number of secondary nuclei becomes greater as the oocyte enlarges (compare figs. 28, 31 and 33). At first the oocyte nucleus always lies very close to the center of the anterior pole of the oocyte and the secondary nuclei form a single layer in contact with the opposite wall of the oocyte nucleus (figs. 27, 28, 29), but in later


518 ROBERT W. HEGNER

stages (figs. 30, 33) the group of nuclei is more often near one side, at the anterior pole, and the oocyte nucleus is entirely surrounded by secondary nuclei, the latter sometimes being several layers in thickness (fig. 34). Hundreds of such groups were carefully examined, beginning with oocytes in Stage D (fig. 14, Z); fig. 26), but iti no case could the origin of the secondary nuclei be definitely determined. As the latter increase in number, they, as well as the oocyte nucleus, tend to lose their spherical shape and become oblong, or indented, or more or less irregular (figs. 28, 29, 34). This may be due* to the action of the fixing solution, or to the pressure of one upon another, but many of them present shapes very suggestive of budding, or of more or less equal constriction into two. Some groups selected from the large number examined are shown in outlme in figure 45. Frequently the space produced by the indentation of one of the nuclei is perfectly clear and resembles a vacuole. This suggests the possibility that the irregularity of the nucleus may be due to the escape of material from it which occupies the space formed by the caving in of the nuclear membrane. If this material were then to become surrounded by a nuclear membrane a secondary nucleus would be the result.

Two other theories have been suggested to account for the formation of secondary nuclei. According to Will ('84) in Hemiptera, Korschelt in Bombus, Henneguy in the honey-bee, and Brunelli ('04) in Hymenoptera they appear to come from follicular epithelial cells. Brunelh thinks they are attracted around the germinal vesicle by chemical action. Gross ('03) likewise believes from his studies on Bombus and other Hymenoptera that they are true nuclei, but that they originate from the epithelial cells which are situated among the nurse cells. This cannot of course be true in forms such as Camponotus where the secondary nuclei appear before a follicle and nurse cells are acquired. The other theory is that advanced by Stuhlmann who says "Ich wiederhole noch einmal, dass ich diese Kerne nur ftir 'Dotterconcretionen' halte."

The investigations of Loyez ('08) upon the 'noyaux de Blochmann' are the most thorough yet pubhshed. She studied these


STUDIES ON GERM CELLS 519

secondary nuclei or 'pseudo-noyaux' as she calls them, in four species of Bombus, two species of Vespa, and one species of Xylocopa. They were found to resemble true nuclei in their fully developed condition, but all stages were observed between these and the very small vacuole-like bodies from which they apparently arise. The theories of their origin by budding off from the germinal vesicle and by the emigration of epithelial cells are considered by Loyez to be untenable. The conclusion is reached that they originate from the germinal vesicle, follicular epithelial cells, and nurse cells not by budding or the emigration of entire nuclei "mais resultent d'une coagulation de substances venues du dehors al'etat fluide on granuleux et modifiees par le cytoplasme de' I'oeuf." (p. 100). In old oocytes the secondary nuclei were found to change in structure so that they resemble nuclei which are undergoing synapsis, and, since all stages between the typical secondary nucleus and a homogeneous globule were observed, Loyez decides that they transform into deutoplasmic spheres.

The presence of these secondary nuclei in certain insects and not in others can be regarded as a sort of precocious diminution of nuclear substances. The loss of chromatin by passage through the nuclear membrane and its identification as chromidia in the cytoplasm has been reported by a number of investigators as taking place in the nuclei of many different kinds of cells during what is known as the resting stage. During ordinary mitosis only a part and sometimes the smaller part of the nuclear chromatin is concerned in the formation of the spireme, the rest being cast out into the cytoplasm with the other nuclear contents when the membrane breaks down. These substances' become scattered and dissolved in the cytoplasm. Just before the maturation divisions occur, it is customary for the germinal vesicle to liberate into the cytoplasm a considerable part of its contents, including granules or small masses of chromatin which become scattered amid the yolk globules and disappear. The diminution of nuclear substance therefore seems to be a widespread process. That the formation of at least part of the secondary nuclei in the oocytes of certain insects is likewise a nuclear


520 ROBERT W. HEGNER

diminution process also seems probable. Each secondary nucleus contains masses of chromatin and in every way resembles a true nucleus, but, as in other cases of diminution, this chromatin and the other contents of the secondary nuclei are lost in the general egg substance. The elimination of this material simply occurs in these species at an earlier stage than in the oocytes of other animals.

The function and fate of the secondary nuclei cannot be stated with any degree of certainty. We have seen that they cease to form a compact group in the older oocytes and become distributed throughout the egg, especially near the periphery (figs. 37 and 39). Later they undergo a process which appears to be degenerative, and, according to those who have studied later stages, finally disappear altogether. The writer suggested a few years ago (Hegner '09) that secondary nuclei of this sort might migrate to the posterior pole and take part in the formation of the germ-line-determinants, but thus far no actual evidence that this occurs has been obtained. Marshall ('07) has expressed the opinion that they may make the substances provided by the nurse cells available for the oocyte. It also is possible, as Loyez claims, that these secondary nuclei may have some function in the formation of yolk.

Summary of Part II. 1. The ovarioles of Camponotus consist of four distinct regions (fig. 13), (a) a terminal filament, (b) a terminal chamber, (c) a zone of growth free from bacteria-like rods, and (d) the posterior part in which the oocytes are arranged in a linear series, are accompanied by nurse cells, and are surrounded and later invaded by the bacteria-like bodies.

2. The bacteria-like rods occupy definite regions of the ovariole. They are absent entirely from the terminal filament, terminal chamber and first zone of growth. In the rest of the ovariole they occur everywhere except in the nurse cells (fig. 25) . The oocyte is at first free from them (fig. 25) but later is invaded (fig. 26) and almost completely filled with them (fig. 29). The rods are arranged at first in bundles (figs. 25, 29), but later become scattered (figs. 35, 36). As the oocyte increases in size and


STUDIES ON GERM CELLS 521

yolk formation proceeds, they gradually disappear until none are visible except near the periphery in the posterior region.

3. Secondary nuclei appear near the oocyte nucleus at an early stage of growth (fig. 27). They increase in number, finally completely surrounding the germinal vesicle (figs. 33, 34). They later become distributed throughout the oocyte especially near the follicular epithelium (figs. 37, 39). Their origin by budding from the oocyte nucleus, or by the iromigration of epithelial cells seems improbable. The conclusion is reached that the oocyte nucleus gives off materials into the cytoplasm which become enclosed by a membrane and develop into nuclear-like bodies. The fate of the secondary nuclei was not determined.

III. HISTORY OF THE NUCLEI AND GERM-LINE DETERMINANTS

IN THE OOCYTES OF CERTAIN PARASITIC HYMENOPTERA

AND HYMENOPTEROUS GALL-FLIES

1. Copidosoma gelechiae

In June, 1914, I published a short account of the growth of the oocyte in Copidosoma gelechiae with special reference to the origin of the germ-line-determinants. Since then two other accounts have appeared on the same subject, one by Martin ('14) on Ageniaspis (Encyrtus) fuscicollis, and the other by Silvestri ('14) on Copidosoma buyssoni. I have also been able to obtain and study a new lot of material. This makes it possible for me to add to my previous account and to clear up certain points about which differences of opinion have arisen. These polyembryonic Hymenoptera are interesting principally because of the peculiarities in their embryonic development. We shall refer in this paper to two of these; (1) the history of the egg nucleus and (2) the origin and fate of the germ-line-determinants.

Silvestri ('06-'08) has shown that a body which he considered the nucleolus of the germinal vesicle is present near the posterior end of the eggs of certain parasitic Hymenoptera. During embryonic development this body is segregated in a single cleavage cell until the seven-cell stage is reached; then, having disintegrated, its substance is divided between two cells. These,


522 ROBERT W. HEGNER

according to Silvestri, are the parents of all of the germ cells, a conclusion that seems justified, since a similar body in monembryonic parasites has been definitely traced until it becomes distributed among the germ cells.

In my preliminary report on Copidosoma gelechiae I pointed out the improbability of the origin of the 'nucleolo' of Silvestri from the nucleolus of the germinal vesicle, and concluded from the material I then possessed that this body consists of all of the chromatin from the oocyte nucleus which had formed into a compact mass. To explain the presence of both this body and an egg nucleus it was suggested that two oocytes might fuse end to end, the posterior one furnishing the 'nucleolo' and the other the nucleus. The eggs of these insects, when ready to be laid, are long, with a very slender bent portion between the two thicker ends, as shown in figure 54. My material consisted only of serial sections cut 2 and 4)u thick, and, as Silvestri ('14) has pointed out, I considered sections through the anterior and posterior ends of an oocyte as sections of complete oocytes. This is a mistake that I now wish to acknowledge, but is one that could hardly be avoided without good in toto preparations. With the aid of such preparations I have been able to confirm Silvestri's account in most respects. My conclusion, however, that the 'nucleolo' of Silvestri is not the nucleolus of the oocyte nucleus is correct, and my account of the history of the oocyte nucleus up to its change into an oval mass of chromatin is also correct, as indicated by the study of new material, and by the confirmatory account by Martin in Ageniaspis. I am indebted to Dr. R. W. Glaser for this new material.

Martin ('14) records the presence of three kinds of cytoplasmic inclusions in the growing eggs of Ageniaspis: (1) a cloud of granules near the posterior end, (2) a 'nucleolus' also near the posterior end, and (3) a few chromatin granules cast out by the nucleus. The 'nucleolus' is of particular interest to us, since it is undoubtedly a body similar to the 'nucleolo' of Silvestri. Martin was able to trace this 'nucleolus' from the young oocytes to the three-cell stage in the cleavage of the developing eggs. It appears fitst as a small group of granules lying in the midst


STUDIES ON GERM CELLS 523

of the cloud of granules mentioned above. It gradually increases in size, reaching its maximum dimensions about the time the egg reaches its full size. Then it becomes vacuolated and loses some of its affinity for stains. When the first cleavage division occurs, it passes entire into one of the two blastomeres. This blastomere does not divide as quickly as the other and a three-cell stage thus results, one cell containing the 'nucleolus' and the other two lacking this body. At this point the 'nucleolus' breaks down and can not be traced further. Regarding the origin of the 'nucleolus' Martin is not certain. He agrees with me that it is not derived from the nucleolus of the germinal vesicle, since, when it first appears, it is at the opposite end of the oocyte. Apparently it is built up by the aggregation of the deeply staining granules among which it lies, but where these granules originate was not determined.

The history of the oocyte nucleus. The ovaries of Copidosoma consist of a number of ovarioles, each of which contains a row of oocytes in various stages of growth, the oldest being situated near the posterior end. It is thus possible to find without much difficulty all stages in the growth period. We shall begin our account with an oocyte (Stage A, fig. 46) which has already acquired an epithelium and is accompanied by a nurse chamber. A close examination of such an oocyte (fig. 55) reveals a very large nucleus, containing an irregular, homogeneous mass of chromatin. A very thin layer of cytoplasm surrounds the nucleus.

The nucleus does not increase much in size during the growth period, but the oocyte enlarges rapidly because of the accumulation of cytoplasm. During the interval between Stages A and B (figs. 46, 47) both the oocyte and the oocyte nucleus become larger and oval. The chromatin now consists of what appears to be a long much coiled thread (fig. 56) and one is led to believe that the homogeneous mass in Stage A is really the same thread much more compactly coiled; in other words, in the condition as synezesis. By the time Stage C (fig. 48) is reached the nucleus has again regained a spherical shape and the chromatic spireme has become spread out as shown in figure 57. Up to


524 ROBERT W. HEGNER

this time the cytoplasm appears to be homogeneous throughout. Martin finds in Ageniaspis at this stage a cloud of granules in the posterior region (fig. 70) but nothing of the sort is present in my preparations, nor were such granules observed by Silvestri in Copidosoma bussyoni. Silvestri ('14), however, thinks he has discovered a group of granules at the posterior end of the nucleus at about this stage (fig. 69) which he suggests may lead to the formation of the oosoma (formerly called by him the 'nucleolo' and designated by me as a keimbahn or germ-linedeterminant) .

The nuclear phenomena are of considerable interest from this time on. The spireme becomes more and more open (Stages D, E, figs. 49, 50) and finally breaks up into thin, chromosomes of irregular shape (Stage F, fig. 51). These chromosomes then become shorter and thicker and appear to unite near their ends (Stage G, fig. 52). At first the pairs are scattered about within the nucleus (fig. 58) but they soon straighten out and become arranged in a parallel series with their points of union lying in the equator (Stage H, figs. 53, 59, 60). Spindle fibers could be seen, but apparently no centrosomes or asters are present. The number of pairs of chromosomes as indicated by cross sections of spindles of this sort seem to be twelve, the same number recorded by Silvestri for C. bussyoni, but several very clear sections contain only eleven (fig. 61).

Soon after the parallel arrangement of the chromosome pairs occurs, the egg reaches its full growth and attains its definite shape (Stage I, fig. 54). The mitotic figure then passes through the stages of condensation, as described in my preliminary report (Hegner '14 b). The chromosomes gradually get closer together and become shorter and thicker (fig. 62). Where their ends meet at the equator a ridge appears, which causes the complex to resemble a maltese cross (fig. 63) . Soon the spaces between the chromosomes are entirely obliterated (fig. 64) and a homogeneous mass of chromatin results (fig. 65).

Silvestri has noted the parallel arrangement of these chromosome rods, but has evidently failed to observe their condensation. Martin, however, has reported a similar phenom.enon in Agenias


STUDIES ON GERM CELLS ' 525

pis, although in this form the rods which condense seem to consist of single instead of double chromosomes (fig. 66). The history of the nucleus as recorded by Martin is as follows:

The chromatin in the very young oocyte is aggregated, at the posterior side of the nucleus. As the oocyte grows, it spreads throughout the nucleus, forming numerous granules which are distributed upon a reticulum. Chromosomes are than formed and soon become arranged on a spindle, which becomes more and more compact until a single mass of chromatin results (fig. 67) . This mass divides in polar body formation (fig. 68) apparently without the presence of spindle fibers or asters.

The germ-line-determinants in Copidosoma. The 'nucleolo' or germ-line-determinant appears in my material at about Stage D (fig. 49) at which time it is perfectly distinct, staining a deep black in iron hematoxylin. From this stage on it is invariably present, increasing in size until Stage F (fig. 51) is attained. Five methods of origin have been suggested for this body. (1) Silvestri's ('06) first idea that it consists of the nucleolus of the germinal vesicle was shown to be incorrect in my preliminary report (Hegner '14 b) and Silvestri has admitted his error (Silvestri '14). (2) My conclusion that it arises from the chromatin of the oocyte nucleus has on the other hand been disputed by Silvestri and I wish here to acknowledge the truth of his observations. (3) In his latest report Silvestri ('14) coins a new name for this body, calling it the 'oosoma,' and thinks that it may possibly arise from a heap of granules at the posterior end of the nucleus (fig. 69). (4) Martin accepts Silvestri's term 'nucleolus' for the body, but claims that in Ageniaspis it is gradually built up by the aggregation of granules which appear in the cytoplasm of the posterior region of the egg (fig. 70). (5) Since , I have been unable to confirm with my material either of the methods of origin suggested by Silvestri and Martin and since this germ-line-determinant appears suddenly at about Stage D (fig. 49), I wish to propose another theory as to its genesis. In Part II of my series of "Studies on germ cells" (Hegner '14 a) I have expressed the following conclusion, after collecting and discussing all the literature on the origin and history of the germ-line-determinants in animals.


526 ROBERT W. HEGNER

The most plausible conclusions from a consideration of these observations and experiments are that every one of the eggs in which Keimbahn-determinants have been described, consists essentially of a fundamental ground substance which determines the orientation; that the time of appearance of Keimbahn-determinants depends upon the precociousness of the egg; that the Keimbahn-determinants are the visible evidences of differentiation in the cytoplasm; and that these differentiated portions of the cytoplasm are definitely localized by cytoplasmic movements, especially at about the time of maturation.

This conclusion still seems to me the only tenable one at the present time and applies, I believe, to the germ-line-determinants in Copidosoma, as well as to those in other animals,

2. Apanteles glomeratus.^

Another Hymenopteron that resembles Copidosoma in some respects is Apanteles, a parasite of the larva of the cabbage butterfly. An abundance of material was obtained in the month of August, 1914. The pupae and recently emerged adults were liberated from the cocoons, and their abdomens were fixed either in Bouin's picro-formol solution or Carnoy's mixture. As in Copidosoma, the ovaries contain oocytes in all stages of growth and hence their history could be traced without much difficulty.

The history of the oocyte nucleus. Oocytes at a very early stage (fig. 71) acquire an epithelium (e) and are accompanied by a group of nurse cells (n) . The chromatin is large in amount and massed into an irregular homogeneous body. As growth proceeds (fig. 72) this chromatin-mass expands, revealing the spireme of which it consists. Soon the entire nucleus is filled with a network of chromatin threads (figs. 73-76), a condition that persists for a considerable part of the growth period. When the oocyte has reached its definitive size (fig. 77), the chromatin threads contract into chromosomes which apparently unite in pairs, as in Copidosoma (fig. 60), and become arranged side by side upon an asterless spindle (fig. 78) . This stage is followed by the condensation of the chromosomes, as shown in figure 79. No later stages in the nuclear history were present in my material,

3 I am indebted to Dr. H. L. Viereck for the identification of this parasite.


STUDIES ON GERM CELLS 527

but it is safe to assume that a further condensation occurs resuhing in an oval, homogeneous mass as in Copidosoma (fig. 65).

The secondary nuclei When a stage about Hke that shown in figure 75 is reached, there appears within the cytoplasm of the anterior one-half of the oocyte a great number of spherical bodies which are arranged as in figure 75, and which resemble small nuclei. Figure 76 is an enlarged drawing of the anterior end of the section shown in figure 75. The secondary nuclei vary considerably in size. The substance within them stains like chromatin and is in the form of one or several small masses from w^hich a few strands of chromatin granules radiate toward the membrane. These secondary nucleoli are present for only a brief period, having all disappeared by the time the chromosomes are formed (fig. 77). Their origin and function are problematical, but it seems hardly possible that they can arise from the germinal vesicle by budding, and hence we are forced to the same conclusions already stated in the case of Camponotus (p. 371).

The germ-line-determinants. The fully grown oocytes of Apanteles contain the most conspicuous germ-line-determinants yet described (fig. A). Although its history during embryonic development is not known, the probability that it plays an important role in the formation of the primordial germ cells is so great that it seems safe to include it among the bodies to which the term keimbahn or germ-line-determinant has been applied.

The first indication of this body occurs in a half-grown oocyte (fig. 73). Here a triangular area at the extreme posterior end may be distinguished from the rest of the egg by a slightly greater staining capacity (somewhat exaggerated in fig. 73). This affinity for basic stains increases as the oocytes grow older, and a network appears (fig. 74) which very much resembles the 'netzapparat' described by many writers both in germ cells and somatic cells (Duesberg '11). In succeeding stages this network condenses into a solid mass (fig. 75), but cavities soon appear again (figs. 77, 80), and the threads become thinner (fig. 81). Finally a condition is reached (fig. 82) in which the threads break

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 3


528


ROBERT ^y. HEGNER





•-.^



m," ►'



opp^


11?^'*".


V



-v^i


r ^ -i;


v-f


m.


Text fig. A Microphotographs of longitudinal sections through the abdomen of Apanteles showing various stages in the growth of the oocytes. The germline determinants appear as distinct triangidar black bodies near the posterior ends of certain of the larger oocytes.


up into a large number of irregular masses, suspended in a homogeneous substance. Near the germ-line-determinant in later stages (fig. 75) are a number of large widely scattered granules which are probably separated off from the main body. The resemblance between this body and the pole-plasm in the egg of Miastor (Kahle '08; Hegner '12, '14 a, '14 c) is quite striking. The pole-plasm in Miastor appears just before the oocyte imdergoes maturation, and apparently does not arise directly from the germinal vesicle, nurse cells, or follicular epithelium, but is a visibly differentiated portion of the cytoplasm that has become localized at the posterior end. What causes this differentiation is not known, but a discussion of the subject will be found in my previous contributions (Hegner '14 a, '14 c). In Miastor the j^ole-plasm never proceeds beyond the granular stage, but in A):)ontFles a rather definite series of conditions


STUDIES ON GERM CELLS 529

ensues during which the granular stage (fig. 73) is succeeded by a heavy network (fig. 74) ; this is followed by condensation into a solid mass (fig. 75), the formation of a heavy network again (fig. SO), the thinning out of this network (fig. 81), and finally the breaking up of the threads into many large irregular granules (fig. 82).

3. Hymenopterous gall-flies

There is still much to be learned regarding the life-cycles of the gall-flies, but what we do know is enough to prove that these insects offer a very profitable field for research. Examinations of the growing oocytes of certain Hymenopterous gall-flies have revealed many interesting structures that have a bearing upon the conditions described in the preceding part of this contribution and, although much more work needs to be done, the data already obtained are included here to indicate the widespread occurrence of phenomena described above. The oogenesis in these insects is more difficult to study than in the parasitic Hymenoptera because fewer stages of growth are represented b}^ the oocytes in a smgle individual.

The maturation spindle in the oak-knot gall-fly , A ndricus punctatus. The oak-knot gall-fly lays a pear-shaped egg (fig. 83), from the anterior end of which extends a long, slender process with an expanded terminal portion. This process resembles those described by Korschelt ('87) in Ranatra linearis. The two long slender processes extending from the anterior end of the eggs of Panatra, arise from a single bud-like protuberance at one side of the anterior end of the oocyte, and their place of origin alternates from one side to the other in the row of oocytes which lie in the lower part of the ovariole. When the eggs are laid, the processes are left extending freely out into the water from the decaying wood in which the rest of the egg is imbedded by the female.

The egg of the oak-knot gall-fly shown in figure 83, was taken from an adult which was just about to emerge from the gall. At one side near the anterior end could be seen a spindle-shaped body — the nucleus of the egg. Several stages in the develop


530 ROBERT AV. HEGNER

ment of this body were found and they seem to indicate a condition similar to that described in Copidosoma and Apanteles. The earUest stage discovered (fig. 84) represents an asterless spindle bearing a number of pairs of chromosomes attached near their ends and drawn out so as to form a more or less parallel series. These pairs then condense, as shown in figures 85 and 86, and finally produce the pear-shaped body mentioned above (fig. 83). Apparently the chromosomes become completely fused in forming this body, since a high magnification (fig. 87) reveals nothing more than a vacuolated mass of chromatin. The nucleus in Copidosoma never seems to undergo vacuolization, nor does the similar body described in Ageniaspis by Martin C14).

No body was found near the posterior end of the oocytes of the oak-knot gall-fly such as occur in those of Copidosoma, Apanteles, and the blackberry-knot gall-fly next to be described.

The maturation spindle ayid germ-line-deter7mnants in the blackberry-knot gall-fly, Diastrophus nebulosus. The eggs of the blackberry-knot gall-fly (fig. 88) resemble in general shape and size those of the oak-knot gall-fly (fig. 83) and the nucleus is in a similar position. This nucleus forms a rather compact body, but not a homogeneous mass. The stage represented in figures 89, 90 and 91 may be one of a series ending in the production of such a mass, but no other stages were found. Figures 89 and 90 were drawn from longitudinal sections and show that the position of the oval nucleus may vary; figure 91 is from a transverse section.

At the posterior end of the egg (fig. 88) is a more or less spherical body to which we are justified, I believe, in applying the name, germ-line-determinant. This body stains black with hematoxylin and is filled with vacuoles (fig. 92). Weismann ('82) described a body near the posterior end of the eggs of Rhodites rosae which he called the 'Furchungskern,' but it is evident from his account and figures that this body is similar to the one I have just described and is not a cleavage nucleus. According to Weismann this body spreads out during cleavage and occupies


STUDIES ON GERM CELLS 531

a large part of the posterior region; at this stage the term 'hinterer Polkern' is applied to it. Its later history was not followed.

It is worth mentioning that the follicle cells of the oocytes divide by mitosis (fig. 93) and not by amitosis as has been described in some insects.

Secondary nuclei in the oocytes of the mealy rose gall-fly Rhodites ignota. The eggs of this gall-fly (fig. 94) possess a very long anterior process, as in the two species already described, and the nucleus is similarly placed, but no body occurs at the posterior end. Of particular interest here is the presence of a large number of secondary nuclei at certain stages in the growth of the oocyte. These secondary nuclei were first observed near the periphery, as indicated in (fig. 95), which is part of a transverse section. They are very small and appear to consist of a single body that stains like chromatin, and are surrounded by a membrane. The occurrence of deeply staining granules without these membranes, and the various sizes of the secondary nuclei formed, lead to the conclusion that chromatin granules from the oocyte nucleus, from the nurse cells, or from the follicle cells, migrate into the cytoplasm and become the center of origin of the secondary nuclei. In later stages these nuclei are all larger and form a layer a slight distance from the periphery of the oocyte (fig. 96). They vary greatly in size as shown in figure 97, but exhibit all the characteristics of true nuclei. No secondary nuclei could be found in older oocytes, but what becomes of them was not determined.

Summary of Part III.^ A. Copidosoma gelechiae. 1. The chromatin in the oocyte nucleus forms chromosomes at an early stage in the growth period (fig. 51). These chromosomes unite near their ends in pairs (figs. 52 and 58) and then become arranged in a parallel series upon an asterless spindle (figs. 53, 59, 60) . Condensation then occurs and an apparently homogeneous oval-shaped mass of chromatin is formed (figs. 54 and 62-65). The number of pairs of chromosomes is eleven (fig. 61) or twelve. The nuclear history is essentially as described in my preliminary

•* Summaries of Parts I and II will be found on pages 505 and 520.


532 ROBERT W. HEGNER

report (Hegner '14 b) and similar to that described by ]\Iartin ('14) in Agenaspis.

2. The germ-line-determinant is not the chromatin from an oocyte nucleus, as stated in my preliminary paper, but it appears to be a differentiated part of the protoplasm which arises at an early stage (fig. 49) near the posterior end of the oocyte.

B. Apanteles. 1. The oocyte nucleus has a history similar to that described for C'opidosoma. Chromosomes are formed at an early period, fuse in pairs, become arranged upon an asterless spindle (figs. 77-78), and undergo condensation (fig. 79). Whether or not they finally form a homogeneous mass could not be determined because of the lack of late stages.

2. Secondary nuclei make their appearance in the almost fully grown oocytes. They are distributed throughout the anterior half of the oocyte (figs. 75-76), but are entirely absent in later stages (fig. 77). Their origin and fate were not determined.

3. The deeply staining substance at the posterior end of the older oocytes is probably a germ-line-determinant. It first appears in a partially grown oocyte as a dark gi"anular mass, which probably represents a differentiated part of the protoplasm (fig. 73). Later it passes through the stages described on pages 527-529 and illustrated in figures 74, 75 and 80 to 82.

C. Gall-flies. 1. The history of the oocyte nucleus of the oak-knot gall-fly resembles very closely that of Copidosoma and Apanteles (figs. 84-87).

2. The oocytes of the blackberry-knot gall-fly contain a chromatin body (figs. 88-91) which probably results from the condensation of chromosomes as in the other forms described above. A conspicuous germ-line-determinant is also present near the posterior end (figs. 88, 92); the follicle cells divide by mitosis (fig. 93).

3. The half-grown oocytes of the mealy rose gall-fly are provided with hundreds of secondary nuclei (fig. 97) which are situated in a single layer equidistant from the periphery at all points (fig. 96). In younger oocytes (fig. 95) these secondary


STUDIES ON GERM CELLS 533

nuclei appear to arise near the periphery from granules which stain like chromatin. These granules may be extruded by the oocyte nucleus, the follicle cells or the nurse cells.

February H), U)lj

LITERATURE CITED

Amma, K. 1911 Ueber die Differenzierung der Keimbahnzellon bei den C'ope poden. Arch. f. Zellforsch., 6. Ayers, H. 1884 On the development of Oecanthus niveus and its parasite,

Teleas. Mem. Boston Socy. Nat. Hist., vol. 3. Balbiani, E. G. 1870 Mc'moire sur la generation des Aphides. Ann. Sci.

Nat.i V S'erie, T. 14. Blochimann, F. 1884 Ueber die INIetamorphose der Kerne in den Ovarial eiern, usw. Verhandl. nat.-med. Vereins zu Heidelberg.

1886 Ueber die Reifung der Eier bei Ameisen und Wespen. Festschr. nat.-med. Verein zu Heidelberg.

1887 Ueber die Richtungskorper bei den Eiern der Insekten. Morph^ Jahrb., Bd. 12.

1892 Ueber das Vorkommen von bakterienahnlichen Gebilden in

den Geweben und Eiern verschiedener Insekten. Centralbl. Bakte riol., Bd. 11. BovERi, T. 1892 Die Entstehung des Gegensatzes zwisehen den Geschlechts zellen und den somatischen Zellen bei Ascaris megalocephala. Stzber.

Gesellsch. Morph. u. Physiol. Miinchen, Bd. 8. Brunelli, G. 1904 Ricerche sull' ovario degli insetti sociali. Rend. Arcad.

Lincei., T. 12. BucHNER, P. 1910 a Keimbahn und Ovogenese von Sagitta. Anat. Anz.,

Bd. 35.

1910 b Die Schicksale des Keimplasmas der Sagitten in Reifung, Befruchtung, Keimbahn, Oogenese, und Spermatogenese. Festschr. R. Hertwig, Bd. 1.

1912 Studien an intracellularen Symbionten. Arch. f. Protistenk.,

Bd. 26. Claus, L. 1864 Beobachtungen iiber die Bildung des Insekteneies. Zeit.

wiss. Zool., Bd. 14. Debaisieux, p. 1909 Les debuts de I'ovigenese dans le Dytiscus marginalis.

La Cellule, T. 25. DuESBERG, J. 1908 Sur I'existence de mitochondries dans I'oeuf et I'embrj'on

d'Apis mellifica. Anat. Anz., Bd. 32.

1911 Plastosomen, apparato reticolare interno und Chromidialapparat. Ergebnisse.

Elpatiewsky, W. 1909 Die Urgeschlechtszellenbildung bei Sagitta. Anat. Anz., Bd. 35.

1910 Die Entwicklungsgeschichte der Genitalproducte bei Sagitta. Biol. Zeitsch., Bd. 1.


534 ROBERT W. HEGNER

VON F^RLANGER, R. 1896 Ueber den sogennannten Nebenkern in den mann lichen Geschlechtszellen der Insecten. Zool. Anz., Bd. 19.

1897 Spermatogenetische Fragen. Zool. Centralbl., Bd. 4. Forbes, S. A. 1891 Bacteria normal to digestive organs of Hemiptera. Bull.

111. St. Lab. Nat. Hist., vol. 4. GiARDiNA, A. 1901 Origine dell' oocite e dell cellule nutrici nel Dytiscus.

Internat. Monatschr. Anat. u. Physiol., Bd. 18. GovAERTS, P. 1913 Recherchessur la structure de I'ovaire des insects. Archiv

de Biol., T. 28. Gross, J. 1903 Untersuchungen iiber die Histologic des Insektenovariums.

Zool. Jahrb. Bd. 18. GtJNTHERT, T. 1910 Die Eibildung der Dytisciden. Zool. Jahrb., Bd. 30. Haecker, V. 1897 Die Keimbahn von Cyclops. Arch. mikr. Anat., Bd. 45. Hasper, M. 1911 Zur Entwickelung der Geschlechtsorgane von Chironomus.

Zool. Jahrb., Bd. 31. Hegner, R. W. 1914 a Studies on germ cells. I and II. Jour. Morph., vol. 25.

1914 b Studies on germ cells. III. Anat. Anz.. Bd. 46.

1914 c The germ-cell cycle in animals. New York. Henneguy, L. F. 1896 Legons sur la cellule. Paris.

1904 Les insectes. Paris. Huxley, T. H. 1858 On the agamic reproduction and morphology of Aphids.

Trans. Linn. Socy. London, vol. 22. Kahle, W. 1908 Die Paedogenese der Cecidomyiden. Zoologica, Bd. 21. Kern, P. 1912 Ueber die Fortpflanzung und Eibildung bei einigen Caraben.

Zool. Anz., Bd. 40. KoRSCHELT, E. 1886 Ueber die Entstehung und Bedeutung der verschied enen Elemente des Insektenovariums. Zeit. wiss. Zool., Bd. 43.

1889 Beitrage zur Morphologic und Physiologic des Zellkernes. Zool.

Jahrb., Bd. 4. Lameere, a. 1890 La raison d'etre des metamorphoses chez les Insectes. Ann.

Soc. Ent. Belg., T. 43. Lee, a. B. 1895 La regression du fuseau caryocinetique. La Cellule. T. 11. Leydig, F. 1850 Einige Bemerkungen iiber die Entwicklung der Blattlause.

Zeit. wiss. Zool., Bd. 4. Loyez, M. 1908 Les 'noyaux de Blochmann' et la formation du vitellus chez

les Hymenopteres. C. R. Assoc. Anat. 10 Reunion, Marseille. Lubbock J. 1860 On the ova and pseudova of insects. Phil. Trans., vol. 149. Marshall, W. S. 1907 Contributions towards the embryology and anatomy of

Polistes pallipes. Zeit. wiss. Zool., Bd. 86. Martin, F. 1914 Zur Entwicklungsgeschichte des polyembryonalen Chal cidiers Ageniaspis (Encyrtus) fuscicollis Dalm. Zeit. wiss. Zool.,

Bd. 110. Mayer, H. 1849 Ueber die Entwicklung des Fettkorpers, etc. bei den Lepi dopteren. Zeit. wiss. Zool., Bd. 1. Maziarski. S. 1913 Sur la persistance de residus fusoriaux pendant les nom breuses generations cellularies au cours de I'ovogenesese de Vespa

vulgaris. Arch. f. Zellforsch., Bd. 10. Mercier, L. 1907 Recherches sur les bacteroides des Blattides. Arch. Pro tistenk., Bd. 9.


STUDIES ON GERM CELLS 535

Meves, ¥. 1895 Ueber eigenthlimliche mitotische Processe in jungen Ovocy ten von Salamandra maculosa. Anat. Anz., Bd. 10. Montgomery, T. H. 1911 Differentiation of the human cells of Sertoli. Biol.

Bull., vol. 21. Paulcke, W. 1900 Ueber die Differenzierung der Zellenelemente im Ovarium

der Bienenkonigin. Zool. Jahrb., Abt. Anat., Bd. 14. Petrunkewitsch, a. 1901 Die Richtungskorper und ihr Schicksal im be fruchteten und unbefruchteten Bienenei. Zool. Jahrb., Abth. Anat.,

Bd. 14. PiERANTONi, U. 1910 Ulterior! osservazioni suUa simbiosi ereditaria degli

Omotteri. Zool. Anz., Bd. 36. Platner, G. 1886 Die Karyokinese bei den Lepidopteren als Grundlage fiir

eine Theorie derZelltheilung. Internat.Monatschr. Anat. Hist., Bd. 3. Prenant, a. 1888 Observations cytologiques sur les elements seminaux des

Gasteropodes pulmones. La Cellule, T. 4. SiLVESTRi, F. 1906-08 Contribuzioni alia conoscenza biologica degli Imenot teri parasitici, i-4, Bollet. Scuola sup. Agric. Portici, T. 1 and 3.

1914 Prime fasi di svillupo del Copidosoma buyssoni (Mayr), Imenot tero Calcidide. Anat. Anz., Bd. 47. Stevens, N. M. 1910 Further studies on reproduction in Sagitta. Jour.

Morph., vol. 21. Stuhlmann F. 1886 Die Reifung des Arthropodeneies nach Beobachtungen

an Insekten Myriapoden und Peripatus. Bericht. Naturf. Gesellsch.

Freiburg i.B.,Bd. 1. SuLC, K. 1906 Kermincola, etc., neue Mikroendosymbionyiken der Cocciden.

Stzber. bohm. Gesellsch. Wiss. Prag.

1910 'Pseudovitellus' und ahnliche Gewebe der Homopteren sind

Wohnstatten symbiotischer Saccharomyceten. Stzb. bohm. Ges.

Wiss. Prag. Tanquary, M. C. 1913 Biological and embryological studies on Formicidae.

Bull. 111. St. Lab. Nat. Hist., vol. 9. Wagner, J. 1896 Einige Beobachtung iiber die Spermatogenese bei den Spin nen. Zool. Anz., Bd. 19. Weismann, a. 1882 Beitriige zur Kenntnis der ersten Entwicklungsvorgange

im Insektenei. Festschr. J. J. Henle. Wheeler, W. M. 1889 The embryology of Blatta germanica and Doryphora

decemlineata. Jour. Morph., vol. 3. WiELOwiEJSKi, H. V. 1886 Ueber den Bau der Insektenovarien. Zool. Anz.,

Bd. 9. Will, L. 1884 Zur Bildung des Eies und des Blastoderms bei den viviparen

Aphiden. Arb. zool. zoot., Inst. Wiirzburg., Bd. 6. VON Winiwarter, H. 1912 Etudes sur la spermatogenese humaine. Arch, de

Biol., T. 27. Witlaczil, E. 1884 Entwicklungsgeschichte der Aphiden. Zeit. wiss. Zool.,

Bd. 40. Zimmer.mann, K. W. 1891 Ueber den Kerntheilungsmodus bei der Spermatogenese von Helix pomatia. Verhandl. Anat. Gesellsch., 5te Versamml.


PLATE 1

EXPLANATION OF FIGURES

Apis mellifica

1 Outline of an ovariole showing the terminal filament, t. a zone containing rosettes of cells, r, a zone of differentiation, d, a region containing a linear series of oocytes, o, and nurse cells, n, accompanying an oocyte. X 110.

2 Part of the rosette region of an ovariole; r, a rosette, the cells (if which are held together bj' deeply staining strands; e, an epithelial cell nucleus. X 650.

3 A single rosette in longitudinal section. X 1900. A A single rosette in transverse section. X 1900.

5 Synchronous division of the cells in a rosette. X 1900.

6 Part of the zone of differentiation of an ovariole; o, oocyte; e, epithelial cell nuclei; n, nurse cell. X 430.

7 Two neighboring oocytes from the zone of differentiation showing the double rings connecting them with each other and with surrounding nurse cells. X 1900.


536


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 1



(Q)


(S>


(§>


(^



n



R. W. HEGNER, del.


537


PLATE 2

EXPLANATION OF FIGURES

Apis mellifica

8 A group of nurse cells surrounding an oocyte. X 1900.

9 An older oocyte with nurse cells and epithelial cells. X 1250.

10 An outline showing the arrangement of an oocyte and its accompanying nurse cells. X 1250.

11 Part of a rather old oocyte, o, still connected with nurse cells, 7i, by means of rings, e, epithelian cell. X 1250.

12 An outline of an older oocyte showing the rings between the nurse cells and oocyte and between neighboring nurse cells. X 430.


538


STUDIES ON GERM CELLS

ROBERT W. HEONER


PLATE 2



8



11



10



R. W. HEGNER, del.


539


PLATE 3

EXPLANATION OF FIGURES

Camponotus herculeanus var. pennsylvanica

13 Outline of an ovariole showing the terminal filanlent, /, terminal chamber, I.e., first zone of growth, g, and later growth zone containing oocytes, o, and nurse cells, n and nc. X 170.

14 Outlines of oocytes in Stages A to G. X 110.

15 Outlines of oocytes in Stages H to L. X 110.

16 Outline of part of the terminal filament, I, and terminal chamber, tc. The numbers 17, 18 and 19 refer to cells shown enlarged in figures 17, 18 and 19. X 620.

17 A single cell from the terminal filament. X 3300.

18 An oocyte from the terminal chamber. X 3300.

19 A nurse cell nucleus from the terminal chamber. X 3300.


640


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 3



nc


O


OqO

ooa



13



R. W. HEGNER, del.


541


PLATE 4

EXPLANATION OF FIGURES

Campanotus herculeanus var. pennsylvanica

20 Outline of the first zone of differentiation, showing the membrane, m, separating it from the terminal chamber, and the oocytes, o, nurse cells, n, and epithelial cells, e. The numbers 21, 22 and 23 refer to cells shown enlarged in figures 21, 22 and 23. X 620.

21 An oocyte from the first zone of growth. X 3300.

22 A nurse cell from the first zone of growth. X 3300.

23 An epithelial cell nucleus from the first zone of growth. X 3300.

24 The posterior portion of the first zone of growth and the anterior portion of the rest of the ovariole containing bacteria-like rods, e, epithelial cell nucleus; n, nurse cell; o, oocyte. X 1250.

25 Part of an ovariole showing two oocytes, Ci and C2, in Stage C n, nurse cell. X 1250.

26 An oocyte in Stage D. The bacteria-like rods have invaded the cytoplasm of the oocyte. X 1250.


542


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 4



R. W. HEGNER, del.


543


JOURNAL OF MORPHOLOGY, VOL. 2tj, NO. 3


PLATE 5

EXPLANATION OF FIGURES

• Camponotus herculeanus var. pennsylvanica

27 An oocyte in Stage E showing three secondary nuclei, s, near the oocyte nucleus, o. e, epithelial cell nucleus; n, nurse cell. X 1250.

28 An oocyte in Stage F. Lettering as in figure 27. X 620.

29 An oocyte in Stage G. Lettering as in figure 27. X 620.

30 Part of an oocyte and two nurse cells, n. Cytoplasm, c, elaborated by the nurse cells is present near the nurse chamber, o, oocyte nucleus; s, secondary nuclei. X 430.

31 Transverse section through the anterior end of an oocyte. X 620.

32 Part of the nucleus of a nurse cell showing vacuoles and deeply staining granules in the thick nuclear membrane. X 3300.


544


STUDIES ON GERM CELLS

nOBERT W. HEGNER


PLATE 5



R. W. HEGNER, del.


545


PLATE 6

EXPLAXATION OF FIGURES

Camponotus herculeanus var. pennsylvanica

33 Part of an oocyte in Stage H showing its connection, a, with the nurse chamber; o, oocyte nucleus; s, secondary nuclei. X 430.

34 An oocyte nucleus surrounded by secondary nuclei from an oocyte in Stage H. X 1250.

35 Part of an oocyte in Stage I. e, follicular epithelium; k, 'Keimhautblastem'; y, yolk globules. X 430.

36 Part of an oocyte in Stage J. X 43C.

37 The anterior part of an oocyte showing the breaking up of the group of secondary nuclei, s. a, connection with nurse chamber, c, cytoplasm; e, follicular epithelium; y, yolk globules. X 430.

38 A single secondary nucleus and three yolk globules in Stage K. X 1900.

39 Part of the edge of an oocyte in Stage L showing the follicular epithelium and the distribution of secondary nuclei and yolk globules. X 430.

40 Two secondary nuclei and two yolk globules, enlarged, from an oocyte in Stage L. X 1900.


546


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 6






3T


■^^'%:)^i'\(,^ '■:■




R. W. HEGNEH, del.



38


547


PLATE 7


EXPLANATION OF FIGURES


41 Longitudinal section through an egg of C. herculeanus var. ferruginous one hour old. X 52 (from Tanquary).

42 Ditto, twenty hours old. X 52 (from Tanquary).

43 Ditto, slightly older. X 52 (from Tanquary).

44 Ditto, two days old. X 52 (from Tanquary).

45 Outlines of ooc3'te nuclei (dotted in) with their accompanying secondary tiuclei of C. herculeanus var. pennyslvanica. X 1250.


548


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 7



44


l;. W. HEGNER, del.



„ ^ ^or,o ^o,

4 ,.^^^^



43



a



45


549


PLATE S

EXPLANATION OF FKiURES

Copidosoma gelechiae

46 Outline of a j'oung oocyte in Stage A surrounded by a follicular epithelium and accompanied by a group of nurse cells. X 1250.

47 Outline of an oocyte in Stage B . The follicular epithelium is shown, but the nurse cells have been omitted. X 1250.

48 Outline of an oocyte in Stage C. X 1250.

49 Outline of an oocyte in Stage D. First appearance of the germ-linedeterminant near the posterior end. X 1250.

50 Outline of an oocyte in Stage E. X 1250.

51 Outline of an oocyte in Stage F. Single chromosomes are present. X 1250.

52 Outline of an oocyte in Stage G. The chromosomes have united near their ends to form pairs. X 1250.

53 Outline of an oocyte in Stage H. The pairs of chromosomes are arranged in a parallel series. X 1250.

54 Outline of an oocyte in Stage I. X 1250.


550


STUDIES ON GERM CELLS

ROfcERT W HEGNER


PLATE S



46




R. W. HEGNER, del.


551


PLATE 9

EXPl^ANATION OF FIGURES

Copidosoma gelechiae

55 An oocyte in Stage A (see fig. 46). X 3300.

56 An oocyte in Stage B (see fig. 47) . X 3300,

57 An oocyte in Stage C (see fig. 48) . X 3300.

58 The anterior portion of an oocyte in Stage G (see fig. 52). X 3300. 59. The nucleus of an oocyte in Stage H (see fig. 53). X 3300.

60 The nucleus of a slightly older oocyte. X 3300.

61 A transverse section through a nucleus in a similar condition. X 3300.

62 to 65 Successive stages in the condensation of a spindle like that shown in figure 60. X 800.


552


STUDIES ON GERM CELLS

HOBEHT W. HEGNER


PLATE 9




60


\


63



65


PLATE 10


EXPLANATION OF FIGURES


66 Ageniaspis; the anterior portion of an oocyte showing the arrangement of the chromosomes on the spindle (after Martin).

67 Ageniaspis; a later stage showing the mass of chromatin resulting from the condensation of the chromosomes (after Martin) .

68 Ageniaspis; the first maturation division of the egg (after Martin).

69 Copidosoma; a young oocyte showing a group of granules near the posterior end of the nucleus (after Silvestri).

70 Ageniaspis; a young oocyte containing a cloud of granules in the posterior portion and a larger body, the 'nucleolus' (after Martin).

71-74 Apanteles.

71 A young oocyte surrounded by epithelial cells, e, and accompanied by nurse cells, 7i. X 1900.

72 An older oocyte. X 1900.

73 An older oocyte showing the first appearance of the germ-line-determinant. X 1900.

74 A still older oocyte. X 1900.


554


STUDIES ON GERM CELLS

ROBERT W. HEGNER


PLATE 10