Paper - The development of the monotremata 4

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Flynn TT. and Hill JP. The development of the monotremata Part IV. Growth of the ovarian ovum, maturation, fertilisation and early cleavage. (1939) Trans. Zool. Soc. Lond. 24: 445- 623.

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This historic 1939 paper by Flynn and Hill describes the early development of echidna.

The Hill Collection contains much histology of echidna and platypus embryonic development.
Modern Notes: Echidna Development Platypus Development

Australian Animal: echidna | kangaroo | koala | platypus | possum | Category:Echidna | Category:Kangaroo | Category:Koala | Category:Platypus | Category:Possum | Category:Marsupial | Category:Monotreme | Development Timetable | K12
Historic Australian Animal  
Historic Embryology: 1834 Early Kangaroo | 1880 Platypus Cochlea | 1887 Monotremata and Marsupialia | 1910 Eastern Quoll | 1915 The Monotreme Skull | 1939 Early Echidna

The Hill Collection contains much histology of echidna and platypus embryonic development.

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Other Marsupials  
Monito del Monte Development | Opossum Development

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Pages where the terms "Historic" (textbooks, papers, people, recommendations) 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, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)

The Development of the Monotremata Part IV. Growth of the Ovarian Ovum, Maturation, Fertilisation and Early Cleavage

James Peter Hill (1873 - 1954)

Prof. T. Thomson Flynn And Prof. J. P. Hill On

FLYNN, ,l).Sc., Queen’s University, Belfastv, and Professor J. P. HILL, F.R.S., F.Z.S., Royal (;‘oll,ege of Surgeons, late of University College, London.


With this part is commenced the systematic description of the developmental material of the Monotremata in our possession. Previous investigators have emphasised the great difficulty of procuring such material, and especially of Platypus. Caldwell (1887) alone seems to have obtained a representative series of early Monotreme stages, mostly of Echidna, gathered in Queensland. However, his published researches into this material did not take him beyond the description of the four-celled stage of the developing egg. The investigations of Semon (1894) and Wilson and Hill (1907) added considerably to our knowledge of Monotreme development, but, even so, we still lack a connected account of the early ontogeny of the Monotremes. It will be our endeavour in the present paper, so far as our material allows, to make good this deficiency in respect of oogenesis, maturation, fertilisation, and early cleavage. In a succeeding part, we hope to give an account of the later cleavage stages and of the formation of the primary germ—layers.


The material in our possession represents the results of collecting carried on from time to time since 1896, and has come from a variety of sources. One of us (H.) has in his possession a-series of stages of Ornithorhynchus and Echidna from New South Wales and Queensland, some of which were used in the preparation of the monograph of Wilson and Hill on “ The Development of Ormlthorhynckus ” (1907). The relevant stages in this collection have been re-examined and incorporated in the present work. More recently, the late Dr. T. L. Bancroft of Queensland was good enough to help in obtaining further stages of Echidna and Platypus, among which are included several of considerable value for the study of late cleavage and of germ-layer formation.

The other author (F.) possesses a fairly complete series of stages of Echidna, the collection of which was commenced by him in Tasmania in 1927 . This material has more recently been added to through the kind help of Dr. V. V. Hickman, C.M.Z.S., Lecturer in Biology in the University of Tasmania. Inclusive of intrauterine and pouch eggs, and pouch young, there are in this collection nearly one hundred and fifty stages. We have therefore altogether an amount of material probably excelling in, number and variety of stages the collections at the disposal of previous investigators into the embryology of this group.

We desire to express our grateful thanks to the Government Grant Committee of the Royal Society, and to the Trustees of the Ralston Bequest (Tasmania) for grants, without the aid of which it would have been impossible to get together this rich collection of Monotreme material. We are also greatly indebted to the Council of the Zoological Society for grants towards the expenses of the investigation.

To the late Dr. T. L. Bancroft and to Dr. V. V. Hickman, we are deeply indebted for the supply of important material. The stages collected by these gentlemen are distinguished in the following pages by the letters TLB and VVH respectively.

To our colleague, Mr. K. C. Richardson, M.Sc., we are indebted for help and advice in regard to special methods of technique. We desire also to express our grateful thanks to Miss F. M. Collinson for the great care, patience, and skill she has displayed in the preparation of the illustrations, to Mr. F. J. Pittock, F.R.P.S., for his untiring enthusiasm and outstanding skill in the preparation of the photographs and photo-micrographs on which the illustrations are based, and to Mr. H. Barker for his excellent work as technician.

We should point out that, in Tasmania, Echidna (as well as Platypus) is a protected animal, and we are grateful to the Tasmanian Animals’ and Birds’ Protection Board, and especially to its Chairman, Colonel J. E. C. Lord, for affording the necessary permission to collect animals for this investigation.



Our records of the methods of fixation of the earlier collected material are not complete, but it would appear that the medium used was usually piero-nitric acid, with or without the addition of osmic acid. In general, the fixation of these eggs is excellent, even in the case of those which had been preserved in alcohol for over a quarter of a century. The Tasmanian Echidna material was fixed in a variety of ways. Bouin’s picro-formol-acetic was used at first as a routine fixative. It gave good preservation of the blastodisc cells, but was found to have a tendency to disintegrate the yolk. Its variation, Bouin-Hollande, is not to be recommended for early stages, although it fixes cytological detail excellently. Bles’ fluid was tried in the case of some eggs, but was soon abandoned as being most unsatisfactory. By far the most useful fixative for this material in our experience is Smith’s bichromate—formol-acetic mixture *. It gives a fine fixation of the yolk and of the nuclei of the blastodisc cells. It tends to exaggerate cleavage furrows (which is not a disadvantage) and, in the early stages, causes the shellmembrane to become distended so that a wide space is left between it and the zona-albumen layer enclosing the egg. The shell is thus easily removable, and

it is then possible to get whole views of the blastodisc such as are shown in fig. 79 (P1. XV.), fig. 87 (Pl. XVIL), fig. 99 (Pl. XVIIL), and others.

Preparation of Sections

  • We are greatly indebted to Dr. Sidney Manton of Cambridge for suggesting the use of this fixative to us.

Previous investigators into the development of this group have agreed that, owing to the presence of a large yolk-mass, good sections can be obtained only with considerable difficulty. Even by the use of the method of double embedding, We have found it almost impossible to prevent the yolk from tearing sections, or from falling bodily out of place or, in innumerable grains, spreading itself through the balsam of the slide and obscuring important details. After some experiment, We have devised a method by which We are now able to make ribbons of sections of Whole eggs without the loss of a section. As this method may be useful in the investigation of other yolky eggs we give it in some detail :—

1. Fix in Smith’s bichromate-formol-acetic fluid, according to directions (B. G. Smith, Journ. Morph. vol. Xxiii. p. 91).

2. Transfer from 5 per cent. formol to 70 per cent. alcohol and grade up to absolute alcohol, and absolute alcohol and ether, equal parts.

3. Infiltrate in celloidin-cedarwood oil, 6 parts to 1, beginning with l per cent., followed by 2 and 4 per cent., several days in each.

4. Harden and clear in cedarwood oil-chloroform 2 parts to 1, changing the fluid once.

5. Transfer to benzol, benzol-paraffin on bath and imbed in paraflin, using air-pump.

6. Sections are best made with a sliding microtome. As each section is cut, paint surface of block with gum-mastic-celloidin solution, rubbing off excess With finger (thick solution of celloidin in absolute alcohol and ether, thick solution of gum-mastic in ether—1—l/10th volume of absolute alcohol, equal parts; for use dilute with ether+a little absolute alcohol until quite fluid (quoted by Minchin, Nat. Sci. vol. iii. p. 122).

7. Coat slide with Mayer’s albumen and float out sections on water. If sections fail to flatten, use Ruyter’s method (Zeitschr. Wiss. Mikr. Bd. 48, p. 226).

8. When sections have dried on, flood slide with 1 per cent. celloidin solution, drain quickly, and place in toluol+10 per cent. chloroform, until the paraffinWax is dissolved.

9. Take down through the alcohols (adding 10 per cent. chloroform to the higher grades) to distilled Water and stain.

The cutting of the sections must be done by two Workers if success is to be assured. One of these paints the surface of the block with gum-mastic solution, the other does the cutting and manipulates the ribbon. It is obvious that, for convenience in painting, that type of microtome is most useful in Which the cutting surface of the paraffin block is arranged horizontally facing upwards. In our work we have used Minot’s Large Precision Microtome, manufactured by Bausch and Lomb, but no doubt other microtomes on the same principle would serve just as well.

Breeding Season

Information so far available on the breeding season of Echidna is confined almost entirely to the variety typical, found on the Australian mainland. Semon’s records (1894, p. 7) indicate that, in the Burnett River district of Queensland, this animal begins to breed towards the end of July. In no season did he find an intra-uterine egg earlier than July 23. He gives no information, however, as to when the season ends. Caldwell tells us very little. He merely states (1887, pp. 464-8) that he collected intra-uterine stages during July and August.

Little is known of the breeding habits of the Tasmanian Eckidna-—in fact, up to the time of publication of Flynn’s paper of 1930, only one record bearing on the subject was in print. This is an account by Morton (1888) of a pouch-egg of Echidna, which was broken in the act of collecting, the embryo and the remains of the shell being now preserved in the Tasmanian Museum. The date of collection is not given, but the paper was read in October of the year 1887.

Climatic conditions might well be expected to retard the incidence of the breeding season in Tasmania to a date much later in the year than is the case in Queensland, but it was found that this is not the case. Collecting was commenced in the month of September 1927, but this proved to be near the end of the season. The succeeding five seasons, during which collecting was carried on, showed that breeding commenced in Tasmania surprisingly early. Thus in 1933 the first fertilized egg of the season was found in the uterus of a female brought in on June 29. This is our only record of a June egg, the earliest previous record being of an egg taken on July 11, 1930.

It would appear, therefore, that the breeding season of Eckidna in Tasmania must commence towards the end of June. The latest date in the year of which we have any record of the discovery of an intra-uterine egg is September 4, 1929, while a pouch-young has been found as late as the middle of that month.

During August most of the females brought in contain intra-uterine eggs.

It is of some interest to note that there is no very great agreement between eggs collected on the same day as regards their degree of development. On August 15, 1930, for example, fourteen eggs were obtained, ranging from an egg with an unsegmented disc, just fertilised, to two eggs measuring 14-80><14-00 mm. and 15-00 X14-10 mm. respectively, the former containing a flat embryo with eleven pairs of somites and the latter, an abnormal embryo.

Ovulation in Echidna

Echidna normally produces one egg at a time. In nearly one hundred and forty records we have found only four exceptions to this rule. In a specimen captured on August 6, 1930, there were present two eggs, one in each uterus. 450 PROF. T. THOMSON FLYNN AND PROF. J. P. HILL ON

That in the left uterus, however, was obviously abnormal, being shrunken and considerably under normal size, its diameter being 280 mm. The ovum found in the right uterus was quite normal, although its shape was elliptical ; it measured 4-60 ><4-20 mm. On August 3, 1931, a female was brought in, in the right uterus contained two eggs ; one measured 4-50 mm. in diameter and was normal, the other 2-00 mm. and was obviously abnormal and degenerate. Since then we have received records from Dr. V. V. Hickman of two females collected on July 6 and 7, 1933, both of which had two eggs in the right uterus. In each case one of the eggs, the smaller, was abnormal. The measurements are as follows : (a) Collected 6. vii. 33, abnormal, 3-00 mm., normal 5-00 mm. (b) Collected 7. vii. 33, abnormal, 2-7 mm., normal, 4-50 mm.

Flynn has already referred to this matter (1930, p. 121) and has drawn attention to Owen’s record (1881) of the discovery of three eggs in the left uterus of Echidna and to that of Broom (1895) in which the latter reports two eggs as having been laid by an Echidna on two successive days. It is possible that one of these eggs was abnormal, as we have had experience in our own material of a perfectly normallooking egg, apparently ready for laying, which, when removed from the uterus, was found to possess not the slightest vestige of an embryo. In still another case, an egg, already provided with a shell, had collapsed in the uterus into a flattened distorted mass.

However, the possibility of more than one egg being developed at one time in Eckidna cannot be entirely ruled out, since Semon (1894, p. 8) records the discovery of a female Echidna with two 69 mm. pouch-young. This, however, is extremely rare and in our own material, apparently much more abundant than Semon’s, we have not met with a single instance.

Reference may be made here to another matter in connection with the phenomenon of ovulation in this animal. An erroneous impression has been created by the statement of Semon to the effect that, in Monotremes, the left ovary is alone functional (1894, p. 62). Flynn (1930, p. 122) has already shown that such a statement is quite wrong in the case of the Tasmanian Echidna, and from the statements of Hill and Gatenby (1926, p. 746) and of Garde (1930, p. 423) it would appear to be also incorrect in the case of the mainland variety. Our records of the uteri in which eggs have been found in the Tasmanian Echddna now number one hundred and thirty-four, and of these eggs 67 prove to have come from the right uterus and 67 from the left—in other Words, when a large series is considered, it is found that the eggs are derived equally from either ovary.

So far as Omithorhynchus is concerned, there seems to be no doubt that the left ovary is alone functional.

Proportion of Males to Females

Both Caldwell (1887, p. 5) and Semon (1894, p. 5) have remarked on the discrepancy which exists in Echidna in the numbers of males and females brought in by their collectors, the former being much in excess of the latter. Wilson and Hill (1907, p. 33) make the following statement with regard to Omitkorkynchus :— “ During the breeding season, however, the pregnant female appears to keep much more closely to the burrow, so that one may then commonly enough shoot five or six males to one female.” Burrell, however, states with regard to Platypus that so far as his observations go, the sexes seem to be numerically equal (1927, p. 151). Semon records the proportion of males to females in Echlalmz as two or three to one. In Tasmania, our records show that this proportion exists there also. As the outcome, the results of collecting are often disappointing and always expensive, since the trapper expects to be paid for all animals sent in irrespective of their sex. The difference in the numbers of the two sexes captured may be due in part to the more retiring habits of the female as suggested by VVilson and Hill. On the other hand, it must be remembered that in Tasmania, for this work, the Echidnas were found by means of hunting dogs, and, as the dogs are not likely to discriminate between the sexes, we must conclude that the males of Echidna largely outnumber the females.

The Intra-Uterine Egg of Eohidna

It is not easy to distinguish between the pregnant and non-pregnant uterus in the early stages of development in Ecktdua, as both oviducts undergo at first a corresponding enlargement. In this respect, Echialua resembles those Marsupials in which only one uterus is pregnant and in which only one young is produced at a birth. Semon (1894, p. 62) and Burrell (1927, p. 178) record that in Platypus the right oviduct also undergoes some enlargement when intra-uterine eggs are present in the left.

The living egg in the early stages is a spherical, pearly object found free in the uterine cavity. The contained yolk is white or greyish white (Flynn, 1930, pp. 123-4). Semon records the yolk as being yellow in the mainland Echidua (1894, p. 68) and pictures it so in an egg taken from the uterus and from which the shell had been removed (Taf. 1, fig. 1). So far as Platypus is concerned, all records describe the yolk as being yellow (cf. Burrell, 1927, p. 179), and our friend Professor Gregg Wilson, who collected material of this Monotreme in Queensland, tells us that this was the case in all the early eggs obtained by him. It is possible therefore that in this respect the Tasmanian Echidna is exceptional. '

In the early intra-uterine egg of Echidna the delicate shell and zona are perfectly transparent, but the albumen is slightly cloudy, so that the position of the disc region in the fresh egg is somewhat obscured. The egg at this stage is very fragile, the yolk being quite soft, and great care in handling is needed. We have never found it necessary to remove the shell in order to allow for complete fixation. Such fixatives as Smith’s fluid, Bouin’s fluid, picro-nitric solution, and others penetrate quite easily in spite of Semon’s statements to the contrary (1894, p. 61).

We have never taken the risk of dissecting a fresh egg, with the possibility of destroying an important stage in development, but the observations we have been able to make serve to support Caldwell in his statement that the albumen in the living egg has the same appearance as the albumen of a hen’s egg (1887, p. 472). Arrived in the uterus, measured in its membranes, the egg of Echidna when fresh has a diameter of 4-00-4-75 mm. The shell after fixation has a thickness of about 0-0013 mm., while the layer of albumen, although variable, seems to be about 0-3-0-4 mm. thick. Semon (1894, p. 8) gives 4-5 mm. as the diameter of an early intra-uterine egg, but whether the measurement is that of the fixed or living egg he does not say.

Caldwell’s measurements are not of great value, since he failed to discriminate between normal and abnormal eggs. He states (1887, p. 473) that the egg on leaving the follicle measures 2-5 mm. to 3 mm. in diameter and in his pl. 31, fig. 1, he shows the median section of an egg which, when complete, had a diameter of 3-2 mm., the disc being unsegmented. On p. 472 he records the finding of two eggs of Ornithorhynchus in the dilated end of the Fallopian tube and he gives a figure of a section of part of one of these (pl. 31, fig. 3). The diameter of this egg is recorded as being 2-6 mm. and it is said to be at the stage of eight segmentation nuclei. In our opinion, Caldwell was here dealing with eggs which were either abnormal or, being unfertilised, were in a condition of degeneration. The ovum, in our experience, does not commence to segment until it has arrived in the uterus.

The formation of the secondary envelopes that are laid down around the ovum in the oviduct (albumen coat and shell) has been described in the papers of Hill and Hill (1933) and is further discussed below (see pp. 540-542).

During the sojourn of the egg in the uterus the shell, it may be noted, undergoes a progressive increase in thickness from an extremely thin membrane measuring only about 0-0013 mm. to a firm resistant structure with a thickness of about 0-2 mm. in the recently laid Echidna egg (Hill, 1933, p. 452).

At the time of ovulation, the zona which encloses the ovum is also an extremely delicate membrane (about 0-0016 mm. in thickness). In sections of the early uterine egg, there is present, below the shell-membrane, a Well-marked layer which Flynn (1930) considered to be the albumen, but which Hill (1933) inadvertently spoke of as the zona. Actually it is a compound structure, formed of the very thin zona, closely adherent to a very much thicker overlying layer of what is apparently condensed albumen. This composite formation we propose to speak of as the zona-albumen layer. Even after fixation it remains completely transparent, so that we have been able to take photographs of surface—vieWs of the blastodisc with the layer still in situ (see, for example, Pl. XV. fig. 79 ; Pl. XVII. fig. 87 ; Pl. XIX. fig. 99).

During the early part of intra-uterine life, the egg, except in rare instances (Pl. XXI. fig. 116), retains its spherical shape, and for a time undergoes no appreciable increase in size. During this period, its yolk-mass remains compact. About the time of the completion of the enclosure of the yolk by the blastodermic membrane, infiltration of the uterine fluid into the egg begins, and from this time forward there is a steady increase in the size of the egg until it attains its maximum. That this growth is really very rapid is shown by the comparative rareness in our collection of stages between about 6 mm. and 14 mm. diameter.

When the egg of Echidna has reached a diameter of about 7~8 mm., the primitive streak has already attained a length of over 4 mm. By the time it has increased to 11-5 mm. diameter, there is present a flat embryo of 3-4 somites. At this stage the egg is still spherical when removed from the uterus. The turgidity of the blastocyst and the tonic condition of the uterine musculature bring about an intimate association of the uterine wall with that of the blastocyst, so that in opening the uterus extreme care has to be exercised to prevent damage to the contained egg.

When the blastocyst has reached a diameter of approximately 14 mm. (or less) the shell begins to become resistant, and the whole egg assumes a permanently ellipsoidal form. Such intra—uterine eggs in our collection measured when fresh (in mm.) 13-20 x 12-00, 14-60 >< 13-00, 14-8 >< 14-00, 15-00 >< 13-80, 15-50 x 13-50, and 15-00 x 14-10. These eggs are now about ready for laying.

The Pouch Egg of Echidna

We have in our possession four laid (pouch) eggs of the Tasmanian Echidna of the following measurements (fresh) :—(a) 24. 8. 30, 15-00 >< 14-50 mm. (b) 28. 7. 33, 15-00><13-00 mm. (c) 31. 7. 33, 14-50 ><13-00 mm. (cl) 21. 7. 33, 17-00 ><14-00 (taken from the cloaca). The colour of these eggs was in all cases a dirty white.

Caldwell (1887, p. 473) states that the Monotreme egg when it is laid measures 15 by 12 mm., and adds in a footnote that the laid eggs of both Echidna and Ornithorkynckus vary somewhat in size and that he obtained “ a normal Eckidna egg as small as 13 mm. by 12 mm.” Semon (1894, p. 8) gives the measurements of the pouch-egg of Eckidna as 16-5 X 15 mm.

Eggs of Omithorhynckus seem to be generally somewhat larger than those of Eckidmz when laid. Thus we have twin-laid eggs of this animal (LL & L) which measure 17 X 14 mm. and 16 X 15 mm. respectively, and we can record further the measurements of twin eggs of 18 X 14 mm. and 16-5 >< 14 mm. respectively (K & KK). ‘

Burrell (1927) gives the average size of the laid egg of Platypus as 17 -25-17-50 >< 14-00-13-80 mm., but records sizes up to 18 X25 and 17 ><26 mm. These latter measurements are far in excess of any that have come Within our own experience, and we suspect the numeral 2 in each case is a misprint for 1.

Oogenesis In The Monotremata

Previous Investigations.

Our existing knowledge of oogenesis in the Monotreme is fragmentary. Caldwell in his paper of 1887 has reviewed the earlier literature dealing with the ovarian ovum, and since that time but few papers on the subject have appeared. Caldwell himself gave a brief account of the growth of the follicle and its contained oocyte. He confirmed the existence of a vitelline membrane or zona round the oocyte as first described by Poulton (1884), and stated that it attains a maximum thickness of 0-016 mm. in the oocyte 0-32 mm. in diameter, and then, as the latter grows, it gradually becomes thinner until in the ripe ovum “ it has no longer a measurable thickness.” He seems also to have observed the “ fibrillar layer” (our striate layer) inside the zona (labelled “ zona radiata ” in his fig. 1, pl. 29). He interpreted its radially striate appearance as being due to “ streams of yolk granules passing into the ovum,” though he also affirmed that protoplasmic processes connecting the follicular epithelium with the egg-protoplasm pass through the zona. He described the growth of the follicular epithelium, and showed that it does not remain a simple epithelium, as his predecessors had believed, but in the course of growth becomes more than one cell thick, “ three to four cells deep ” in the oocyte of 0-2 mm. diameter according to his description, a curious misstatement which is not borne out by his own figure (pl. 29, fig. 1), nor by our observations, which show that in the oocyte of the diameter mentioned the follicular epithelium is still one cell thick. But it does eventually become two to three cells deep in much later stages in the growth of the oocyte as his fig. 4, pl. 29, demonstrates. Altogether, Caldwell’s account of the follicular epithelium in the Monotreme is of very great interest. He showed that it attains its maximum thickness and also its maximum activity at the stage of the full-grown oocyte, inasmuch as it functions at this time as an actively secretory layer and produces a dense homogeneous material which forms a continuous layer, situated between the inner surfaces of the follicular cells and the zona. As the result, the full-grown egg becomes, as he says, “suspended ” in a continuous dense layer of secretory material (see his fig. 4, pl. 29, and our fig. 65 (Pl. XIII.) and fig. 70 (Pl. XIV.). This layer he termed the “ pro—albumen,” on the supposition that it gives rise to the albumen layer of the uterine egg. We are able to confirm this most interesting discovery of Caldwell’s, though our interpretation of its significance is quite different from his (vide p. 530).

Caldwell makes no mention of the nuclear changes in the ovarian oocytes, and his account of yolk-formation is now only of historical interest.

From the date of publication of Caldwell’s paper in 1887 up to 1922, not a single paper dealing with the Monotreme ovary seems to have been published. Semon (1894) in his monograph on the development of the Monotremes confined himself to the uterine egg, and his collection of ovarian material has apparently never been described. In 1922 this sterile period (so far as the Monotreme ovary is concerned) of thirty-five years was brought to an end by the publication of a paper by Gatenby, dealing with gametogenesis in Platypus and based on the study of material in the possession of one of us (H.). In this paper the author gave a brief account of the structure of the ovary of Platypus. He noted the absence of oogonia in the adolescent and adult ovary, and described the structure of the early oocytes in some detail, emphasising the peripheral position of the nucleus and the presence of a centrally situated centrosphere. He also described the structure of the full-grown egg and discussed briefly yolk-formation, the origin of the latebra, the formation of the zona, and the history of the constituent layers of the follicular Wall. This paper marked a definite advance in our knowledge of the Monotreme ovary.

In 1925 Kolmer, in a paper dealing with the anatomy and histology of various organs of Proechidna (Zaglossus) Bruymll, gave an account of the structure of the ovary in this genus (1925, pp. 470~47 8), but he makes no reference to the Work of Gatenby. Both ovaries, he states, were equally developed, but the largest follicles they contained did not exceed 1-5 mm. in diameter. Like Gratenby, he failed to find oogonia. His description of the structure of the smallest oocytes (0-08 mm. in diameter) is in agreement with that of Gatenby for the Platypus oocyte, in so far as concerns the peripheral position of the nucleus and the presence of a centrally situated sphere, surrounded by a darkly staining zone of cytoplasm, and containing a spherical (centrosome) granule. In connection with yolk-formation in the larger oocytes, he described the occurrence in the cytoplasm of fat-containing vacuoles and of proteid granules, and states that the yolk-spheres resemble in their arrangement those of the Reptilian egg. He also recorded the presence of atretic follicles.

The next paper dealing with the Monotreme ovary is that by Hill and Gatenby (1926). Although this paper is primarily concerned with the formation of the corpus luteum, it also includes a more detailed account of the general structure of the ovary than had hitherto appeared. The structure of the follicular Wall and the histological changes in the follicular epithelium and thecal coats during the growth of the oocyte are also described in some detail. We shall have occasion to refer to the foregoing papers later on in this work.

The paper of Garde (1930) provides a detailed account of follicular atresia in the ovary of Platypus, but makes only brief reference to the structure of the normal follicle, whilst the paper of van den Broek (1931) on the structure of the internal genital organs of the Monotremes deals only quite briefly with the structure of the ovary.

Our own study of Monotreme oogenesis Was originally undertaken with the limited objective of elucidating the condition of the germinal disc and nucleus in oocytes immediately prior to the onset of maturation, since we found we had amongst our ovarian material a small number of oocytes at the stage of polar body formation, but as our investigation of that problem progressed we found it impossible to limit our observations to the conditions in the full-grown oocyte only, and so we were led on to attempt a study of the growth of the oocyte beginning with the earliest stage available in the adult ovary. Our observations are presented in the succeeding pages, but we make no pretension of having been able to provide even a reasonably complete account of the various complicated occurrences involved in oogenesis. We have in mind, in particular, the intricate histo—chemical processes of yolk—formation, the details of which can only be satisfactorily studied when fresh material is available and special methods of fixation and staining are employed. For this part of our work we have had at our disposal a large number of series of sections of ovaries of Platypus and Echiolna, mostly fixed in picronitro-osmic acid (PNO) and Bouin’s Fluid, but we also had available a few ovaries of Echtdua, fixed in Mann’s Corrosive-osmic Fluid (for which we have to thank Professor F. Wood Jones, F.R.S.) and in Flemming’s Fluid (F.W.A.), which have proved invaluable for the study of the distribution of mitochondria and fat in the early oocytes.

In the Monotreme ovary, follicular atresia (Kolmer (1925), Hill and Gatenby (1926), Garde (1930)) is a very common phenomenon. It can set in in the quite early oocyte, and we have found it necessary to be constantly on our guard when selecting our series of growth-stages, not to include oocytes from such atretic follicles. We hope we have succeeded.

The Ovary

As is known (ante, p. 450), in Platypus the left ovary is alone functional and usually two follicles attain maturity at the same time, whilst in Echidua, both are capable of functioning, though not normally together, since as a rule only one follicle ripens at any one time.

In both Monotremes the ovary is an irregularly ovalish body which, as is well known, resembles in appearance that of Reptiles and Birds, since the follicles, as they grow in size, come to form large spherical swellings which project from its deeply grooved and irregularly corrugated surface.

P1. I. fig. 1, shows the left ovary of Platypus XV (16-25 X 10-5 mm. in diameter), with three large maturing follicular swellings and several smaller ones projecting from its surface, the middle swelling of the three measuring about 4-5 mm. in diameter. Though the contained oocytes are not yet full-grown (vide Pl. VII. fig. 34, and p. 490), it is a fact of interest, which we owe to Dr. C. J. Hill, that numbers of sperms are already present in the uterine lumen and in the lumina of the uterine glands. The white spots visible on the follicular swellings and elsewhere on the surface mark the sites of atretic follicles.

P1. I. fig. 2 is a corresponding view of the left ovary of Echidna B. 11. 8. 30, measuring 11-5 X8-5 mm. in diameter. This ovary shows one large follicular swelling 4-75 ><4-5 mm. in diameter, its contained oocyte being full-grown and 4-1 X 3-4 mm. in diameter, and a smaller one about 3-25 mm. in diameter. Below the large swelling, the characteristically corrugated surface of the ovary is clearly seen. The ovaries of Echidna vary in diameter from 10 X 8 mm. to 18-5 X 7-5 mm., the average diameter of eight ovaries measured being 13-6 X9-1 mm.

For a general account of the structure of the ovary, the reader is referred to the paper of Hill and Gatenby (1926, pp. 718-727). Here we may mention that the germinal epithelium on the surface of the ovary varies from a quite thin layer of flattened cells, through cubical to a high columnar epithelium which may be thrown into folds, especially where it lines the surface-grooves. In the cubical epithelium we have observed in an Echidna ovary what we take to be “ primordial ova ” or oogonia in the form of clear spherical cells 0-02-0-023 mm. in diameter, which bulge down into the underlying stroma and sometimes possess more than one nucleus, whilst nests of similar clear cells in and immediately below the germinal epithelium are also met with. It would seem that we have here to do with an abortive attempt at the formation of oogonia. The smallest oocytes we have encountered in the adult Echidna ovary have a diameter of 0-042 mm., but that size is exceptional, mostly they measure about 0-06 mm. Careful search through Platypus and Echidna ovaries has failed to reveal transitional stages between the supposed oogonia in the germinal epithelium and these small oocytes which are already enclosed by a thin flattened follicular epithelium.

Gatenby (1922) also records that he failed to find oogonia in the adult and adolescent ovary of Platypus, and we agree with his conclusion that all the oogonia originally present in the foetal ovary “ seem to have undergone their maturation prophases and to have become oocytes certainly long before the animal is full grown.” In this respect the Monotremes differ from the Reptiles (Loyez, 1905) and certain higher mammals (Gérard, 1920, 1932, Evans and Swesy, 1931) in which oogonia continue to be produced throughout life.

We should perhaps point out that all our ovarian material was collected either just before or during the breeding season.

The existence of a rete ovarii in the Monotreme ovary has, so far as we know, not been recorded. In the hilar region of an Echidna ovary (B. 21.7.30) we have observed a large, quite conspicuous rete. It occupies an area about 1-35 x 0-75 mm. in maximum diameter, and is composed of a network of large tubules with wide lumina, lined by a layer of columnar epithelium.

The Growth of the Oocyte

The oocyte during its period of growth from a relatively minute cell, about 0-06 mm. in diameter to its full-grown condition of a relatively huge yolk-laden cell which may attain a diameter of 4-3 mm. in Platypus and 3-9 mm. in Echidna, undergoes a highly complicated series of changes affecting both the nucleus and the cytoplasm, as the outcome of which the germinal disc is established and a rich store of food-yolk is laid down in the cytoplasm. After a detailed survey of these changes we find it possible to recognise three clearly defined stages or phases in the growth of the oocyte, each of them characterised by the presence of certain definite structural features, and to these growth-phases We can add a fourth or final ovarian phase, during which the full-grown oocyte enters on the completion of the maturation process. resulting in the formation of the 1st polar body and the 2nd polar mitotic figure.

First Phase

Echidna Oocyte. Diameter 0-10 ><0-08 mm.

In this first phase we include the smallest oocytes of the adult ovary, ranging in diameter from about 0-06 mm. or less to just over 0-15 mm. They occur, usually in considerable numbers, in the superficial portion of the cortical zone of the ovary, close below the germinal epithelium. They have been briefly described and figured by Gatenby (1922, p. 486, pl. xiv. figs. 7 & 8), but he had no fully osmicated ovaries at his disposal. We provide two figures to illustrate their general structure (Pl. I. figs. 3 & 4). As Gatenby has pointed out, the nucleus is already excentric in position and lies close below the future upper pole. The most striking feature of the oocyte at this stage, however, is the presence in the cytoplasm of numerous coarse fat-droplets. These are very evident in P1. I. fig. 3, which illustrates an oocyte of Echiolna 0-10><0-08 mm. in diameter, with a nucleus O-04><0-027 mm. in diameter, after fixation in F.W.A. Here the fatdroplets ( ft.) (blackened by the osmic acid) are seen to occupy a broad horseshoeshaped peripheral zone of the egg-cytoplasm, the nucleus filling the opening of the horse-shoe. Internally to the fat-laden zone and the nucleus, the central region of the oocyte is occupied by a dense finely granular mass of cytoplasm which we may distinguish as the central zone (mt.z.). It contains only a few fine fatdroplets, but situated near its centre, shortly below the nucleus, there is present a definitely limited ovalish body (0-O09 mm. in diameter), containing a number of small fat-droplets (cs.). This is the centrosphere or so-called “ yolk-body ” of Balbiani, the existence of which in the early Monotreme oocyte was first observed by Gatenby (1922) in Platypus and later by Kolmer (1925) in Proechidna. According to Gatenby, centrioles or small granules can be made out in some cases within the centrosphere. Kolmer describes the cytocentrum (as he terms it) as being very finely granular and surrounded by a progressively less deeply staining area of cytoplasm, which is frequently prolonged as fine processes to the periphery of the egg. In all small oocytes the sphere contains a homogeneous deeply staining granule which he compares to the centrosome of the Ascaris egg. Neither THE DEVELOPMENT OF THE MONOTREMATA. 459

observer makes mention of the occurrence of fat-droplets in the sphere. We may mention that we have observed oocytes in which the latter is represented by a centrally situated group of small fat-droplets, quite devoid of any differentiated area of cytoplasm around them. It is now recognised that the centrosphere is a structure of constant occurrence in the early oocyte. It has been described in detail, with a full discussion of the earlier literature, by 0. Van der Stricht (1904, 1905, 1923) as well as by others, in the oocyte of Mammals, by Loyez (1905) in that of certain Reptiles and by various observers (D’Hollander (1905), van Durme (1914), Brambell (1925), and others) in the oocyte of Birds.

The central zone (mt.z.) owes its dense appearance to the presence in it of innumerable very fine granular mitochondria, which tend to be arranged in rows simulating short filaments. Mitochondria are also present in the peripheral cytoplasm, but are much less numerous, so that the latter stains more lightly than the central.

This central mitochondrial zone (mt.z.), enclosing as it does the centrosphere, clearly corresponds to the ‘ couche vitellogene ou mitochondriale ” described by van der Stricht in the early human oocyte and that of Vespertilio noctula, by Loyez in the oocyte of Reptiles, by van Durme in the Bird oocyte, and by Brambell (1925) in the same, under the names of mitochondrial cloud and mitochondrial yolk-body. Gatenby (1922) also recognised the occurrence of a cloud of mitochondria round the centrosphere in the youngest oocytes of Platypus.

Echidna Oocyte. Diameter 0-O83 ><0-079 mm.

In Pl. I. fig. 4 we illustrate a comparable but slightly smaller oocyte of Echidmt after Bouin fixation. This oocyte measures O-083><0-079 mm. The fat having been dissolved, the peripheral cytoplasm now appears as a richly vacuolated zone (more coarsely so on the left in the figure than on the right), which in this oocyte extends across below the nucleus, separating the latter from the central zone. The space around the nucleus is evidently a contraction-space, since it is not present after corrosive—osmic and F.W.A. fixation. The central zone (mt.z.) presents the same dense granular appearance as in the preceding oocyte, but the centrosphere (cs.) situated in it is a much more conspicuous structure. It appears as an irregularly rounded, rather deeply staining body (0009 ><0-008 mm. in diameter) of definite contour, the interior of which is vacuolated (indicative, no doubt, of the former presence in it of fat-droplets). Surrounding the central body is a light staining zone of cytoplasm (0-0126 mm. in diameter), not always recognizable. After Bouin fixation, the central body usually appears vacuolated as just described, but sometimes it is perfectly homogeneous and it may contain one or two minute basophil granules (centrioles). In many oocytes, otherwise Well preserved, it is irregular in outline and stains very faintly, and in yet others we have failed to find it. Possibly in some oocytes it early undergoes fragmentation. Apart from the presence in it of minute fat-droplets, the centrosphere of the Monotreme oocyte is evidently Very similar to that of the early human oocyte as described by 0. van der Stricht (1904, 1905). According to his account the “ corps vitellin ” consists of one, two, or several central corpuscles, surrounded by a medullary zone or centrosome (“ la couche médullaire de Ed. van Beneden ”), outside which is a clearer, more lightly staining cortical zone (“ la couche corticale de la sphere attractive de Ed. van Beneden ”). In his papers van der Stricht provides a very complete review of the earlier literature relating to the “ corps vitellin de Balbiani.”

Structure of the Nucleus.

We have made a careful study of the nucleus of these early oocytes partly in the hope of being able to locate the chromatin, but our results in this regard are not Very conclusive.

The finer details of the nuclear structure are Well seen after Bouin and corrosiveosmic fixation, whereas in our F.W.A. material, the nuclei of these early oocytes are Very poorly fixed.

In Pl. I. fig. 5 we illustrate the nucleus of an oocyte 0-104><0-O88 mm. in diameter, the nucleus measuring 0-037 ><0~03l mm., after corrosive—osmic fixation and staining by Feulgen’s method. In this figure the nuclear space inside the thin nuclear membrane is seen to be occupied by a Very delicate alveolar matrix or reticulum (hardly stained), in the strands of which are present numerous very fine granules. These, we at first supposed, might be dispersed chromatin granules, but, since they do not stain with iron-haematoxylin after Bouin fixation, We presume they cannot be such. Situated in this nuclear matrix is a conspicuous formation composed of a more or less loose tangle of darkly staining strands of Varying length, which cross and interlace, forming What we may term, following D’Hollander (1905) and van Durme (1914), a pseudo-reticulum. The strands for the most part have a wavy convoluted form, but sometimes they are straight and extend out from the main tangle towards the periphery of the nucleus (Pl. I. fig. 4). Sometimes also minute cleft-like or rounded spaces are present in them at intervals, so that at these places they appear double or even bead—like (figs. 4 & 5). Their margins are not sharply defined, but are wavy and irregular, sometimes indeed after Bouin fixation the strands appear plumose (Pl. I. fig. 4). Structurally the strands consist of a matrix containing fine granules comparable in size with those in the nuclear reticulum as well as slightly larger granules, some of which have stained a bright red With the Feulgen stain. Possibly these larger granules are chromatinic.

Lying in the matrix, in close association with the pseudo-reticulum, there is constantly present a large rounded body which has stained of a dull brownish red and is vacuolated internally. This we regard as of the nature of a nucleolus.

In addition, there are numbers of smaller nucleolar granules in the matrix, some of which have stained of a dull red colour, others lightish brown.

Essentially the same features are seen in P1. I. fig. 6, depicting the nucleus of an early oocyte after Bouin fixation and staining with Mann’s methyl blueeosin. The nuclear matrix iscoarser and perhaps not so well preserved as that of P1. I. fig. 5, and the fine granules shown in the latter figure are not visible. The delicate strands of the pseudo-reticulum, for the most part wavy and irregular in disposition, are very obvious in the figure. They contain fine basophil granules, which do not stain very sharply or very intensely with the methyl-blue, and are present in such numbers as to give the strands a finely granular appearance. There is no definite evidence of the presence of such granules lying free in the matrix between the strands. The main nucleolus has stained intensely red with the eosin, and contains bright refractive vesicular inclusions, though in some cases these are replaced by larger homogeneous granules. The smaller nucleolar granules, associated with the pseudo-reticulum and also present below the nuclear membrane, are markedly acidophil like the main nucleolus. They vary in size and in _the majority of cases are perfectly homogeneous, but they may contain granular inclusions like the main nucleolus.

The main nucleolus,it may be noted, varies in form; sometimes spherical, it is frequently irregularly angular, and occasionally little bud-like masses can be seen projecting from its surface, suggestive of the origin from it of the smaller nucleoli by a budding process. The nucleus in Pl. I. fig. 4 (Bouin fixation) is of interest as showing the characteristic beaded appearance often exhibited by the strands of the pseudo-reticulum; the hollow, bead-like enlargements may occur singly, or several may occur in close succession in a strand. The supposed chromatinic granules in the strands stain with iron-haematoxylin, whilst the fine granules in the nuclear matrix fail to do so, but are slightly coloured by eosin.

We have not encountered in the literature any descriptions of early oocyte nuclei quite comparable with that given above for the nucleus of the early Monotreme oocyte. De Winiwarter (1900) and de Winiwarter and Sainmont (l908—9‘), in their classical studies of the oogenesis of the Rabbit and Cat, describe the nuclei (“ noyaux dictyés ”) of the oocyte of the primordial follicles as being in process of transformation into the resting condition. Some of the double filaments characteristic of the preceding diplotene stage are still recognisable, though no longer quite typical. Most of them have broken down to form irregular masses of granular material enclosing coarse chromatin particles, the nucleus appearing as if it possessed a genuine reticulum. The nucleolus is large and is filled by clear refringent vacuoles such as we have described above. The relationship of our pseudo-reticular strands to the paired filaments of the diplotene stage and the significance of the beaded character of the former can only be determined when the early phases in the oogenesis of the Monotreme have been investigated.

In the nucleus of the oocyte of the Bird at the beginning of intra-follicular growth van Durme (1914) states that there are present a nucleolus and chromatic rings (“ anneaux chromatiques ”) connected by undivided filaments. These rings become stretched, and by branching and superposition give origin to a pseudoreticulum composed of thin varicose segments, striated transversely. These segments would seem to correspond to our pseudo-reticular strands, but, judging from van Durme’s account, their subsequent history is much more complicated than that of the strands in the Monotreme, since they give origin to plumose segments which eventually undergo a process of chromatolysis, their fragmentation resulting in the formation of numerous very chromatic granules. Unfortunately the details of the changes she describes cannot be followed in her figures.

Loyez (1906) has provided a very detailed account of the structure of the nucleus and its transformations during the growth of the intra-follicular oocyte in the Reptilia. She maintains that in the Squamata the chromosomes persist throughout the entire period of the growth of the oocyte as discrete structures, which in the early oocyte take the form of plumose filaments (Lacertilia) or of diffuse granular cords (Ophidia).

Echidna Oocyte. Diameter 0-128 ><0-124 mm.

We figure next an oocyte (Pl. II. fig. 7) from an ovary of Echidna, fixed in corrosive-osmic, which is somewhat larger than that of P1. I. fig. 3. The oocyte measures 0-128 ><0-124 mm. in diameter, its nucleus 0-048 X0-036 mm. The peripheral cytoplasm is densely laden with fat-droplets, which are much smaller and much more numerous than in oocytes fixed in F.W.A. Possibly this is a varietal difference, since the C-0. ovaries were obtained by Professor Wood Jones from the mainland variety of Eckidna (actually from Kangaroo Island), whilst the F.W.A. ovaries are from the Tasmanian variety. The fat-droplets extend across below the nucleus, so that the central zone is completely enclosed, and they also occur in small numbers above the nucleus, which is more deeply situated than is that of P1. I. fig. 3.

The central zone (mt.z.) presents a homogeneous finely granular appearance and is extremely rich in mitochondria. The yolk-body has now disappeared, though we have occasionally found it in oocytes somewhat larger than this. At the surface of the oocyte there is as yet no continuous peripheral cytoplasmic layer free from fat-droplets, though it is indicated here and there and especially above the nucleus. The oval nucleus possesses a finely granular matrix in which are situated the main nucleolus containing deeply staining granules, a number of small nucleolar granules, and traces of the pseudo-reticulum.

All these early oocytes are enclosed in a single-layered follicular epithelium, varying from flattened to low cubical as in P1. II. fig. 7, where it has a thickness of 0-006 mm. Outside the epithelium a thin layer of flattened, concentrically arranged cells is becoming recognisable——the thecal primordium.

Zona pelluctda.

In the later oocytes belonging to this first phase, the zona is easily recognisable as a homogeneous membrane lying immediately internal to the follicular epithelium, but it is by no means easy to determine the moment of its first appearance. Fortunately, in our sections of the ovary of Ecktdna XVIII fixed in F.W.A., early oocytes ranging between 1 and 2 mm. in diameter are to be met with, in which the follicular epithelium is not, as it usually is, in close and intimate apposition with the periphery of the oocyte, or with the zona when that is present, but is separated therefrom, no doubt as the result of contraction, by a very narrow cleftlike space, and from such oocytes we have prepared a series of four figures (Pl. II. figs. 8-11) which we think are of interest in connection with the much-discussed question of the mode of formation of the membrane in question. Pl. II. fig. 8 represents a sectional View of the periphery of an oocyte of Echiclna 0-l03>< 0-0'78 mm. in diameter. The follicular epithelium (f.e.) has a thickness of from 0-0056-0-006 mm., and is composed of a single layer of cubical cells, with large spherical or oval nuclei, the limits between the cells being indistinct, as Gatenby (1922, p. 488) also found to be the case in the early oocyte of Platypus. Between the sinuous deep surface of the epithelium and the extremely thin but quite definite pellicle (em.) bounding the cytoplasm of the oocyte, and which we regard as the egg-membrane, there are present minute isolated masses of a light, practically unstained material (zp.s.) which We take to be a secretory product of the follicular cells. It is most obvious where it occupies the depressions marking the cell-limits, but it also occurs opposite the cell-surfaces. Gatenby (1922) has de scribed and figured (pl. xiv. figs. 10 & 12) what he terms a “ pre-zona ” material situated in the cytoplasm of the inner part of the follicular epithelium of the early Platypus oocyte, but this We have never been able to detect.

In Pl. II. fig. 9, from an oocyte 0-13 ><0-094 mm. in diameter, it will be seen that the cleft between the follicular epithelium and the egg-membrane (em.), wider than in P1. II. fig. 8, is partially occupied by a continuous layer of homogeneous material (zp.) varying somewhat in thickness from place to place and so presenting an uneven contour. This is clearly the zona, formed, we suggest, by the joining up of the above-described isolated masses of secretion. We estimate its thickness at i0-0007 mm. Here and there it lies in direct contact with the inner surfaces of the follicular cells, whilst in’ between these areas of contact it is separated from the latter by a narrow cleft, which in places appears quite empty, but over most of its extent seems to be occupied by a light staining material like a coagulum. On the opposite side between the zona and the egg-membrane (here less distinct than in P1. II. fig. 8) is a similar cleft also occupied by a light staining material, but presenting faint traces of a radial striation. In later oocytes this radial striation becomes quite pronounced, and is due to what appear to be very fine fibrils extending more or less radially between the zona andthe egg-membrane. They were observed by Gatenby (loc. cit.) in the Platypus oocyte and termed by him the “ cortical fibrillae.” We shall return later (vide pp. 474—475) to a consideration of these “ fibrils,” meantime we shall refer to the layer in which they occur as the “ striate layer ” (Pl. II. fig. 10, s.l.).

Pl. II. fig. 10 illustrates the condition of the egg-envelopes in an oocyte 0-179 >< 0-148 mm. in diameter. The follicular epithelium is somewhat thicker than in the preceding oocytes, measuring 0-0058-0-0065 mm., whilst the zona (zp.) is now definitely recognisable as such, and is, on the average, about twice as thick as that of the preceding oocyte. Its lower surface is smooth and even, its upper surface, facing the follicular epithelium, on the other hand, appears roughened and irregular as if it were being increased by the addition of material on this side.

The limits between the follicular cells are now much more evident than in the preceding oocytes. In places, indeed, the cells are separated by conspicuous intercellular clefts (Pl. II. fig. 10), straight or slightly curved, which begin shortly below the bases of the cells and at their opposite ends seem to open directly into the cleft between the surface of the epithelium and the zona. They appear to be filled by a non—staining material, presumably secretory and apparently destined to be added to the zona. Occasionally, as in P1. II. fig. 10, an intercellular cleft, bulbously enlarged and filled with the same material, is encountered. We have been unable to find any evidence of the existence of closing terminal bars in any part of the follicular epithelium.

Pl. II. fig. 10 further shows that the surfaces of the follicular cells are connected here and there with the zona by extremely short and delicate processes, often spike-like, which seem to be cytoplasmic, though the possibility of their being composed of secretory material cannot be wholly excluded. Whatever their nature, they terminate at the zona and are not in any way related to the “ fibrils ” of the striate layer. The latter (s.l.) is now distinct, its “ fibrils ” being clearly apparent, and the egg-membrane is also well marked.

In Pl. II. fig. 11 we illustrate part of the periphery of a slightly larger oocyte (0-17 ><0-16 mm. in diameter). The zona (21).) has a thickness of 0-0021 mm., and is about half as thick again as in the preceding oocyte, whilst the striate layer (s.l.) has acquired a thickness of about 0-0007 mm. In the portion of the periphery figured, the follicular cells are again seen to be connected with the zona by clear bridges, here more suggestive of bridges of secretion than of cytoplasm, but over most of the periphery they are new little in evidence.

We may summarise the foregoing observations as follows :——(l) The zona first appears in oocytes of Echidna just under 0-10 mm. in diameter in the form of minute isolated masses of secretion elaborated by the follicular epithelium. (2) In the oocyte just over 0-10 mm. in diameter, a continuous very thin zona is present. It increases in area and in thickness through the continued secretory activity of the follicular cells. The secretion may be passed directly through the free surfaces of the latter or indirectly by way of the intercellular clefts. There THE DEVELOPMENT OF THE MONOTREMATA. 465

is no evidence of the participation of terminal bars in the formation of the zona at the time of its first appearance, nor is there any evidence that cytoplasmic processes of the follicular cells are prolonged through the zona.

(3) The striate layer develops outside the egg-membrane in the space between the latter and the zona, its striations only becoming evident after the zona is established as a continuous layer.

We may mention that in later oocytes (Eckidna oocytes of about 0-6 mm. in diameter) we have sometimes observed traces of a darkly staining material between the follicular cells, identical in all respects with the substance of the zona and traceable into direct continuity with it, and we have seen a similar material with identical relations between the follicular cells in atretic follicles, only here it is much more richly developed, in correlation, no doubt, with the much greater thickness frequently attained by the zona in such follicles.

We are therefore in agreement with those authors who, like 0. van der Stricht (1923) for higher mammals and Thing (1918) for Chelonia, hold that the zona is exclusively a secretory product of the follicular cells. According to these two authors the zona is really of the nature of an intercellular cement substance, which first appears in the form of a network or fenestrated membrane produced by the extension of the cuticular substance constituting the terminal bars over the free surfaces of the follicular cells. Thing in the Turtles further postulates the existence of what she terms a “secondary network apparently produced directly by the superficial cytoplasm of the epithelial cells ” which, she says, seems capable of secreting a cement similar to that forming the terminal bars. We interpret this to mean simply that the follicular cells are also capable of pouring out secretory material from their free surfaces, which is added to the similar material composing the terminal bar network, and both together form the zona. Further, both authors describe the existence of fine cytoplasmic prolongations of the follicular cells (one from each, according to van der Stricht), which pass through the zona (at first through the perforations in the fenestrated membrane) and connect with the surface of the oocyte, Thing stating that in the Turtle they end in knob-like enlargements. It is these prolongations which, according to Thing, form the fibrils of the radially striate layer which underlies the zona.

As mentioned above, we have never been able to observe terminal bars in our material, nor have we seen the just-mentioned cytoplasmic prolongations. These latter have also been described and figured by Mjassojedoif (1923) in the zona of oocytes of the Cat. This author holds that the zona is of dual origin and formed partly by the follicular epithelium, partly by the cytoplasm of the oocyte. His observations are referred to below (pp. 476 & 527).

Various authors have cited the case of bi-ovular follicles, in which the two oocytes are separated by a common zona as favouring the idea that the zona is entirely derived from the oocyte. We have observed in one particular ovary of Echidna. (B. 21.7.30) numbers of such follicles, -and whatever may be the explanation of this condition in other mammals, it is perfectly clear that here the bi-ovular condition is secondary and the result of two actively growing follicles coming into apposition with subsequent atrophy through pressure of the apposed portions of the follicular epithelium and fusion of the related portions of the two zonae.

It is a fact of some interest, previously commented on by Gatenby (1922), that the early oocytes of this phase already exhibit the definite polarity which characterises the full-grown oocyte, the upper pole being clearly distinguishable from the lower by the proximity of the nucleus to it as well as by_ the relative poorness of the perinuclear region in fat—globules.

Particular attention may also be called to the remarkably close resemblance in general structure which exists between the early Monotreme oocyte and those of Birds and Reptiles as described by van Durme (1914), Konopacka (1933), Bulliard (1924) *, and others. Van Durme’s pl. v. fig. 25 representing a section through an early oocyte of the fowl is, apart from the absence of the centrosphere, a very fair replica of our Pl. I. fig. 3. We observe in both the same polar position of the large nucleus, the numerous coarse fat-droplets in the peripheral cytoplasm, and the central mitochondrial zone (“couche vitellogene ”) enclosed between the latter and the nucleus.

Second Phase

We pass now to a second distinctive phase in the growth of the oocyte, comprising oocytes ranging from about 0-2 mm. to about 0-5 mm. in diameter, in which the thecal primordium is distinct, the follicular epithelium is cubical, the zona has attained some thickness and is easily recognisable, whilst the fat-droplets have taken up a more definitely peripheral position so that we can recognise in the oocyte a cortical fatty zone and an extensive central or medullary zone relatively free from fat, and in addition a peripheral cytoplasmic zone also free from fat intervening between the cortical fatty zone and the egg—membrane. In this phase the primordia of the yolk-spheres make their first appearance.

Echidna Oocyte. Diameter 0-21 X0-20 mm.

An oocyte of Echidna, 0-21 X0-20 mm. in diameter, after F.W.A. fixation, may be taken as typical of the conditions at the commencement of this phase. The nucleus (somewhat shrunken) has a diameter of 0-055 X0-038 mm. This oocyte exhibits a well-marked advance on those of the first phase (Pl. I. fig. 3 and P1. II. fig. 7) inasmuch as the fat-globules are now aggregated to form a definite cortical fatty zone (of. fig. 12, Pl. II. (vf.z.)), which in this oocyte happens to be thickest over the lower polar region and at the upper pole, is interrupted by the nucleus. On its outer side, between it and the zona, is a narrow belt of cytoplasm, the peripheral cytoplasmic zone, largely free from fat except over the nucleus and containing rather coarse non-osmicated granules.

The fat-droplets of the cortical zone vary in size, and many of them present a shrivelled appearance, indicative, no doubt, of their commencing utilisation in the vitellogenetic processes, though there is as yet no definite indication of yolk-sphere primordia.

Within the cortical zone the medullary cytoplasm is finely granular and uniform in character throughout and contains fine disseminated fat-spherules.

The follicular epithelium is formed of a single layer of cubical cells, averaging 0'0079 mm. in thickness, whilst the zona has a thickness of 0-004 mm.

Echidna Oocyte. Diameter 0-29 x 0-27 mm.

A slightly later oocyte of this phase, measuring 0-29 X0-27 mm. in diameter, is illustrated in P1. II. figs. 12 & 13. The cortical fatty zone (vf.z.) has a similar disposition to that of the preceding oocyte. ‘On its outer side the fat-droplets are mostly small and often shrivelled. Within these occur much larger droplets, many ofithem angular and shrunken, and on its inner side numbers of quite minute droplets lie dispersed in the cytoplasm. Also situated here and there in this deep part of the zone are pale—staining homogeneous spherical bodies varying from about 0-005 to 0-008 mm. in diameter. These are the primordia of the first yolk-spheres (Pl. II. fig. 13, ys.p.), but they are as yet few in number, and, judging from their fragile appearance and delicate staining, have only recently been formed. The cortical fatty zone thus becomes the cortical vitello-fatty zone.

Throughout the extent of the cytoplasm of the oocyte (Pl. II. fig. 13) there are present granular mitochondria similar to those in the follicular cells, but perhaps rather finer. They occur singly, in little groups and in short rows, and are not appreciably more abundant in the peripheral cytoplasmic zone than elsewhere. Furthermore, in the vitello-fatty zone, and especially in the cytoplasm deep to the large fat-droplets where the yolk-sphere primordia occur, there are present numbers of minute bodies measuring about 0-003 mm. and less in diameter and irregularly oval or rounded in form (Pl. II. fig. 13). They stain intensely With acid-fuchsin just like the mitochondria, and, since they often appear to be composed of discrete granules, they‘may possibly represent aggregations of swollen mitochondria. It is suggestive that they should be most numerous in the deep part of the vitello-fatty zone just where the yolk-sphere primordia are making their appearance.

The medullary cytoplasm (m.z.) is free from fat except round its rather indefinite periphery, Where minute dispersed droplets are present. It is uniform throughout and very finely granular in appearance, owing to its mitochondrial content.

The follicular epithelium (Pl. II. fig. 13, f.e.) averages 0-008 mm. in thickness and is composed of a single layer of cubical to low columnar cells with large spherical or ovalish nuclei. In the cytoplasm of the cells there are present granular mitochondria (mt.) which are not uniformly dispersed but are aggregated in an irregular fashion around the nuclei. Minute fat-droplets, distinctly larger than the mitochondria and usually clustered together, are also present in or near the mitochondrial groups (cf. also Pl. IV. fig. 28). The zona (21).) has attained a thickness of 0-006 mm., so that its growth has been fairly rapid. Below it the very thin striate layer (s.l.) and the egg-membrane are clearly visible (Pl. II. fig. 13). This figure, it may be noted, is taken from a part of the periphery of the oocyte where the fat-droplets of the vitello-fatty zone are still fairly intact ; they have mostly lost their smooth spherical contours, but are not markedly shrivelled.

Echidna Oocyte. Diameter 0-296 X0-284 mm.

We figure another oocyte (Pl. III. fig. 14) comparable in size with the preceding, but differing from it in showing an asymmetrical disposition of the vitello-fatty zone, a condition which is not uncommon and which may be due to a primary asymmetry in the distribution of the fat-droplets, or more probably to their earlier dissolution in one particular region. The oocyte measures 0-296 X 0-284 mm. in diameter, the zona 0-005 mm., and the follicular epithelium 0-009 mm. in average thickness.

As will be seen from P1. III. fig. 14, the cortical vitello-fatty zone (vf.z.) is confined to the apparent left half of the oocyte, where it forms a broad crescent, whilst in the opposite half there are present only minute disseminated fat-droplets and the shrivelled remnants of such, together with a number of large spherical bodies situated in vacuolar spaces in the cytoplasm; these are yolk-sphere primordia. The fat-droplets of the vitello-fatty zone are mostly spherical in its lower half, but markedly shrivelled and irregular in its upper half. Yolk-sphere primordia appear to be absent from the zone itself, but they occur in small numbers (ys.p.) in the rest of the cortical cytoplasm, as well as in the cytoplasm on the inner side of the zone, and, as just mentioned, some of them reach a considerable size. The details of the peripheral region are shown in P1. III. fig. 15, taken from another oocyte of about the same size as the above, its diameter being 0-31 ><0-25 mm. The follicular epithelium (f.e.) measures 0-10 mm., and the zona (21).) 0-006 mm. in thickness.

The vitello-fatty zone (vf.z.) resembles in its disposition that of the abovedescribed oocyte, but the fat-droplets are best preserved in its upper portion adjacent to the nucleus, and it is from this region that Pl. III. fig. 15 is taken. The figure shows the follicular epithelium (f.e.) composed of low columnar cells, with the mitochondria characteristically grouped around the nuclei, the relatively thick zona (zp.) (its apparent division into two layers being of no morphological significance), the extremely thin striate layer (s.l.), and the egg—membrane. It also shows below the latter the peripheral cytoplasmic layer of the oocyte containing mitochondria (p.c.z.), followed by the outer part of the vitello-fatty zone (vf.z.), with its conspicuous fat-droplets, spherical and irregularly angular in outline. Pale-staining spherical yolk sphere primordia of varying size are present amongst the fat-droplets, but as yet in no great abundance. The larger of these primordia mostly appear homogeneous, but the smaller may contain minute spherical granules or sometimes appear to be composed of such. In addition to mitochondria numbers of pale spherical granules (varying in size from about 0-0007 to 0-002 mm.) occur throughout the cortical cytoplasm of this and the preceding oocyte, and most abundantly where the fat-droplets are in process of dissolution. Their significance is unknown.

Echidna Oocyte. Diameter 0-368 ><0-328 mm.

A further stage in the formation of yolk-sphere primordia and in the correlated utilisation of the fat-droplets of the vitello-fatty zone is seen in the oocyte represented in P1. III. fig. 16. This oocyte measures in diameter 0~368 ><0-328 mm. ; the follicular epithelium (f.e.) averages 0-010 mm. in thickness and the zona (23).) 0-011 mm., a noteworthy increase. The vitello-fatty zone (vf.z.), compared with that in P1. II. fig. 12, is no longer such a conspicuous formation, though it has increased in width. In the upper polar region below and peripherally to the nucleus, the fat-droplets large and small are mostly still spherical and Well preserved, and, as a consequence, the zone stands out prominently, but over the remainder of its extent it is much less marked, being more diffuse and finer-grained in texture. Here the fat-droplets are represented by shrivelled remnants, mostly small, and intermingled with them are numerous yolk-sphere primordia. These latter are now much more abundant than in preceding oocytes, and over the greater part of the zone constitute the main bulk of its thickness, the larger spheres lying in its inner half, whilst the smaller are mainly located in its outer half. The large spheres (g/8.10.) attain a diameter of about 0-0056 mm., though much larger spheres are occasionally met with.

Echidna Oocyte. Diameter 0-448 ><0-36 mm.

The details of the vitello-fatty zone are shown under higher magnification in P1. III. fig. 17, which represents part of the periphery of a slightly larger oocyte, 0-448><0-36 mm. in diameter, the zona measuring 0-01 mm. and the follicular epithelium O-012 mm. in thickness.

The vitello-fatty zone (vf.z.) is again broad (its average width being about 0-09 mm.), and throughout the greater part of its extent the fat-droplets are shrivelled and greatly reduced. On the other hand, yolk-sphere primordia (3/s.p.) are now more numerous than in the preceding oocyte, the larger being situated in the inner two-thirds of the zone, the smaller mainly but not exclusively in the outer third (Pl. III. fig. 17). They vary in size, but the largest attain much the same diameter (00056 mm.) as in the preceding oocyte. The majority (comprising all sizes down to spheres 0-001 mm. in diameter) appear perfectly homogeneous, the remainder (comprising spheres of medium to small size) are more or less completely filled by spherical granules, which, like the homogeneous spheres, have stained of a dull reddish tint with the acid-fuchsin or they show traces of such granules. Possibly of significance from the point of View of vitellogenesis is the fact that enlarged mitochondria, two or three times the size of the normal and still staining bright red with the acid-fuchsin, are of frequent occurrence throughout the cytoplasm. They occur as isolated granules and as definite aggregations, and occasionally such groups appear as if enclosed in a common envelope or matrix, but with our limited technique we must leave the question open as to whether or not mitochondria become directly transformed into yolksphere primordia, as maintained by van Durme (1914), Brambell (1925), and others. After careful study of our material, all We can say is that such a transformation suggests itself as a possibility. It remains to be mentioned that the peripheral cytoplasmic zone (p.c.z.) is distinct and free from yolk-sphere primordia. Its superficial portion, owing to its greater content of mitochondria, now stains rather more intensely than its deeper portion.

The immediately succeeding stages in the growth of the oocyte are illustrated by a series of eggs selected from ovaries of Platypus, fixed mostly in picro-nitric acid. These suffer from the disadvantage that the fat has not been preserved, but, on the other hand, the fixation of nuclear structure is much better than in the osmicated oocytes of this phase.

Platypus Oocytes. Diameter 056 X0-50 mm. and 0-53 ><0-43 mm. respectively.

We provide two figures of Platypus oocytes (Pl. III. figs. I8 & 19) to illustrate the condition attained towards the end of phase 2.

Pl. III. fig. 18 represents a section of an oocyte, 0-56 ><0~50 mm. in diameter, the zona and follicular epithelium both measuring about 0006 mm. in thickness. It links up directly with the preceding Echldna oocyte (Pl. III. fig. 17), but the zona and follicular epithelium are only about half as thick as in that. The peripheral cytoplasmic zone is distinct and very finely granular. As in preceding oocytes, it is continuous over the nucleus. The cortical yolk-zone (c.y.z.) (as We now term the vitello-fatty zone) exhibits the typical disposition and is not greatly in advance of that of the preceding oocyte. It is formed in the main by rather light staining eosinophil yolk-sphere primordia, varying in size from spheres -0045 mm. in diameter down to quite small spherules, the smaller lying mainly towards its outer side, the larger in its inner half. These spheres are quite numerous and lie in vacuoles which they only partially fill. Frequently several small spheres, aggregated together, occur in one vacuole. "

In addition to these eosinophil spheres, there are present in sparse numbers in the innermost part of the zone quite small spheres which have stained with the haematoxylin. They are situated like the other spheres in vacuoles, the larger occurring singly, but more usually several occur together forming simple aggregations or rings.

The central cytoplasm (m.z.) is uniformly finely granular throughout its extent.

Pl. III. fig. l9 illustrates an oocyte slightly smaller than the last (its diameter being 053 X043 mm.), but definitely more advanced, the yolk-zone (c.y.z.) being a much more prominent formation (about 0-07 mm. in thickness). Throughout its thickness, but especially numerous in its outer half, there are present small yolk—sphere primordia, now evidently somewhat altered in chemical composition since they stain of a light violet tint with haematoxylin. Many of them are filled with spherules or are formed of aggregations of such. In addition, there occur in its inner half, in considerable numbers, larger spheres which stain intensely black with haematoxylin. These we regard as definitive yolk-spheres, though they have not yet attained their full size. Some of them are spherical and homogeneous (with a diameter up to about 0-006 mm.), others, still larger and more irregular in outline, are composite and composed of aggregations of smaller spheres.

Immediately to the inner side of the zone, the central cytoplasm (m.z.) over a depth of about 0-03 mm. is now richly vacuolated, the vacuoles containing very small darkly stained spheres which occur singly or more often two or more together. In this oocyte the vacuoles with their contained spheres extend across below the nucleus. Dispersed vacuoles have also now appeared deeper in, in the central cytoplasm, but there are very few in the actual central area itself.

The nucleus in these two oocytes occupies its normal position below the upper pole, and measures in each 0-l0><0-09 mm. in diameter. It contains a delicate nuclear reticulum, irregularly arranged pseudo-reticular strands supporting fine granules, a large intensely stained nucleolus, with vesicular inclusions in the case of that of P1. III. fig. 19, and a number of small nucleolar granules.

Third Phase

This third phase of Monotreme oogenesis includes oocytes ranging from about 0-6 mm. in diameter up to the stage of the fu1l—grown oocyte, with a maximum diameter in Platypus of 4-3 mm. and in Eckidmt of 3-9 mm., all characterised by the possession of a germinal disc or its primordium and of definitive yolkspheres. It is the phase in which yolk-formation is most active and during which the oocyte undergoes its maximum growth. Whether this growth is a slow process extending from one breeding-season to the next, or is mainly effected in the weeks or months immediately preceding that season, as is more probable, we cannot determine from our material. We may mention that in ovaries with full-grown follicles there are usually present one or two partly grown follicles (2-3 mm. in diameter), but whether these will eventually attain maturity or are destined to undergo follicular atresia before the next breeding-season We cannot say.

We recognise two sub-phases in this third phase. Sub-phase A includes oocytes 0-6 to 0-7 mm. in diameter, characterised by the presence of a germinal discprimordium, a peripheral cytoplasmic zone, a cortical yolk-zone with definitive yolk-spheres, and a central or medullary zone distinguishable into a vacuolated outer zone and a central non-vacuolated core. In the course of this sub—phase, yolk-sphere primordia appear in the vacuoles of the outer zone. Sub-phase B comprises oocytes ranging from about 0-8 mm. in diameter up to and including those which have attained the full-grown condition.

Sub-phase A.

Platypus Oocyte. Diameter 0-62 ><0-61 mm.

The oocyte illustrated in P1. IV. fig. 20 and P1. V. fig. 21 represents a highly characteristic stage in the vitellogenesis of the Monotreme. Its measurements are as follows: diameter 0-62 ><0-61 mm., nucleus 0-109 ><0-09 mm., follicular epithelium 0-0056 mm., and zona 0006 mm. in thickness.

The peripheral cytoplasmic zone (Pl. V. fig. 21, p.c.z.) is distinct, but thin (0003 mm.), and passes over internally into a thin zone of vacuolated cytoplasm, mostly free from yolk-spheres, and this on its" inner side merges without limit into the much more coarsely vacuolated cytoplasm of the cortical yolk-zone (c.y.z.).

As the peripheral cytoplasmic zone approaches the upper pole it gradually thickens, and over and around the nucleus gives origin to a definite localised thickened area, which is none other than the primordium of the germinal disc (gd.p.), really of the formative cytoplasmic zone of the latter, which is here seen for the first time. Above the nucleus the disc-primordium has a thickness of 0-017 mm. It extends beyond the periphery of the same for a short distance, but its peripheral limits are rather indeterminate, a condition which persists throughout the growth—period of the oocyte, and it also extends down around the equatorial region of the nucleus as a quite thin investment.

The cortical yolk-zone (c.y.z.), very evident in P1. IV. fig. 20 and P1. V. fig. 21, has a thickness of about 0-05 mm. Definitive yolk-spheres are now the most conspicuous and most abundant elements in it. They are spherical in form, are situated in the vacuoles, mostly singly, stain deeply and homogeneously with haematoxylin, and vary in size from small to large, the maximum diameter attained being about 0011 mm. In the peripheral part of the zone especially, occur numbers of eosinophil yolk-sphere primordia (3/3.19.). They are comparable in size-with the medium-sized definitive spheres and mostly appear homogeneous, though occasionally they contain included granules. They occur singly or as small aggregates in the vacuoles. In addition, vacuoles containing small violetstaining spheres, similar spheres with included granules and groups of the same are met with throughout the zone and especially in its deep region. THE DEVELOPMENT OF THE .\1ON()'l‘Rl*JMATA. 473

)-Vithin the cortical yolk-zone the central or medullary cytoplasm (m.z.) is constituted by a broad outer zone (vmz.) (about 0-14 mm. in thickness), which is richly and fairly uniformly vacuolated, and a central finely granular core (cmz.) (about 0-172 mm. in diameter) with only traces of vacuolation.

A comparable vacuolation of the central cytoplasm of the oocyte of the Fowl at the corresponding stage of vitellogenesis has been described by various observers~ Loyez (1906), van Durme (1914), B. Konopacka (1933), Marza and Marza (1935). According to Konopacka, the vacuoles are occupied by clear homogeneous droplets composed of very liquid protein. She regards these droplets as constituting the actual primordia of the yolk-spheres.

The nucleus is now ovoidal in form, and has attained its definitive peripheral position. In the nuclear reticulum there are present ill-defined pseudo-reticular strands, a large nucleolus, and numerous nucleolar granules.

Marza and Marza (1935, p. 165) comment on the fact that in the Fowl’s oocyte the follicular epithelium attains a great thickness at the time the above-mentioned clear vacuoles become numerous in the cytoplasm, but this does not seem to hold good for the follicular epithelium of the Platypus oocyte, which at this stage has a thickness of only 0-0056 mm. In two Echidna oocytes (0-60 and 0-63 mm. in diameter respectively), on the other hand, the follicular epithelium reaches a thickness of 0-014 mm., whilst in a third with a diameter of 0-625 mm. it averages 0-010 mm. These oocytes are definitely more advanced in yolk~formation than the

Platypus oocyte under description.

Platypus Oocyte. Diameter 0-66 ><0-61 mm.

The periphery of a slightly larger oocyte from the same ovary as the preceding is depicted in P1. V. fig. 22. This oocyte measures 0-66><0-61 mm. in diameter, the nucleus 0-107 ><0-O5 mm. in diameter, the follicular epithelium 0-003 mm., and the zona 0-0056 mm. in thickness.

The cortical yolk-zone, averaging about 0-068 mm. in thickness, is even more conspicuous than in the last oocyte, owing to the larger size attained by the yolkspheres (maximum diameter 0-017 mm.). Specially noteworthy is the fact that many of the larger spheres are now more or less completely enclosed by a narrow definitely contoured layer or “ mantle ” (often thicker on one side), composed of finely granular eosinophil material. Once laid down, it constitutes an integral part of the yolk-sphere and persists throughout later stages, when the spheres are no longer enclosed in discrete vacuolar spaces. It apparently corresponds to what Brambell (1925) in the oocytes of the Fowl has termed the M.C. yolk (mitochondrial-ground-cytoplasmic yolk), and which he believes is derived from the contents of the vacuole surrounding the yolk-sphere. Konopacka (1933) describes the white yolk-spheres of the Fowl as “ globules vitellins contenant des globules proteins-phosphatiques.” Her vitelline globule corresponds to our “ mantle ” layer, and her protein-phosphatide globule to what we call the “ yolk sphere ” (vide Konopacka’s pl. iv. fig. 14). On the basis of Konopacka’s observations we recognise that our terminology is inexact, as the whole yolk—structure should be called the yolk-sphere, but we shall continue to use it as a matter of convenience.

It is also worthy of mention that in this oocyte eosinophil yolk-sphere primordia are not recognisable as such in the cortical zone. Owing, no doubt, to some alteration in their chemical constitution, they have become converted into ordinary basophil spheres, though the very small spheres and some of the larger are still apparently in a transitional condition, staining violet with haematoxylin instead of deep black as do the large spheres. Vacuoles each containing a number of these small light-staining spheres are not uncommon. 2


We may interpolate here a brief account of the egg—envelopes of the two Platypus oocytes under description as illustrative of their condition at this stage of growth (Pl. V. figs. 21 & 22).

The follicular epithelium (f.e.) is relatively thin, measuring 0-0056 mm. in the smaller of the two and only 0-003 mm. in the larger, and is composed of a single layer of cubical cells, with indistinct cell-limits, and their oval nuclei crowded together and disposed with their long axes tangential to the zona. The zona (zp.) has a thickness of 0-0056-0-006 mm. It appears as a perfectly homogeneous membrane, which stains intensely with haematoxylin, and shows not the slightest evidence of the passage through it of cytoplasmic fibrils derived from the follicular cells, as is said to be the case in the Reptilian oocyte (Loyez (1906), Thing (1918)), and also in that of higher mammals (van der Stricht (1923), Mjassojedoff (1923)).

Within the zona the striate layer (s.l.) (ante, p. 464) is clearly visible in P1. V. figs. 21 & 22 (its thickness in the former figure being about 0-002 mm.), but it has become separated from the zona through the appearance of a narrow cleft, clearly an artefact, due to shrinkage. As the result of this artificial separation the layer is seen to comprise a very thin but perfectly definite wavy membrane, forming the inner boundary of the contraction space, in addition to the very delicate fibril-like strands which constitute its main thickness. Whether these latter are true fibrils or not is difficult to determine, meantime we shall refer to them as “ fibrils.” At their outer ends they are directly continuous with the wavy membrane and at their inner ends with the egg-membrane, and as testimony to the structural continuity of these parts we may mention that we have observed the striate layer (wavy membrane and “ fibrils ”) together with the egg-membrane separated off as a single band-like formation, lying quite free in the space between the zona and the superficial cytoplasm of the oocyte.

The egg-membrane, though pellicle-like when first observable, does appear to acquire in the Monotreme oocyte the status of a definite detachable membrane.

In well-fixed oocytes the membrane of the striate layer, closely applied as it is to the zona, is usually difiicult to distinguish, but in favourable sections, after staining with haematoxylin and eosin, it can be seen as a thin dark line, stained by the haematoxylin, on the inner surface of the red-stained zona, and it may also be demonstrated by staining with J. F. Robinson’s triple stain (phloxine, orange, methyl-blue), when it colours of a blue tint, in striking contrast with the Vivid orange-red coloration of the zona. \Ve think it must be regarded as forming an integral part of the striate layer.

As concerns the so-called “ fibrils,” these are disposed for the most part radially and so give to the layer its characteristic striated appearance, but they are not always straight, frequently they are curved or disposed obliquely, and sometimes are connected with each other by one or other of their extremities. Moreover, in sections which pass obliquely to the zonal surface the layer no longer exhibits its striate character, but presents the appearance of a thin vacuolated layer, the apparent vacuoles being separated by thin partitions, presumably formed by the “ fibrils.” We are accordingly inclined to doubt whether these latter are really independent fibrillar structures.

In addition to the outer membrane and the “ fibrils,” the striate layer comprises a third constituent, viz., a non-staining homogeneous matrix substance which l.ies between the “ fibrils ” and which is comparable with the fundamental substance described by Thing (1918) as present in the inner layer of the zona pellucida (our striate layer) in the oocytes of Turtles.

Loyez (1906) gives an account of the structure of the membranes which enclose the early oocyte in Reptiles and Birds. In agreement with the majority of previous workers, she describes the presence of two membranes round the oocyte, an outer finely striate membrane, which she designates the vitelline membrane and which corresponds to our zona, and an inner more coarsely striated membrane, the zona radiata, which corresponds to our striate layer. Although she points out that the striae of the latter are not in continuity withthose of the vitelline membrane, being less numerous, wider apart, and more readily visible, she still seems to think that the striae in both layers are cytoplasmic, in View of the existence of thin filamentous prolongations of the large (glandular) follicular cells in lizards and snakes, which pass through definite pores in the two layers to reach the surface of the oocyte. She attempts to trace the origin of these two layers, but her account (p. 63) is quite unconvincing.

Thing (1918), in the oocytes of Chelonia, recognises a zona pellucida, distinguishable into an outer denser and thicker layer (our zona) and an inner narrower clearer striated layer (our striate layer), and, like Loyez, she considers that both layers are penetrated by filamentous prolongations of the follicular cells which are connected with the surface of the oocyte. In her view, both layers take origin from the intercellular cement-substance secreted by the follicular cells, the inner layer representing the oldest part of the zona and being originally composed of the same substance as the outer layer. ‘

We have shown (cmte, p. 463) that the striate layer only appears after the zona has been laid down as a continuous membrane and at a time when the eggmembrane is already distinct. It seems to be formed from the transparent material which occupies the narrow space between the zona and the egg-membrane, but whether this material really represents the inner portion of the follicular secretion which also furnishes the zona, as Thing believes, or whether it is to be regarded as a secretory product of the cytoplasm of the oocyte, we are unable to decide. In this connection it is perhaps worthy of mention that Mjassojedoff (1923) supports the view that the zona in the Cat is a joint derivative of the follicular epithelium and the egg-cytoplasm, but in no higher Mammal, so far as we are aware, has any layer comparable with the striate layer of the Monotreme and Reptilian oocyte ever been described.

Lastly, we would emphasise our conclusion that the so-called “ fibrils ” of the striate layer of the Monotreme oocyte are not formed by cytoplasmic fibrils prolonged from the cells of the follicular epithelium, and accordingly we are unable to accept the view of Gatenby (1922) that the “ cortical fibrillae,” which he was the first to describe and so name in the Platypus oocyte, act “ as living protoplasmic connexions between the nutrient bringing follicle and the receptive interior of the egg.”

Echidna Ooeyte. Diameter 065 X060 mm.

The immediately succeeding stage in the growth of the oocyte is exemplified by the two oocytes of Echidmz represented in Pl. IV. figs. 22 A & B. The first of these oocytes, a sectional View of which is shown in P1. IV. fig. 22 A, measures 0-65 ><0-60 mm. in diameter, and is thus of practically the same size as the preceding Platypus oocyte (Pl. IV. fig. 20), but is definitely more advanced in respect of yolk-formation, since the vacuoles of the outer portion of the medullary cytoplasm now contain yolk-sphere primordia as well as definitive spheres, though in no great number. The zona is 0-004:3 mm. thick, whilst the follicular epithelium averages in thickness 0-10 mm. (maximum 0-012 mm.). The nucleus is much contracted and the germinal disc is poorly preserved, but the general disposition of the yolk can readily be made out from the figure.

Forming the periphery of the oocyte is a narrow zone of cytoplasm, corresponding to that of P1. IV. fig. 20, and devoid of yolk-spheres. It passes over internally into the coarsely vacuolated cytoplasm of the yo1k—containing zone, which has a width of about 0-18 mm. and encloses the central cytoplasmic core, measuring 0-20 X0-19 mm. in diameter. The core is composed of finely alveolar cytoplasm, except round its periphery, where it is coarsely vacuolated, the vacuoles each containing one or sometimes two minute basophil yolk-granules (Pl. IV. THE DEVELOPMENT OF THE MONOTREMATA. 477

fig. 22 B, lg/.z.). This vacuolated periphery constitutes the primordium of the latebral yolk-zone as we term it, which is definitely established in the succeeding Platypus oocyte (Pl. IV. fig. 23).

The yolk-zone (y.z.) is distinguishable into an outer part characterised by the presence in its vacuoles of deeply staining basophil yolk-spheres and an inner part, the vacuoles of which are mainly occupied by much smaller, light-staining yolksphere primordia, though small basophil spheres also occur in it in small numbers. The outer part of the zone occupies the site of the cortical yolk-zone of the preceding oocyte, but owing to the formation of yolk-sphere primordia in the medullary cytoplasm lying immediately internal to it, the cortical zone, though still distin~ guishable, no longer exists as a separate entity. If Pl. IV. fig. 22 A be examined, it will be seen that the nucleus is surrounded peripherally by deeply staining basophil yolk-spheres, but such spheres, it will be noted, are not present below the central region of its lower surface. Instead of extending across below the nucleus, the basophil spheres are seen to continue directly downwards to the periphery of the central core. In this way they form the boundary of a narrow axial tract of vacuolated medullary cytoplasm (l.'r1,.) running down from below the nucleus to join the central core. In the vacuoles of this tract there are present minute basophil yolk-spheres identical with those in the peripheral vacuoles of the central core, and, although a few somewhat larger spheres are also present, spheres of the size of those in the outer part of the yolk-zone do not occur in it. This tract is the primordium of the neck of the latebra which, like the latebral yolk-zone already mentioned, is clearly marked in the succeeding Platypus oocyte (Pl. IV. fig. 23, l.n.).

Echidna Oocyte. Diameter 0-64 X0-56 mm.

The details of the yolk-zone are shown under higher magnification in P1. IV. fig. 22 B, taken from another oocyte in the same ovary as the above and of the corresponding stage of growth. It measures 0-64 X0-56 mm. in diameter. The zona has an average thickness of 0-0078 mm. and the follicular epithelium a thickness of 0-014 mm. It is now one to two cells thick. The egg-membrane is exceptionally distinct, and the striate layer is also well marked. '

The peripheral cytoplasmic layer (p.c.z.) is thin and poorly marked off from the underlying finely vacuolated layer, which merges without limit into the much more coarsely vacuolated cytoplasm of the yolk-zone. Both layers contain numbers of very fine refractive granules of unknown significance, which tend to be aggregated together in little groups.

The cytoplasm of the yolk-zone takes the form of a finely granular network, composed of Well-marked thickish strands and enclosing vacuolar spaces, which are larger in its outer part, Where they are occupied by the large definitive yolkspheres, than in its iimer part, in which the small immature spheres mainly occur. The definitive yolk-spheres vary in size from quite small spheres to large spheres 0-011 mm. and more in diameter (maximum 0-014 mm.). They appear perfectly homogeneous, stain deeply and uniformly with haematoxylin, and are usually situated singly in the vacuoles, although occasionally two, three, or more small spheres of uniform or varying size may occur together in one vacuole. The larger spheres‘ as well as some of the smaller possess enclosing mantle—layers (wide p. 473) composed of a lightly staining, very finely granular material in which the sphere is excentrically situated. The large definitive spheres with intermingled small spheres occupy, as already indicated, the peripheral part of the yolk-zone. Within this is a region occupied by small definitive spheres, and Within this again is the innermost part of the zone, containing almost exclusively immature spheres. We may conclude accordingly that in oocytes at this stage of growth yolkformation proceeds, in the main, from without inwards, 23. e., in the centripetal direction.

The immature spheres (yolk-sphere primordia, yap.) stain of a light dull reddish tint after haematoxylin and eosin, and are definitely smaller than the definitive spheres, the great majority of them measuring from about 0-0056 to 0-007 mm. in diameter, but they vary not only in size but in form and appearance. What we take to be the youngest primordia occur in the vacuoles adjoining the vacuolated periphery of the central core (ly.z.). These appear as faintly staining spheres, mere shadows, with a light centre and a darker periphery, and measuring from 0-0035 to 0-0056 mm. in diameter. Then we find throughout the zone slightly larger spheres which stain uniformly and rather more deeply with eosin, and intermingled with these, spheres containing granules which stain more deeply than the matrix of the sphere, but with varying degrees of intensity, and which vary in number and size in the different spheres. Thus we meet with spheres containing numerous fine granules, spheres with fewer and coarser granules, which may be of fairly uniform size, or they may vary in size, a large spherical granule being accompanied by smaller ones, or there may be only a single large spherical granule occupying most of the matrix. Also to be met with are larger bodies of irregular form, composed of several small spheres containing granules, aggregated together.

The transformation of these primordia into definitive spheres appears to proceed along two slightly different lines. Primordia containing a single large granule, as well as those with numerous minute granules dispersed through the matrix, seem to become transformed more or less directly into small spheres, which stain deeply and uniformly with haematoxylin. On the other hand, primordia which contain a small number of coarser granules in their matrix behave quite differently. As they increase in size, so also their granules enlarge, and from being eosinophil they begin to stain with haematoxylin. Finally, by the time the granules have attained some size and have acquired the capacity to stain deeply and uniformly with haematoxylin, the matrix-substance of the primordium has disappeared, possibly having been utilised in the growth of the granules, and so the latter, now isolated, form a more or less loose aggregate of small yolk-spheres, occupying the interior of a vacuolar space. They may be fairly uniform, or may vary considerably in size. In this way, a single primordium is capable of giving origin to a number of yolk-spheres (up to six or more).

The periphery of the central cytoplasmic core (which in this oocyte measures 0-16 ><0-15 mm. in diameter) is occupied by a ring of conspicuous vacuoles (lg/.z.), arranged two or three deep, which contain very fine spherical basophil granules (about 0-0021 mm. in diameter), usually one, sometimes two, granules occurring in each vacuole. This ring, as already mentioned, marks the site of the future latebral yolk-zone, and its early differentiation may perhaps be regarded as indicative of the important part this zone is destined to play in the growth of the

oocyte. Sub-phase B.

This sub-phase, as already indicated, includes oocytes ranging from about 0-8 mm. in diameter up to the full-grown condition. In the earliest oocytes belonging to this sub-phase, definitive yolk-spheres are present in the outer zone of vacuolated medullary cytoplasm, and so a continuous yolk-zone is established, in which the original cortical zone is incorporated. The central core of medullary

cytoplasm undergoes vacuolation and, concomitantly, the latebra with its latebral.

yolk-zone is definitely established and becomes active in the production of small yolk-spheres which are added in the centrifugal direction to the yolk-zone, with the result that the latter increases in thickness, and so the oocyte as a whole grows in diameter. The yolk—bed is formed relatively early below the nucleus and the germinal disc-primordium. The latter increases in area and in thickness and gives origin relatively late to the definitive disc of the ovarian oocyte, composed of a superficial formative layer and a deep vacuolated layer rich in fine yolk-granules. As the oocyte grows, so the nucleus increases in size and assumes at first a plano— convex and eventually a saucer-shaped form. It is rich in nucleolar granules, and in the full-grown oocyte extremely fine linin threads, supporting minute chromatin granules, make their appearance in it. During this phase yolkformation attains its maximum, and consequently it is the phase of most active growth of the oocyte.

Platypus Oocyte. Diameter 0-86 ><0-81 mm.

The earliest oocyte of this sub-phase we have examined is the Platypus oocyte illustrated in P1. IV. fig. 23 and P1. V. fig. 24. It measures 0-86><0-81 mm. in diameter, and has already been described and figured by Gatenby (1922, pl. xiii. fig. 4 D). Although only about 0-2 mm. larger in diameter than the immediately preceding oocytes (Pl. IV. figs. 20 & 22 A), it is strikingly in advance of these. The nucleus measures 0-098 ><0-051 mm. in diameter. The follicular epithelium is about 0-007 mm. in thickness (average) and the zona 0-0056 mm. The striate layer is distinct, but very thin over the disc. '

The peripheral cytoplasmic layer (averaging about 0007 mm. in thickness) now contains numbers of small yolk-spheres. These become sparse as the layer begins to thicken to form the germinal disc-primordium (gd.p.), and are not found in its main central portion. The disc has increased in area and in thickness, and its peripheral limits are rather better defined than in the preceding oocytes. It has an approximate diameter of 0-21 mm. and a thickness over the nucleus of 0-020 mm.

The nucleus (Pl. V. fig. 24) lies in contact with the under surface of the central portion of the disc, and is no longer spherical but ovoidal, its long axis being disposed tangentially to the surface. In the fine nuclear reticulum are situated the irregular, poorly defined strands of the pseudo-reticulum containing deeply staining granules, and also a large vesiculated nucleolus (0019 mm. in diameter) and a number of smaller nucleolar granules.

The body of the oocyte (Pl. IV. fig. 23) inside the thin peripheral layer consists of a broad zone (y.z.) (averaging about 022 mm. in width), containing conspicuous yolk-spheres and a central coarsely vacuolated core (l.b.) (0-37 ><0~34 mm. in dimeter) entirely devoid of yolk. As we shall show, the latter is destined to form a with its surrounding yolk-zone the body of the latebra.

The broad yolk~containing zone, as inspection of P1. IV. fig. 23 demonstrates, does not form a complete circle, but is interrupted at the upper pole owing to the presence of the nucleus and so is horseshoe-shaped. Furthermore, it is evident from the figure that it is not a single uniform formation, but really consists of two concentrically arranged parts, viz., (a) a broad outer region (y.z.) comprising roughly about three-quarters of its thickness, characterised by its coarsely vacuolated cytoplasm and its content of large yolk-spheres, and (b) a narrow inner region (ly.z.) forming the remaining quarter, which surrounds the central coarsely vacuolated cytoplasm (except over a small area on its upper side), and which consists of more finely vacuolated cytoplasm containing numerous yolk-spheres much smaller than those of the outer region. The outer region is evidently the same as the yolk-containing zone of the preceding Eckidna oocyte, which we have spoken of as the “ yolk-zone,” whilst we shall distinguish the inner region as the “ latebral yolk-zone.” Its primordium, as we have seen, is already recognisable in the preceding oocyte of Echidna (Pl. IV. fig. 22 B). The yolk-zone comprises the original cortical yolk together with the later~formed medullary yolk. With the additional yolk added to it during the growth of the oocyte, it is destined to form the main bulk of the yolk-mass of the full-grown oocyte. The latebral yolk-zone includes yolk which has been formed at the periphery of the central vacuolated core of the oocyte, and as Pl. IV. fig. 23 shows it is prolonged up to enclose the latebral neck. With its parent matrix, the cytoplasmic THE DEVELOPMENT OF‘ THE .\'l0l\70'I‘R-EMATA. 481

core, it persists throughout the growth-period of the oocyte and is still recognisable in the uterine egg. It is the source of the additional yolk which is added to the yolk-zone during the growth of the oocyte, as we hope to demonstrate in the sequel. The latebral yolk-zone, as its name implies, forms an integral constituent of the latebra, the parts of which, already indicated in the preceding Echidna oocytes, are now definitely established. I

The occurrence of a latebra in the Monotreme egg has been recorded by previous observers (Caldwell (1887), Semon (1894), Gatenby (1922), Gatenby and Hill (1924)), and has been shown to have much the same form and relations as that of the Sauropsidan egg. We regard the latebra as a formation of fundamental importance from the point of View of yolk-formation and the growth of the oocyte. It represents a localised persistent portion of the medullary cytoplasm, occupying the central region of the oocyte and the upper half of the egg-axis directly below the germinal disc and nucleus, in which only fine yolk-spheres are produced. In its completed condition, as seen in later oocytes, it appears as a club-shaped formation composed of a centrally situated expansion, the latebral body, and of a thin stalk continuous with the same and constituting the latebral neck which runs up in the egg-axis to terminate in the saucer-shaped yolk-bed underlying the germinal disc. The latebral body consists of vacuolated cytoplasm distinguishable into a central core, and an enclosing layer rich in fine-grained yolk, which forms the above-described latebral yolk—zone, the latter merging on its outer side into the surrounding yolk-mass. The neck has a similar constitution, consisting of a cord of more or less vacuolated cytoplasm richly laden with very fine yolk-spheres and surrounded by a continuation of the latebral yolk-zone.

Here inthis oocyte the future latebral neck (l.n.) is represented by a tract of vacuolated cytoplasm (about 0-128 mm. in height and 0-14 mm. in Width), which leads down from below the nucleus to become continuous with the more coarsely vacuolated central cytoplasm. This latter is destined to form the central core of the latebral body, and is already surrounded by the latebral yolk-zone as above described. As concerns its formation, the latebra, as we have already remarked, is simply the persistent axial portion of the medullary cytoplasm lying directly below the nucleus, in which, as Gatenby (1922) has pointed out, coarse yolk-spheres are never formed. In fig. 20 (Pl. IV.) of the preceding oocyte it will be seen that the continuity of the cortical yolk-zone is interrupted by the nucleus, whilst the vacuolated medullary cytoplasm continues uninterruptedly below the latter. Now, in the later stage, fig. 22 A (Pl. IV.), when yolk-formation progresses inwards from the cortical zone, it does so in such a way as to leave an axial tract of vacuolated cytoplasm running vertically downwards from below the nucleus, entirely free of coarse spheres, and so in this way the latebral neck is defined right down to its junction with the body, represented by the vacuolated central core of the oocyte. 482 PROF. T. TH,()Nl.\‘()N FLYNN AND PROF. J. P. HILL ON

The yolk in this oocyte is definitely in advance of that of the preceding Echiclna oocytes, inasmuch as definitive spheres are now present throughout the yolkzone, but between them, especially in the inner part of that zone, there are still present numbers of yolk-sphere primordia in various phases of differentiation. The majority of the spheres stain deeply and uniformly with haematoxylin, but there are also present in small numbers spheres which, instead of being homogeneous, are densely packed with refractive spherules. They vary in size from conspicuously large spheres measuring up to 0-034 mm. in diameter to quite minute spheres 0-004 mm. in diameter. Most of the larger spheres are enclosed in well-developed mantle-layers.

The yolk-sphere primordia are similar to those of the preceding oocyte of Echidna, and comprise small eosinophil spheres (0-006 to 0-008 mm. in diameter), some homogeneous, others containing granules, larger spheres (up to 0-013 mm. in diameter) with violet-staining granules, and still larger spheres (0-017 mm. in diameter) with light-staining centres and peripherally situated basophil spherules which may reach a diameter of 0-004 mm., and often smaller Violet-staining spherules as well. As in the preceding oocyte, so here, these basophil spherules, increasing still further in size, eventually become free on the disappearance of the matrix-substance, and so we find here and there in the inner part of the yolkzone vacuoles containing groups of small yolk-spheres, each group the product of a single parent sphere, so that here also we meet with the same multiple production of small yolk-spheres from a single primordium that we encountered in the preceding oocyte of Echidna. Not all the eosinophil primordia, however, take this course, others of them become transformed directly into basophil spheres.

The latebral yolk-zone (Z7/.z.), now, as we have seen, definitely established, has already taken on its yolk-forming function, although it is only in later oocytes that it attains its full activity, and it is an interesting fact that it is in this zone that the mode of vitellogenesis described above, viz., the production by a single primordium of a number of yolk-spheres, is most strikingly manifested. It is, unquestionably, the chief method of yolk—formation in the latebral region."

The definitive spheres of the zone are small and of the deeply staining homogeneous type, and merge without limit into those of the yolk-zone. They Vary in size from spheres with a maximum diameter of about 0-017 mm. down to quite minute spheres 0004 mm. in diameter, and it is significant that the smaller spheres exhibit a marked tendency to occur in groups, each group in a vacuolar space of its own, just like the similar groups in the yolk-zone. Eosinophil yolk-sphere primordia (0006 to 0-008 mm. in diameter) are present throughout the zone, but in this oocyte are not very numerous, and are more abundant in its continuation around the latebral neck. Along with them occur rather larger primordia (about 0-01 mm. in diameter) with granules in their matrix-substance, usually arranged peripherally and varying in size and in their staining reactions; they may be eosinophil or they may stain in varying degrees of intensity with haematoxylin.

Yolk-vesicles.~In addition, there are present throughout the zone, and especially in its nner portion Where it merges into the vacuolated latebral core, highly characteristic primordia which we propose to term “ yolk-vesicles.” Such “ vesicles ” are also present in the vacuolated cytoplasm of the latebral neck, where indeed they attain their richest development (fig. 24, 3/v., Pl. V.). They are rather remarkable structures, distinctive of the latebra of the oocyte of Platypus and not normally found in that of Eckidna, and, so far as we know, nothing quite like them has ever been described. In reality, they are vesicular yolk-sphere primordia, specially adapted for the production of relatively large numbers of small yolk-spheres. In section (fig. 24, 3/22., Pl. V.) they appear as vesicular bodies, more or less spherical and with smooth contours, which are situated in vacuoles just like the other primordia. In the latebral yolk-zone they vary in diameter from about 0-013 to 0-021 mm., but in the vacuolated cytoplasm of the neck, where they are richly developed (fig. 24, Pl. V.), they attain a somewhat larger size, their diameter being mostly about 0-025 mm., but exceptionally they may reach a diameter of 0-030 mm. They appear to be bounded by an extremely delicate pellicle-like layer, and on the inside of this are situated in some Vesicles fine, slightly eosinophil yolk-spherules, arranged one or two deep, in other vesicles somewhat larger basophil spherules are found intermingled with them, and in yet others the spherules are all basophil and some of them may reach a diameter of 0-004 mm. The interior of the vesicle, no doubt fluid-filled during life, either appears empty or it may contain small numbers of free eosinophil or basophil spherules.

These basophil spherules represent the final product of the yolk-vesicles, and are destined to furnish the groups of small yolk-spheres that occur in the vacuoles of the latebral yolk-zone, as well as the fine spheres that are characteristic of the latebral neck. In this connection it is worthy of mention that the spherules in the vesicles of the latebral yolk-zone attain a larger size and are less numerous than those of the vesicles of the latebral neck, which latter, it will be remembered, are larger than the former. '

As concerns the origin of the yolk-Vesicles they would seem to be formed by the enlargement and vacuolation of the granule-containing eosinophil primordia that are present in the zone, and in the immediately succeeding oocytes we have observed what we regard as transitional stages between the two.

It remains to be mentioned that the latebral neck is surrounded by What appears as an upward continuation of the latebral yolk-zone, in which are present small yolk-spheres similar to those in the latter, occasional small yolk-vesicles, and numbers of eosinophil yolk—sphere primordia.

As can be seen in fig. 24 (Pl. V.) the coarsely vacuolated cytoplasm of the latebral neck reaches up to within a short distance of the nucleus, where it passes into a thin layer of more finely vacuolated cytoplasm (yb.p.) (containing sparse fine yolk-spheres), on which the nucleus rests and which extends up around the nucleus into continuity with the granular cytoplasm of the disc. This layer marks the site of the future yolk~bed.

Platypus Oocyte. Diameter 1-10 x 1-0 mm.

We pass next to an oocyte 1-1 X 1-0 mm. in diameter, the latebra of which is illustrated in fig. 25 (Pl. V.) and fig. 26 (Pl. VI.). In its general configuration it is very similar to the preceding one, but the latebral yolk-zone is now thicker and richer in yolk-spheres.

The germinal disc primordium has increased in area, its diameter being 0-24 mm. and its thickness over the nucleus 0-02 mm. The nucleus (diameter 0-136 mm., thickness 0-06 mm.) is now definitely plano-convex in form (of. fig. 27, Pl. VI.). The zona has a thickness of 0-0043 mm. (striate zone 0-O02 mm.) and the follicular epithelium a thickness of 0-0087 mm.

The spheres of the yolk-zone exhibit the same dispersed arrangement as in the preceding oocyte, but are more numerous and slightly larger, their average diameter (exclusive of their mantle-layers) being about 0035 mm., though some reach 005 mm. The enclosing mantle-layers of the large spheres are thick and not infrequently are composed of a light-staining finely granular material, suggestive of a coagulum (Pl. V. fig. 25). The delicate cytoplasmic reticulum between the spheres is well marked, and in it occur numbers of quite small spheres. These become more numerous as the latebral yolk-zone is approached and are specially abundant in that zone itself.

The central core of the latebra (Pl. V. fig. 25, l.b.), still circular in outline, is very similar to that of the preceding oocyte and of about the same diameter (0-35 mm.). Its latebral yolk-zone (lg/.z.), however, is more prominent, owing to its greater richness in yolk-spheres. These comprise deeply staining spheres varying in size from small (0017 mm. in diameter) to minute (0-003 mm. and less), the larger spheres lying towards the outer side of the zone, as well as numbers of eosinophil spheres (referred to below), whilst yolk-vesicles are present in its inner part adjoining the core.

The latebral neck (l.n.) has increased in length, now measuring about 0-28 mm., but its width is much the same as in the preceding oocyte. It is now also richer in yolk. In addition to fine yolk-spheres, there are present in its lower part numerous yolk-vesicles reaching up to 0-21 mm. in diameter, and specially abundant at and just above its junction with the central core (Pl. VI. fig. 26, 3/12.). Their contained spherules vary in size from quite minute granules up to basophil spheres 0-004 mm. in diameter. Its surrounding latebral yolk-zone (lg/.2.) contains, besides small yolk-spheres, numbers of homogeneous eosinophil spheres (about 0008 mm. in diameter), similar spheres with peripheral granules staining more deeply than the matrix, slightly larger vesicular spheres (about 0-012 mm. in diameter) with coarser peripheral granules, some of them staining a violet tint, and what We take to be transitional stages between these latter and typical yolk-vesicles. The cytoplasm of the upper part of the neck is more finely vacuolated than the remainder, and contains mostly fine basophil spheres. As in the preceding oocyte it passes over without a break into the vacuolated cytoplasmic bed containing similar fine yolk-spheres, which underlies the nucleus and surrounds its periphery, but the yolk-spheres are not yet sufficiently abundant to justify us in speaking of a yolk~bed. Such a bed is, however, definitely indicated in P1. VI. fig. 27, yb.p., representing another Platypus oocyte of this same stage and measuring 1-09 ><0-96 mm. in diameter. In this oocyte, as a curious variation, the latebral neck (l.n.) is exceptionally thick and yolk-formation has been retarded, and so We can see quite clearly that the yolk-spheres of the yolk-bed originate in situ, quite independently of those of the latebral neck. The yolk~bed accordingly cannot be regarded as a mere expansion of the upper part of that structure as it appears to be when fully established.


Echidna Oooyte. Diameter 1-0 ><0~95 mm.

The structure of the periphery of the oocyte and the egg-envelopes at this stage of growth is illustrated in P1. IV. fig. 28, taken from an Echidna oocyte l~0><O-95 mm. in diameter, fixed in F.W.A. The follicular epithelium (f.e.) has a thickness of 0-015 mm. and is thus much thicker than that of the abovedescribed Platypus oocyte. It is composed of a single layer of columnar cells, their broader basal surfaces resting on the thin membrana propria and their narrower apical surfaces on the zona (21).). The spherical or oval nuclei are situated in the thicker basal portions of the cells, whilst Well-marked intercellular spaces occur between their thinner apical portions. Occasionally cells are met with in mitosis, the plane of division being tangential to the zona so that the daughter cells are super-imposed (Pl. IV. fig. 28).

Of special interest from the point of view of yolk-formation is the occurrence in the cytoplasm, especially of the apical portions of the cells, of numbers of very fine fat-granules, Whilst similar but somewhat coarser granules are present in the cytoplasm of the thecal cells (th.i.) as weH as in the cytoplasmic reticulum between the yolk-spheres. Since fat-granules cannot pass through the zona, We must suppose they are broken down in the follicular cells and the difiusible products, passing through the zona, are reconstituted in the cytoplasm of the oocyte and utilised in the building up of the yolk-spheres.

The zona appears perfectly homogeneous, and has a thickness of O-0045 mm. On its inner side is the very thin striate zone (s.l.) (O-0015 mm. thick).

The peripheral cytoplasmic layer (p.c.z.) of the oocyte, containing small yolkspheres, is distinct, and is seen to be connected at intervals with the very delicate cytoplasmic reticulum, which lies between the large yolk-spheres and in which are situated numbers of fat-granules, larger than those of the follicular epithelium and often aggregated into little groups.

The follicular wall of the Platypus oocyte of corresponding size (10 X0-97 mm. in diameter), but fixed in PNO, has an essentially similar structure, except that fat- granules are not preserved and the follicular epithelium, measuring 0-0086 mm., is much thinner.

The thecal investment of the follicle in these oocytes is distinguishable into a fibrous theca externa, the fibroblasts of which possess elongated flattened nuclei and a theca interna, composed in Platypus of several layers of small flattened cells with ovalish nuclei. It contains sparse capillaries, and is much better developed in Platypus than in Echidna.

Platypus Oocyte. Diameter 1-35 x 1-14 mm.

VVe call attention here to P1. VI. fig. 29, which represents a low power View of a horizontal section through an oocyte of the stage of growth under consideration and measuring 1-35 ><l-14 mm. in diameter. The figure shows very clearly the dispersed arrangement of the yolk-spheres of the yolk-mass (y.z.) at this stage, the clear vacuolated cytoplasm of the body of the latebra (l.b.), and, in particular, the remarkable richness of the latebral yolk-zone (ly.z.) in fine yolk-spheres, and the Way they extend outwards to intermingle with the coarse spheres of the yolk-mass, this extension being here much more marked than in the oocyte above described.

Platypus Oocyte. Diameter 1-90 X 1-80 mm.

The next oocyte in our series, measuring 1-9 X 1-8 mm. in diameter, exhibits a progressive advance on the preceding in its yolk-content and in the condition of the latebra.

The formative cytoplasm of the disc shows a further increase in surface area, with a slight decrease in thickness, measuring 0-27 mm. in diameter><0-012 mm. in thickness. The follicular epithelium is slightly reduced in thickness to 0-007 mm., but the most noteworthy reduction is in the zona, which has now a thickness of only 0-0014 mm., as compared with 0-0043 mm. in the preceding oocyte. It looks as if the follicular epithelium had suddently slowed up its production of secretion, and so the zona, as the result of the increase in size of the oocyte, had undergone stretching and consequently thinning so as to cover a larger area.

The nucleus, rather obliquely cut, appears roughly triangular in section, its broad base adjoining the disc cytoplasm. It rests on a thin, slightly vacuolated yolk-bed containing numerous, mostly very fine, eosinophil granules identical with those in the upper part of the latebral neck, which is more coarsely vacuolated than the yolk-bed. The peripheral cytoplasmic layer with its contained small yolk-spheres is thin, but distinct, and here and there is still connected by delicate strands with the cytoplasmic reticulum between the underlying large yolk—spheres, but this reticulum is now much reduced, and is only recognisable in places.

The yolk-spheres of the yolk-mass, though still well dispersed, are much more numerous than in the preceding oocyte. Round the periphery of the oocyte the spheres, two to three deep, are smaller on the average than those more deeply situated, some of which may reach a diameter of 0-04 mm. Around the latebral yolk-zone the spheres are again small, ranging in diameter from about 0-017 mm. down to quite small spheres 0-004 mm. in diameter. It is significant that these small spheres are much more numerous in the inner part of the yolk-mass than in its outer part, being specially abundant around and in the latebral yolk-zone.

The latebra (figs. 30 and 31, Pl. VI.) has now assumed its definitive club-shaped form, possessing an elongated narrow neck (l.n.) about 0-52 mm. in vertical height and 0-05 mm. in width, and a body (l.b.) no longer spherical, but pyriform. Its central core of vacuolated cytoplasm, 0-36 mm. in vertical height and 0-23 mm. in transverse width, is somewhat reduced as compared with that of the preceding oocyte, whilst its neck is much in advance of that of the latter, and is now a definitely organised formation. It consists of a narrow tract of cytoplasm containing coarse vacuolar spaces, and densely laden with Very fine, mostly eosinophil yolk-granules. It is surrounded by a zone of fine-grained yolk continuous below with the latebral yolk-zone. The body of the latebra and its junctional region with the neck are shown in section in fig. 30 (Pl. V1,), whilst the detail of a tangential section of the latter region is shown under higher magnification in fig. 31 (Pl. VI.). Round the periphery of the central cytoplasm, the vacuoles are much larger than elsewhere, and many of them contain yolk-Vesicles, mostly with small eosinophil spherules. The latebral yolk-zone itself (lg/.2.) has much the same constitution as in the preceding oocyte, but is definitely richer in small yolk-spheres. Pale-staining basophil spheres varying in size from 0-002 mm. upwards are numerous in its inner part adjoining the central cytoplasm, and here also occur numbers of homogeneous eosinophil spheres, 0-008-0-011 mm. in diameter, as well as what we regard as further developmental stages of these, viz., spheres with peripheral granules, Vesicular spheres, and large yolk-Vesicles (yv.) with walls formed of eosinophil, as well as coarser light-staining basophil spherules. The outer part of the zone, as indicated above, contains numerous small and medium-sized basophil spheres, and merges without limit into the yolk-mass. The continuation of the latebral yolk-zone which surrounds the neck has a similar constitution, but yolk-Vesicles (311).) are remarkably numerous round the junctional region (fig. 31, Pl. VI.).

Platypus Oocyte. Dia/meter 2-0 X 1-8 mm.

The next oocyte, 2-0><l-8 mm. in diameter, the latebra of which is seen in sectional View in fig. 32 (Pl. VII.), is only slightly in advance of the preceding one.

Measurements: nucleus 0-129><0-038 mm. ; formative cytoplasm of disc about 0-43 mm. in diameter ><0-011 mm. in thickness ; follicular epithelium 0-0058 mm., and zona about 0-0014 mm. in thickness (the latter slightly thicker over the disc).

The nucleus is now somewhat saucer-shaped, but with a prominent projection from the central region of its under surface. The slight concavity on its upper side is occupied by rather loose disc-cytoplasm, containing sparse fine yolk-spheres. They appear here for the first time in this position, and in later oocytes are constantly present. The formative cytoplasm of the disc shows a definite increase in area, and appears dense and rather coarsely granular. The yolk-bed below the nucleus is distinct, and contains numerous fine yolk-spheres similar to those in the latebral neck.

The central cytoplasm of the latebra (Pl. VII. fig. 32, Lb.) has much the same volume as in the preceding oocyte, but the neck (0-8 mm. in height >< about 0-04 mm. in width) is somewhat longer and narrower. The latebral yolk-zone (fig. 32, lg/.z., Pl. VII.) presents a more massive appearance than that of the preceding oocyte owing to the greater abundance in it of medium-sized spheres with a diameter of about 0-014 mm., whilst in between these are numerous quite small spheres 0003 mm. and less in diameter. The larger of these small spheres are basophil, the smaller eosinophil, whilst others possess a basophil core and an eosinophil periphery. In view of the relative abundance of these small spheres, it is interesting to find that in the latebral yolk-zone itself yolk-vesicles are now not nearly so conspicuous, and though they are still obvious in the junctional region (fig. 32, yo.) they are here also less numerous. In this region they contain, in addition to coarser basophil spherules, very large numbers of quite minute eosinophil spherules. Such spherules are also abundant in the cytoplasm of the latebral neck, and have, no doubt, been derived from the yolk-vesicles. Small yolkspheres are again fairly numerous between the coarse spheres in the inner part of the yolk-mass adjoining the latebra, but are very sparse in its peripheral region. The yolk-spheres of the yolk-mass average about 0-025 mm. in diameter (maximum about 0-036 mm.).

Platypus Oocyte. Diameter 2-70 ><2-50 mm.

Our next oocyte, measuring 2-7 ><2-5 mm. in diameter, shows definite progress towards the attainment of the full-grown organisation, especially as regards the yolk-mass, which is now markedly increased in bulk.

The follicular epithelium is thicker than in the preceding oocyte, averaging about 0-008 mm., but the zona is now reduced to an extremely thin membrane (i0-0001 mm.). The peripheral cytoplasmic layer with its fine yolk-spheres, though thin, is still quite distinct.

The yolk-mass, as stated above, shows a very definite increase in bulk as compared with the preceding oocyte, yolk-spheres of medium to large size being now more numerous and more compactly arranged than in the latter. Furthermore, it exhibits a very striking grouping of the yolk-spheres into two concentric layers or zones—an arrangement we have not hitherto encountered. This zonal arrangement results from the fact that the large spheres (averaging 0-03 mm. in diameter, maximum 0-048 mm.) occur in the inner part of the mass, where they form an inner zone of somewhat varying width surrounding the latebral yolk-zone, whilst the smaller spheres (averaging about 0.025 mm. in diameter) constitute a peripheral or outer zone, with a width varying round about 0-6 mm. We have encountered this zonal arrangement of the yolk in later oocytes of both Platypus and Echidna (cf. fig. 34, Pl. VII.), and although it is not always recognisable, we regard it as a phenomenon of considerable significance, as we hope to show.

The central core of the latebra (fig. 33, l.b., Pl. VII.) measures about 0°29>< 0-17 mm. in diameter, and is thus reduced as compared with that of the preceding oocyte, no doubt as the result of its gradual incorporation in the latebral yolkzone as the oocyte increases in size. Fine pale-staining yolk-spherules, possibly liberated from broken-down yolk-vesicles, occur in some of its more peripheral vacuoles. In the latebral yolk~zone, yolk-vesicles are sparse and quite small; they are also present, but in no great numbers, in the junctional region and base of the neck. The zone is crowded with small basophil spheres of varying size, intermingled with which are numerous minute faintly eosinophil spheles, and here again the basophil spheres extend well out amongst the coarse spheres of the yolkmass. The axial cytoplasm of the neck contains large vacuolar spaces and is densely laden with fine spherules. Its enclosing yolk-zone consists of small spheres similar to those of the latebral yolk-zone.

The oocyte just described, measuring as it does just over 25 mm. in diameter, is rather more than half-grown, and so far as the yolk is concerned may be regarded as a miniature of the full-grown oocyte with a diameter of slightly over 4 mm. Our observations lead us to conclude that the progressive increase in the size of the oocyte as a whole is due only in small part to the increase in size of the yolk-spheres first laid down in the cortical and medullary zones of the early oocyte, and that by far the greater part of its growth is to be ascribed to the progressive increase in the number of its constituent yolk-spheres until a maximum is attained. From the stage of the 1 mm. oocyte onwards throughout the greater part of the growth-period, the oocyte grows in size as the result of the addition to the yolk-mass of small spheres which themselves subsequently increase in diameter, and so the yolk-mass as a whole increases in bulk. The evidence we have brought forward in the preceding pages demonstrates, we think conclusively, that the seat of origin of these small spheres is to be found in the latebra (including its latebral yolk-zone). From the stage of the 1 mm. oocyte onwards, the primary function of the latebra is, in our opinion, that of yolk-production. It is the central yolk-factory of the oocyte.

Furthermore, if we consider the process of yolk-formation as a whole in the light of these conclusions, it becomes evident that we can distinguish two phases in the process, viz., a first or initial phase occurring in the earlier oocytes in which yolk-formation proceeds in the main from without inwards, 2'. e., centripetally, and a second phase, involving oocytes from about 1 mm. in diameter onwards, in which the process proceeds from within (from the latebra) outwards, i. e., centrifugally. Herein, we think, lies the explanation of the above-described zonal arrangement of the yolk.

VVe conclude our account of the growth of the oocyte with a brief description of the yolk in two later oocytes, viz., an oocyte measuring 3-2 X 3-1 mm. in diameter from Platypus XV, and a full-grown oocyte from Platypus oz, 19. 8. 01, with a diameter of 4-4 X 4-2 mm., the largest oocyte we have encountered in the Platypus ovary.

Platypus Oocyte. Diameter 3-20 >< 3-10 mm.

The follicular epithelium, germinal disc, and nucleus of the Platypus X V oocyte are described below (p. 498). P1. VII. fig. 34 is a low-power View of a section of the entire egg, from which it will be seen that we have here a second and very striking instance of the zonal arrangement of the yolk referred to above, a denser, more darkly stained central region being readily distinguishable from a looser, lighter—stained peripheral region. Closer examination shows that the more deeply staining character of the central mass is due in the main to the more compact grouping of its yolk-spheres as compared with the more dispersed arrangement of those of the peripheral zone, but partly also to the fact that the mantle-layers of the central spheres (stained a light Violet with the haematoxylin) are much better developed than those of the peripheral spheres (which appear shrunken), and so enhance the staining of the central mass. But, apart from these features, measurements show that the central spheres, as in the preceding oocyte, are slightly larger, on the average, than the peripheral (0024 mm. average diameter as compared with 0-023 mm.), whilst in the proximity of the latebra the coarse spheres there situated average about 0-032 mm. in diameter. It is evident then that we have here the same zoned configuraton of the yolk-mass that we encountered in the preceding oocyte, and it is significant that the width of the peripheral zone in the two oocytes is fairly comparable, its width in the present oocyte Varying from 0-4 to 0-6 mm. as compared with 0-6 mm. in the preceding oocyte. From these measurements we conclude that the main increase in the yolk-mass has occurred in the central area surrounding the latebra.

The latebra in this oocyte is much reduced as compared with that of the fullgrown oocyte (see below) and is situated excentrically in the central yolk-mass. The latebral yolk-zone also appears reduced, and there is no trace of yolk-vesicles, but the surrounding yolk for a distance of from 0-4 to 0-6 mm. outwards is permeated by large numbers of minute yolk-spheres evidently of latebral origin. THE l)E\'ELOPMENT OF THE .\lONOTREl\lATA. 4,91

Platypus Oocyte. Diameter 4-40 >< 4-20 mm.

In the full-grown oocyte (4-4 X4-2 mm. in diameter) the zoning of the yolk-mass is much less obvious than in the preceding oocyte, though in a full-grown oocyte of Echidna (B.21.7 .30) it is equally as striking as in the latter. The peripheral zone of the yolk-mass, roughly about 0-5 mm. in width consists of spheres averaging about 0-022 mm. in diameter and less regularly spherical than those of the central mass, which average about 0-024 mm. in diameter. The mantle-layers of the spheres in this oocyte appear to be greatly reduced, and particularly so in the case of those of the peripheral zone. Below the zona a row of minute yolk-spheres is present, but the peripheral cytoplasmic layer can hardly be said to exist.

The latebra, a horizontal section of which is shown in Pl. VII. fig. 35, is remarkably well developed. It consists of a light-staining cytoplasmic mass (l.b.) with a diameter of 0-28 ><O-25 mm., surrounded by a darkly staining latebral yolk-zone (ly.z.) about 0-028 mm. in width. The cytoplasmic mass is distinguishable into a central region (about 0-17 ><O-16 mm. in diameter) in which the cytoplasm is practically free from granules and is irregularly vacuolated, some of the spaces being quite large, and a peripheral zone, less coarsely vacuolated and containing numerous basophil spherules, mostly minute, though they increase in size towards its periphery. The latebral yolk-zone is crowded with fine-grained yolk-spheres, varying in size from about -003 to -005 mm. and upwards, the larger spheres extending outwards for a short distance between the adjoining coarse spheres of the yolk-mass.

In connection with the above-described zoning of the yolk-mass, We may mention here that we have occasionally met with oocytes in which the central zone of the yolk-mass is separated from the peripheral zone by a ring of small yolk-spheres. Such a ring (s.y.s.) is well seen in the photomicrograph of a section of a Platypus oocyte (2-18><2 mm. in diameter), reproduced as P]. VII. fi.g. 36. A possible explanation of this condition that occurs to us is that the ring marks a break in yolk-formation. We suggest that the small spheres of the ring represent the product of the latebral yolk-zone and originally lay in the periphery of that, a cessation in the growth of the oocyte then supervened and the small spheres failed to increase in size. Subsequently growth recommenced and the ring of small spheres was carried out to its mid-way position by the crop of new spheres originating from the latebral yolk-zone. Whether or not this be the true explanation, we regard the “ zoning ” of the yolk-mass as of considerable significance, inasmuch as we hold that the peripheral zone represents yolk originally formed from without inwards in the cortical and medullary zones of the early oocyte, Whilst the central zone represents yolk formed secondarily from the latebral yolk-zone and therefore laid down in the direction from within outwards.

Yolk- formation in late Oocytes of Echidna.

We have not followed the later stages in the growth of the oocyte in Echidna in detail, but, so far as we have observed, yolk-formation proceeds along the same lines as in Platypus, except for certain differences in the details of the formation of the latebral yolk, which we think are deserving of mention.

In Platypus, as we have seen, the body of the latebra is characterised by the presence of a central cytoplasmic core, uniformly coarsely vacuolated and devoid, at all events at first, of yolk-spheres or other granules. In Echidna the same vacuolated central core occurs, but its cytoplasmic trabeculae tend to be thicker and, instead of being devoid of granules as in Platypus, are densely laden already in the oocyte of 1 mm. or so in diameter with large numbers of eosinophil spheres varying in diameter from about 0-003 to 0-005 mm. (Pl. VIII. fig. 37). These spheres are at first homogeneous, then granules of varying but quite minute size appear in them. These granules are strongly eosinophil, but eventually they become intensely basophil, appearing as minute black spherules in the fightstaining matrix.

Echidna Oocyte. Diameter 2-60 X2-20 mm.

The further history of these eosinophil spheres and their enclosed spherules can be followed in the latebra of the Echidna oocyte, approximately 2-6 X 2-2 mm. in diameter, which is illustrated in P1. VIII. figs. 38 & 39. Pl. VIII. fig. 38, which should be compared with Pl. VII. fig. 33, of the nearly corresponding stage in Platypus, shows very. clearly the vacuolated central core, prolonged up to enclose the base of the latebral neck (a feature characteristic of Echidna and not found in Platypus), and also the surrounding fine-grained latebral yolk-zone. The central core is now more uniformly vacuolated than in the earlier oocytes, and the trabeculae of its central region contain numbers of the above-described spheres, though they are not so abundant as in the earlier stages. They range up to about 0-0056 mm. in diameter, and contain minute basophil spherules. In the peripheral zone of the core and in the base of the neck, on the other hand, the spheres in question are present in large numbers (Pl. VIII. fig. 39). Here they attain a larger size than centrally (measuring up to 0-0084 mm. in diameter), and their contained basophil spherules are also larger. Each sphere contains a varying number of the latter, they may be fairly uniform in size or one or more may be larger (up to 0-0056 mm. in diameter) and the remainder quite small. From the peripheral zone they spread out amongst the yolk-spheres of the latebral yolk-zone, which vary in diameter from about 0-0056 to -0084 mm. It is thus evident that the small yolk-spheres of the latter zone are comparable in size with the largest of the basophil spherules, and there can be no doubt that here in Echidna these latter constitute the main source of supply of fine yolk-spheres to the latebral yolk-zone and to the base of the latebral neck. That this is so is THE DEVELOPMENT OF THE MONOTREMATA. 493

borne out by the frequent occurrence in the inner part of the latebral yolk-zone of aggregates of small yolk-spheres, all enclosed in a common matrix, presumably derived from that of the parent eosinophil sphere (Pl. VIII. fig. 39). Such aggregates are very variously constituted; they may, for example, consist of one or two larger spheres with a number of minute spherules distributed in the matrix or of a number (up to seven or so) of spheres of fairly uniform but small size. In the latter case, the aggregate doubtless breaks up later and its spheres become isolated, whilst round the base of the neck the appearances suggest that the eosinophil spheres themselves may break up and liberate their basophil spherules as minute yolk-spheres.

We have already recorded the occurrence in the latebra of the Platypus oocyte of granule-containing eosinophil spheres similar to those just described in the Eckidna latebra, but, Whereas in Platypus they would seem in part to give origin to the characteristic yolk-producing structures We have termed “ yolk-vesicles,” in Eckiolna these vesicles do not normally occur, and the eosinophil spheres produce yolk-spheres so to speak directly, without the intervention of a vesicular phase. VVe have, however, observed small yolk-vesicles (yen) in the vacuoles round the central cytoplasmic core of an abnormal (atretic) Echidna oocyte (0-72 ><O-62 mm. in diameter), in which there is present an excessive amount of fat ( ft.) (Pl. VIII. fig. 40). They are accompanied by solid spheres with included granules and numerous aggregates of small spherules.

Echidna Oocyte. Diameter 4-0 ><3-4 mm.

To conclude our observations on the latebra of the ovarian oocyte, We illustrate in P1. IX. fig. 41 a horizontal section through the body of the latebra in a fullgrown Eclmldna oocyte (4-0 ><3-4 mm. in diameter) for comparison with the corresponding stage in Platypus (Pl. VII. fig. 35). The central core differs from that of P1. VIII. fig. 38 in being larger and much more coarsely vacuolated, resembling in this respect that of Platypus. Its cytoplasmic network contains numbers of rather ill-defined eosinophil spheres with basophil granules, similar to those of the earlier oocyte above described and unrepresented in the corresponding Platypus latebra. They are more numerous and larger at its junction With the latebral yolk-zone, and in the latter aggregations of small yolk-spheres like those described above are not uncommon. The latebral yolk-zone (ly.z.) is well marked, but, as compared with the preceding oocyte, yolk-formation is not apparently in very active progress.

The Latebra in the I ntra-uterine Egg.

Having reached the conclusion that the primary function of the latebra in the growing oocyte is that of yolk-production, we were interested to see in how far it retained its integrity in the uterine egg and whether its structural condition indicated a continued activity of a more localised nutritive character in relation to the formation and growth of the blastodisc. We have accordingly examined its condition in a series of uterine eggs of Platypus and Echidna, and append a brief account of our observations.

Gatenby and Hill (1924) have already given an account of the structure of the latebra in a uterine egg of Platypus (egg C) which they believed to be unsegmented, but we are now satisfied that in the sections they studied the disc is no longer present. Apparently the disc (probably at the blastodisc stage) had become adherent to the zona as we have found to be the case in other eggs, and was lost when that membrane accidentally became detached during the preparation of the egg for imbedding. As the result, these authors unwittingly arrived at certain erroneous conclusions, e. g., that the Monotreme egg is polysperrnic, which we now know is not normally the case, and apposite in the present connection they mistook the yolk-bed, which is very fine-grained in Platypus, for the germinal disc. Apart from this misinterpretation, We can confirm their description of the latebra, which is the only detailed account extant of this structure in the Monotreme, Caldwell (1887) and Semon (1894) merely recording its existence.

We have examined the latebra in four uterine eggs of Platypus (including egg C above mentioned). It is very similar in all four and only differs from that of the ovarian egg in certain details. The coarsely vacuolated core of the latebral body contains fine yolk-spheres throughout its extent, but they are specially abundant in its less vacuolated periphery (of. P1. VII. fig. 35). The axial cytoplasmic core of the slender neck is also laden with fine yolk-spheres and contains large, more or less spherical vacuoles so close-set that the cytoplasm between them is reduced to thin cross-strands, giving the neck a ladder-like appearance in section, as described by Gatenby and Hill. About 0-3 mm. or so below the surface the neck widens out into a curious, clear, coarsely vacuolated expansion (about 0-20 mm. in width), suggestive of a reservoir and well seen in Gatenby and Hill’s text-fig. 1). Like the rest of the neck, it is permeated by minute yolk-spheres. In egg R (with a unilaminar blastodermic membrane) a corresponding expansion is present, but it is thinner, much less vacuolated, and more densely laden with fine yolk-spheres than in the other three eggs. This expansion is continued upwards as a narrower axial plug—like mass composed of compactly arranged fine-grained yolk, which spreads out into the similarly constituted yolk-bed.

The latebra in the uterine eggs of Echidua we have examined exhibits curious individual differences. In the 4-celled stage (VVH 1), the central core (Pl. XVII. fig. 88, l.b.) is richly and fairly uniformly vacuolated throughout, and its cytoplasm is crowded with fine yolk-spheres, intermingled with which are numbers of eosinophil spheres containing basophil spherules. The latebral yolk-zone (ly.z.) is very wide and formed of a narrow inner zone of rather large, loosely arranged spheres, and a wider outer zone of smaller more closely packed spheres—a quite exceptional arrangement. The latebral neck is very slender, not obviously Vacuolated, and crowded with fine yolk-spheres. It is not visible in Pl. XVII. fig. 88, but the conspicuous wide continuation of the latebral yolk-zone (l.n.), which encloses it, is well seen. Its junction with the yolk-bed is marked by the presence in the latter of a group of vacuolar spaces.

In the 16-celled stage (VVH 38), the latebra is in a more interesting condition. A small portion of its central core, including the base of the neck, is illustrated in Pl. IX. fig. 42. The core is irregularly Vacuolated, and its cytoplasm, except for the small area seen in the figure, is crowded with granules, comprising small yolk-spheres, spheres with basophil yolk-spherules, and curious irregular vesicular bodies (0-008 mm. in diameter), which distantly recall yolk-vesicles. Eosinophil spheres with their enclosed basophil yolk-spherules are specially abundant in the periphery of the core, in the base of the neck, and in the latebral yolk-zone where they are larger than elsewhere. The neck is poor in vacuoles, but is easily traceable to its junction with the yolk-bed as a narrow cord containing fine yolk—spheres, the junctional region being again coarsely Vacuolated.

In the 31-celled stage (Pl. XIX. fig. 104) eosinophil spheres without enclosures are alone present in the Vacuolated cytoplasm of the core, and such spheres, but containing minute basophil spherules, also occur amongst the fine yolk-spheres of the latebral yolk-zone. The neck is Vacuolated, and its contained fine spheres become still finer in its upper part before it joins the yolk-bed. The junctional region is again vacuolated, and there is also a clear Vacuolated area, containing sparse very fine spheres, in the yolk-bed itself just above the junction.

In egg VVH 3 (with a blastodermic membrane, incomplete over the lower pole, and sparse primitive endoderm cells (Pl. XXI. fig. 117)), the latebra is still a very striking structure when seen in vertical section. The uniformly Vacuolated central core is crowded with fine eosinophil spheres, mostly somewhat shrunken, whilst the latebral zone is very wide and composed of small yolk-spheres, but spheres containing basophil spherules are no longer present in any number. The basal part of the neck is, as is usual in Echidna, enclosed in the upper part of the central core, and is composed of Vacuolated cytoplasm laden with fine spheres. The neck proper is of similar constitution and remarkably distinct. In its upper part it contains minute eosinophil granules, and just above its junction with the yolkbed a Vacuolated area is again present in the latter.

Lastly, in egg A,TLB (completely enclosed and with a very short primitive streak) the body of the latebra is still intact. It is coarsely Vacuolated and crowded with small yolk-spheres, together with eosinophil spheres, mostly minute, and occasional spheres with basophil spherules. Of the neck only a minute remnant of its upper part remains. The yolk-bed, though displaced, is still clearly recognisable. It is richly Vacuolated and densely laden with fine granules. In the slightly later egg, VVH 19, the latebral body is still well preserved and indeed is very conspicuous, since it contains numerous fine deeply stained eosinophil granules, in striking contrast with the surrounding black-stained yo1k—spheres. Round the periphery of the body is a quite thin layer of small spheres.

In these eggs with a complete cellular wall, and therefore capable of increasing in size, the peripheral yolk-spheres have become dispersed owing to the active absorption of the nutritive fluid secreted by the uterine glands which is now going on, but the central part of the yolk-mass is still intact and so the latebral body has been preserved. Evidently, then, the latter persists until the yolk-mass finally breaks up. But it is difficult to arrive at a definite conclusion as to whether or not the latebra remains in any way active in the uterine egg. The occurrence of vacuolation in the junctional region of the latebral neck and yolk-bed, as well as in the latter where it is especially well marked, suggests the utilization of the fine yolk through enzymatic action, but are the vacuoles of the neck and body also the expression of the same activity ? We cannot express a definite opinion nor can we say whether or not there is an active transference of fine-grained yolk by way of the latebral neck to the yolk-bed, but there is fairly definite evidence that the material of the latter is utilized in building up the substance of the germinal disc (mlde pp. 501, 536, 545, 548).

The Nucleus, Germvlnal Disc and Egg-envelopes during the Later Stages in the Growth of the Oocyte.

In the preceding section we have made reference to the condition of these structures in dealing with the growth of the oocyte. Here we propose to give a more detailed outline of the progressive alterations they undergo during the latter half of phase 3. For this purpose We have selected for description a series of oocytes mainly from ovaries of Echidna, since our maturation stages are also from the same.

Up to the stage of the oocyte 2-2-5 mm. in diameter the nucleus undergoes, so far as we have observed, no very striking change apart from a progressive increase in size and an alteration in form from spherical to plano-convex, and the formative cytoplasm of the germinal disc, apart from an increase in area and in thickness, also exhibits no essential alteration.

Echidna Oocyte (C.20.7.29). Diameter about 2 mm.

This oocyte follows fairly closely on the Platypus oocyte of 2 X 1-8 mm. diameter, the nucleus and disc of which are described on p. 488'.

The nucleus, plano-convex in section (Pl. IX. fig. 43), measures 0-124 X0-l3 mm. in diameter and 0-034 mm. in thickness. Its upper surface, apart from a slight wrinkling of the nuclear membrane, is flat and lies in contact with the disccytoplasm, Whilst its convex under surface rests on the yolk-bed (yb.) composed of small spheres of varying size, except on the right side of the nucleus (in the figure), where the disc-cytoplasm is continued down around the periphery of the nucleus and contains only very fine yolk-spheres. The yolk-bed extends for a short distance peripherally to the nucleus where it lies below, and is in continuity with the disc-cytoplasm.

In the nuclear matrix are Visible (Pl. IX. fig. 43) the irregular strands of the pseudo-reticulum, in which are situated numbers of small eosinophil granules and there is also present a large spherical nucleolus (0021 mm. in diameter) which is markedly basophil and contains clear spherules of varying size in its substance. The formative disc-cytoplasm (gd.p.), below which the nucleus is excentrically situated, has a diameter of about 0-48 mm. and a thickness over the nucleus of 0-01 mm. In it are situated sparse, very fine basophil yolk-spherules, Which, however, are absent over the nucleus, though quite minute granules occur in the cytoplasm filling the slight infoldings of the nuclear membrane. The superficial cytoplasmic layer, into Which the disc passes at its periphery, is extremely thin and contains fine yolk-spherules like those of the disc.

The follicular epithelium (f.e.) is composed of a single layer of low cubical cells, and has a thickness of about 0-0084 mm. The zona is extremely thin, as also is the egg-membrane, which is clearly recognisable over the disc, and in this region, crossing the very narrow space between it and the zona, traces of the “ fibrils ” of the striate layer can be made out.

Echidna Oocyte (B.3.8.29). Diameter 2-6 X2-7 mm.

The nucleus (Pl. IX. figs. 44 & 45) is now somewhat larger, measuring 0-138 X 0-13 mm. in diameter x 0-038 mm. in thickness, and its upper surface is no longer flat, since the nuclear membrane is in places wrinkled and infolded. Most of the surfacedepressions so produced are slight, but an especially wide and deep one appears in the section figured (Pl. IX. fig. 44). They are occupied by disc-cytoplasm, containing fine to medium-sized yolk-spheres, the occurrence of which in this position we have noted above. Except in the region of these infoldings and round its periphery, the nucleus lies so close to the egg-membrane that very little disccytoplasm intervenes between the two. Dispersed throughout the nuclear matrix are irregular string-like rows and groups of fine eosinophil granules, marking the pseudo-reticular strands, the granules being more abundant and slightly larger than those in the nucleus of the preceding oocyte. A spherical, intensely basophil nucleolus (0-018 mm. in diameter) is also present, with, around its periphery, a number of small basophil spheres, possibly budded off from it (Pl. IX. fig. 45).

The formative disc-cytoplasm (gd.p.) has a diameter of about 0-50 mm. and a thickness, just outside the nucleus, of 0-017 mm. and is almost f_ree from yolkspheres. Underlying it is the yolk-bed, composed of a delicate cytoplasmic reticulum continuous with the disc-cytoplasm and enclosing mostly small basophil yolk-spheres. Below the nucleus these are less compactly grouped than in the rest of the bed.

The follicular epithelium (f.e.) is definitely in advance of that of the preceding oocyte. It averages in thickness about 0-01 mm., but is somewhat variable, being thinnest over the nucleus (0-008 mm.) and thickest peripherally to the same (0014 mm.). It is composed of a single layer of cubical to columnar cells, with occasionally two cells superimposed. The spherical or oval nuclei are large and active-looking, and basally situated. Between the cells are well-marked intercellular spaces, filled by a secretion which, especially above the disc, stains intensely with hsematoxylin (Pl. IX. fig. 44).

The zona is rather thicker than that of the preceding oocyte, especially over the disc, where it measures about 0001 mm., but elsewhere it is much thinner. The theca interna is present as a discontinuous layer of small flattened cells varying greatly in thickness, the maximum (0-025 mm.) being attained over, and in proximity to, the disc.

Platypus Oocyte (Platypus XV.). Diameter 3-2 X3-1 mm.

We interpolate here a description of this Platypus oocyte, since it is intermediate in size between the preceding and succeeding oocytes of Eckidna and, moreover, differs from both these oocytes in the remarkable development attained by the theca interna (a curious and interesting feature which distinguishes the Platypus oocyte from that of Eckidna), whilst the follicular epithelium has already attained the condition only reached in the later Echidna oocyte. Furthermore, the fixation (by PNO.) of the nucleus is excellent, and nuclear details are more clearly seen than in the preceding oocytes of Echidna.

The nucleus (Pl. X. figs. 46 & 47) has the same plano—convex form as that of C.20.7.29 (Pl. IX. fig. 43), and though of less diameter is of greater thickness, its measurements being 0-1 1 ><0-10 mm. in diameter x 0-054 mm. in thickness. The nuclear membrane on its upper surface is thrown into irregular sinuous folds, occupied by disc-cytoplasm containing minute yolk-spheres. The nuclear matrix is very finely alveolar, and situated in its central region is what appears to be a wide-meshed irregular network composed of very thin thread-like strands. These We take to be the pseudo-reticular strands, here much better defined than those of the preceding oocytes. They contain numbers of minute eosinophil granules dispersed along their length at varying intervals. Accompanying the strands, but not actually imbedded in them, are peculiar spherical bodies, presumably of nucleolar nature and mostly about 0-0025 mm. in diameter, though exceptionally they may attain twice that size (Pl. X. fig. 47). They possess a darkly stained periphery enclosing a clear centre, in which are situated one or more basophil granules. It is only in this nucleus that we have encountered nucleolar spherules of this type. In addition, a number of large homogeneous basophil nucleoli are present in the matrix.

The disc (gd.p.) is very similar to that of the preceding oocyte. Its formative cytoplasm has a diameter of 0-48 mm. and a thickness just outside the nucleus of 0-0058 mm. It passes over below into the uniformly fine-grained yolk-bed, which thickens around the nucleus and continues down to form the coarsely vacuolated fine—grained bed on which the nucleus rests.

The follicular epithelium (Pl. X. fig. 46, f.e.) is markedly in advance of that of the preceding Echidna oocyte. Over the disc it attains an average thickness of 0026 mm. and elsewhere of 0-028 mm. It consists of a single layer of cubical to columnar cells, though two super-imposed cells are not infrequently met with. The cytoplasm is greatly vacuolated and the nuclei, large and deeply staining, are mostly situated adjacent to the zona, with their long axes tending to be disposed tangentially to the latter. In the intercellular spaces, especiallybetween the outer ends of the cells, are to be found irregular little masses of a homogeneous deeply staining secretion.

The theca interna (th.i.) is noteworthy for the degree of development it has already reached. It forms a thick continuous layer all round the oocyte, attaining over the disc an average thickness of 0-047 mm. and elsewhere of 0-034 mm. It is composed of polyhedral cells, arranged up to five or six cells deep. The cytoplasm is alveolar and the nuclei, oval or spherical, are centrally situated. The membrana propria (mp), which separates the layer from the follicular epithelium, is rich in capillaries.

Echidna Oocyte (B.l9.7 .30). Diameter 4-l >-:3-l mm.

This oocyte, which is approaching its full size, shows conspicuous advances on oocyte B.3.8.29 in respect of the nucleus, germinal disc, and follicular epithelium. The nucleus (0-l44><0-18 mm. in diameter><0-026 mm. in maximum thickness) is now of greater diameter and more flattened than that of B.3.8.29 (Pl. X. figs. 48 & 49). On its upper surface the nuclear membrane is very irregular in contour. In addition to smaller infoldings, there is a large central depression (Pl. X. fig. 49) 0-037 mm. in width, filled by greatly vacuolated disc-cytoplasm in which are situated fine yolk-spheres. The nuclear matrix is formed by a finely alveolar ground-substance, in which there are now situated numerous minute eosinophil and rather larger basophil nucleolar spherules, varying in size, but mostly about 0-0014 mm. in diameter, and so abundant that the nucleus under low magnification appears coarsely granular. The pseudo-reticular strands are no longer obvious as such, but in place of them, and possibly derived from them, there are now present extremely fine threads, in which are situated at intervals minute basophil granules. We regard these threads as linin threads and their granules as chromatinic. In this nucleus they are not easily seen owing to the abundance of the nucleolar granules, but in later oocytes they are much more obvious (of. P1. XI. fig. 53, and P1. XII. fig. 55). In the matrix there are also present two large basophil nucleoli, as well as a few smaller spheres of similar appearance, which are distributed amongst the nucleolar granules. 500 PROF. T. THOMSON FLYNN AND PROF. J. P. HILL ON

The germinal disc exhibits what is perhaps the most striking of the advances observable in this oocyte, since it has now attained its definitive condition so far as the ovarian oocyte is concerned. In the preceding oocytes it will be remembered the nucleus rests directly on the fine-grained yolk-bed, but here in this oocyte it has become separated from the latter through the appearance of a vacuolated cytoplasmic layer (vcd.), which continues out below the formative layer (fcd.) and contains minute yolk-spheres only, so that the germinal disc as now completed may be described as consisting of two zones, a superficial zone of homogeneous formative cytoplasm and a deep zone of vacuolated cytoplasm, rich in fine yolkspheres, below which the yolk—bed (3/b.) is situated (Pl. X. fig. 48, cf. also Pl. XI. fig. 50, from the succeeding oocyte).

The formative cytoplasm of the disc ( fad.) has an approximate diameter of 0-48 mm. Peripherally it is quite a thin layer, containing sparse, very fine yolkgranules. It merges below into the just-mentioned layer of vacuolated cytoplasm (vcd.), which is much thicker than the formative layer and is laden with numerous small yolk-spheres. Traced towards the nucleus, both layers thicken, the deep zone becoming much less rich in yolk-spheres. The superficial layer continues as an attenuated layer above the nucleus, and at the same time is prolonged down along with the deep layer to underlie the nucleus, and so we find the latter enclosed peripherally and below by a cytoplasmic investment consisting of a quite thin, finely granular layer lying in contact with the nuclear membrane and a much thicker underlying layer of vacuolated cytoplasm containing numbers of very fine basophil yolk-granules. Below the nucleus the deep zone of the disc so constituted has a thickness of about 0-013 mm., whilst just outside the nucleus the two zones have a combined thickness of 0-038 mm. To attain the condition here described, it is evident that a considerable increase in the amount of cytoplasm below and peripherally to the nucleus must have taken place, and it is of interest to note that this increase is accompanied by an increase in the size of the nucleus and in the number of its nucleolar granules and by a striking increase in the thickness of the follicular epithelium.

The follicular epithelium (f.e.) is definitely in advance of that of B.3.8.29, and is now a conspicuous layer, recalling that of the Platypus XV oocyte. Over the disc it has an average thickness of 0-024 mm. and elsewhere of about O-O23 mm. Its cells are large, often arranged two deep, and intercellular spaces, sometimes containing a secretory coagulum, are well marked (Pl. X. figs. 48 & 49). The theca interna is still inconspicuous and occurs in disconnected patches, in marked contrast with its condition in the Platypus X V oocyte.

Echidna Oocyte (D.15.8.29). Diameter 3-9 ><3-6 mm.

This oocyte is in most respects very like the preceding, and need not be described in detail. We provide, however, a figure of a section through the disc (Pl. XI. fig. 50), which shows its relations to the adjacent yolk more clearly than does Pl. X. fig. 48.

The nucleus (0-136 ><0-14 mm. in diameter ><0-032 mm. in maximum thickness) generally resembles that of B.19.7.30, and shows a similar deep infolding of the nuclear membrane on its upper surface. It contains numerous nucleolar spherules, coarser on the whole than those of the latter, the smaller eosinophil, the larger (up to 0-0029 mm. in diameter) basophil, whilst others possess a basophil core and an eosinophil periphery. Linin-threads were not observed.

The disc (Pl. XI. fig. 50) has the same constitution as in the preceding oocyte and the same diameter (0-48 mm.). Its superficial formative zone (fcd.) is very thin, measuring only 0-0086 mm. in thickness just peripherally to the nucleus, whilst its deep vacuolated zone (vcd.) is much thicker (0034: mm.) and is very rich in minute yolk-spherules, the smaller eosinophil, the larger (up to 0-002 mm. in diameter) basophil. The total thickness of the disc just outside the nucleus is about 0-043 mm., which compares with 0038 mm. in the preceding oocyte. The deep zone merges below into the underlying yolk-bed (3/b.) containing small deeply stained yolk-spheres, varying in size up to a maximum diameter of about 0-007 mm., intermingled With which are minute spherules identical with those of the deep zone.

Below the nucleus, however, the yolk-bed is interrupted by what appears to be a localised thickening of the deep zone in the form of a conspicuous mass composed of cytoplasm more coarsely vacuolated than the rest of the zone, but laden with similar fine yolk-spherules. This mass has a diameter of about 0-146 mm. and a thickness below the nucleus of about 0-05 mm., and its deeper portion, in which vacuolation is most marked, actually lies in the yolk-bed immediately above the junction of the yolk of the latebral neck (l.n.) with the latter, the latebral yolk being much coarser than that of the yolk-bed. The appearances strongly suggest that the central part of the yolk-bed, directly above the latebral neck, is being utilised in the growth of the deep zone of the disc.

The follicular epithelium (f.e.) is irregularly 2-layered ; it is exceptionally thin over the disc (0016 mm.), but elsewhere averages in thickness about 0-026 mm.

Echidna Oocyte (2.8.28). Diameter 3-7 ><3-4 mm.

This oocyte is also very similar to the preceding two, but is noteworthy for its remarkably well-developed follicular epithelium.

The nucleus (0-165 ><0-14 mm. in diameter ><0-025 mm. thick) (Pl. X. fig. 51) is thin and flattened, and shows two large infoldings of the nuclear membrane on opposite sides of its upper surface close to the margin. The nuclear matrix is crowded with eosinophil and faintly basophil nucleolar granules measuring up to 0-0022 mm. in diameter, and there are also present in it two large basophil nucleolar spheres. The linin network is indicated, but difficult to see owing to the abundance of the nucleolar granules. .

The disc (about 0-51 mm. in diameter) is generally similar to that of the preceding oocyte, but its deep zone is richer in small basophil yolk-spheres.

The follicular epithelium (Pl. X. fig. 51, f.e.) averages about 0-04 mm. in thickness and is now a conspicuous layer mostly two cells thick. The cells are large, cubical, columnar, or polyhedral in shape, with large deeply staining spherical or oval nuclei. Vacuolar spaces, mainly intercellular, are common and sometimes contain a shrunken darkly staining coagulum. The theca interna (th.i.) is also somewhat better developed and may attain a thickness of 0-012 mm.

Echidna Oocyte (B.11.8.30). Diameter 4-l ><3-4 mm.

The nucleus (0-146 ><0-15 mm. in diameter ><0-023 mm. in maximum thickness) is asymmetrically saucer-shaped (Pl. XI. figs. 52 & 53) owing to the presence on its upper surface of a wide and deep depression, which is not quite central, so that the surrounding fiat rim is of unequal width. The depression as usual is occupied by vacuolated disc-cytoplasm containing numerous fine yolk-spheres. In the nuclear matrix, the nucleolar granules present in the nuclei of preceding oocytes are here represented by numerous much larger nucleolar spheres, varying in diameter from 0-002 to 00036 mm., which have stained of a violet tint with haematoxylin, and much less deeply than the true nucleoli. Of the latter, one is large (0-011 mm. diameter) and irregularly rounded, whilst several smaller spheres lie in contact with it. They are all intensely basophil. This nucleus is of special interest, since the linin-threads, only observable with difficulty in the nuclei of the preceding oocytes, are here very obvious and are seen to form a very delicate Wide-meshed network, situated in the central region of the nuclear matrix (Pl. XI. fig. 53). The very fine threads forming the network exhibit at intervals minute basophil thickenings which, as already indicated, we regard as formed of chromatin.

The disc (Pl. XI. fig. 52) measures in diameter about 0-58 mm. and just outside the nucleus has a thickness of about 0-030 mm. In its structure it differs in no essential respect from those of the preceding oocytes.

The follicular epithelium (P1. XI. fig. 53, f.e.) is less advanced than that of oocyte 2.8.28. Over the disc its measures 0-023 mm. in thickness and elsewhere varies from 0-020 to 0-046 mm. It is two cells thick over the disc and for a short distance peripherally thereto, but over most of its extent it is only a single cell in thickness.

Echidna Oocyte (A.20.'7.29). Diameter 4-3 X3-63 mm.

This oocyte, with a diameter averaging 3-96 mm., represents the stage of the full-grown oocyte, and possibly the same is true for preceding oocytes with a diameter of 3-75 mm. It is of special interest as being one of the most advanced THE DEVELOPMENT OF THE MONOTREMATA. 503

oocytes with an intact nucleus that we have encountered prior to the onset of maturation.

The nucleus (0-159 X0-ll mm. in diameter X0-0258 mm. in thickness) resembles that of the preceding oocyte in shape, having the form of a shallow oval saucer with a thick, slightly asymmetrical rim, its concavity being occupied by the usual vacuolated cytoplasm containing numerous small yolk-spheres (Pl. XI. fig. 54, and P1. XII. fig. 55). The linin-network is again clearly visible in the central (medullary) region of the matrix. In Pl. XII. fig 55, its very fine threads (l.t.) can be seen running along the length of the nucleus and connected here and there by short oblique branches, so that the network is a very loose open one. In the threads there occur at intervals very minute granules, presumably of chromatin. The conspicuous granules, which in the figure are seen to occur along the fine lininthreads, must not be confused with the presumed chromatin granules which are very much finer and actually lie in the substance of the threads. The large granules in question, which lie in contact with the threads, often in irregular rows, are nucleolar granules, apparently of the same nature as the granules we have described in preceding nuclei. These nucleolar granules are minute spherical bodies, slightly eosinophil and measuring about 00014 mm. and under in diameter. They occur not only along the threads but also in the matrix between them, and are specially numerous in the central region of the nucleus, though they are also present in small numbers in its peripheral region and below the nuclear membrane. Also in the same region there occur larger nucleolar spheres, strongly basophil and measuring up to 0-0045 mm. in diameter. Three of them appear in P1. XII. fig. 55, and altogether about a dozen or so are present.

The germinal disc (Pl. XI. fig. 54, and Pl. XII. fig. 55) resembles that of the preceding oocytes in all essential respects. It has an approximate diameter of about 0-6 mm. and a thickness just outside the nucleus of 0-06 mm., whilst its subnuclear bed has a thickness of about 0-03 mm. As in previous oocytes it is difficult, if not impossible, to determine the precise limits of the disc, since its superficial formative layer (fcd.) thins out quite gradually, whilst its deep vacuolated layer (vcd.) likewise merges quite gradually into the ring of fine—grained yolk, which encircles the disc and which is directly continuous with the yolk-bed underlying the latter.

The follicular epithelium (Pl. XII. fig. 55, f.e.) is now better developed than in any of the preceding oocytes. It has an average thickness of 0-048 mm. and is two cells deep throughout. The cells are large, of very varying shape, and are enclosed by well-marked exoplasmic membranes. Their nuclei are also large, ovalish in form, and rich in nuclear contents, so that they stain deeply. Between the cells occur intercellular spaces of varying size and shape, which are often subdivided by partitions of exoplasmic substance and are best developed between the basal ends of the cells. Except for occasional traces of a granular material, they are devoid of stainable contents.

The cytoplasm, finely granular to alveolar in texture, now presents definite evidence of secretory activity on the part of the follicular cells. The secretory material takes the form of minute flaky-looking bodies, which do not occur dispersed through the cytoplasm, but are loosely grouped together to form what appears in section as a horseshoe-shaped, or less frequently as a ring-shaped, figure. Examination of the sections shows that the actual form of this figure is that of a more or less spherical shell, usually incomplete on one side where the nucleus is situated, enclosed in which is a mass of cytoplasm free from granules. The secretory bodies, flake-like in appearance, vary in size and possess quite irregular contours. They stain rather diffusely with haematoxylin, and are so related as to produce the appearance of a loose network. Intermingled with them are a few minute rounded granules.

We regard these secretory bodies as the precursor of the follicular secretion or follicular fluid as we propose to term it, which forms a continuous layer surrounding the oocyte at the time of maturation (vide Pl. XIII. figs. 62 & 68). It remains to be mentioned that the position of the aggregation of secretory bodies in the cell is variable and is apparently determined by the position of the nucleus, and that again determines which region of the cell contains most cytoplasm. When, for instance, the nucleus is basal, as is frequently the case, the apical region of the cell is richest in cytoplasm, and there the aggregation is situated in contact with the apical pole of the nucleus, but it may be found in contact with the basal pole or with the equatorial region of the nucleus, in accordance with the position of the latter and the shape of the cell.

In view of the intimate relationship known to exist between the Golgi apparatus and the secretory process, it is of interest to note that these aggregations of secretory bodies occur in the position that apparatus might be expected to occupy, and it is possible that the very minute vacuoles which occur amongst the secretory granules mark the sites of the now dissolved out Golgi bodies as suggested to us by our colleague, Mr. K. C. Richardson. In this connection it is pertinent to mention that Solomons and Gatenby (1924, p. 4), in describing the follicular epithelium of the human Graafian follicle, state that the cells of the basal layer next the membrana propria are all so orientated that their nuclei lie adjacent to the latter, 13. e., are basal, whilst the small Golgi apparatus is situated at the apical pole of the nucleus away from the membrana propria. They further state that the remaining follicle cells show no definite orientation. According to Brambell (1925) the Golgi apparatus in the follicular epithelium of the Fowl occupies an excentric perinuclear position, being “ situated at the side of the nucleus or between it and the surface of the oocyte.”

The theca interna (Pl. XII. fig. 55, tk.9}.) appears to be more extensive than in the preceding oocytes, but is still an insignificant layer as compared with that of the full-grown Platypus oocyte (Pl. XII. fig. 58), the maximum thickness it attains being about 0-012 mm.

The zona is extremely thin and over the disc is separated from the still more delicate egg-membrane by the striate layer, which has the appearance of a very narrow cleft, occupied by a homogeneous material and crossed here and there by very fine fibrils.

Echidna Oocyte (C.20.7.29). Diameter 4-0 X3-4 mm.

This oocyte, though of smaller diameter than the preceding one, belongs to the same stage of completed growth and in most respects closely resembles it. Its claim to mention rests on the evidence it presents of increased secretory activity on the part of the follicular epithelium. The nucleus (0-144 ><0-13 mm. in diameter

><0-024: mm. in thickness) is somewhat bowl-shaped with a deep central concavity on its upper side. The linin-network is distinct and presents the same appearance and disposition as in the preceding oocyte, and the same holds true for the eosinophil nucleolar granules, which are numerous in the central region around the linin—threads and sparse elsewhere. But, i11 addition, this nucleus contains a number of larger perfectly homogeneous spherules, lying mostly in contact with the nuclear membrane, which have stained of a pale Violet colour with the haematoxylin and which vary in size from 0-003 to 0-01 min., and there is also present a marginal group of basophil nucleoli varying in size up to 0-004 mm.

The disc has an approximate diameter of 0056 mm., and resembles in all essentials that of the preceding oocyte.

The follicular epithelium (0048 mm. in thickness over the disc and elsewhere varying from 0-024 to 0-032 mm.) is, so far as secretory activity is concerned, greatly in advance of that of the preceding oocyte, as inspection of P1. XII. figs. 56 & 57 readily shows. An intercellular follicular secretion (f.s.), of which sporadic traces only have been observed in preceding oocytes (e. g., in P1. XI. fig. 51), is now present in considerable abundance and, in addition, intra-cellular secretory material still occurs in the cytoplasm.

As in the preceding oocyte, the follicular cells are large and possess correspondingly large deeply staining nuclei, rich in contents. Whilst the perinuclear cytoplasm is rather dense and homogeneous in appearance, the peripheral cytoplasm is finely alveolar and has present in it numerous extremely minute, poorly defined, flaky bodies which stain lightly with haematoxylin. They only differ from the aggregated secretory bodies in the follicular cells of the preceding oocyte in being smaller and in their diffuse peripheral arrangement, so that, evidently, the aggregations therein described break up and their constituent secretory bodies become dispersed and take up a peripheral position in the cell prior to being shed. The intercellular follicular secretion is very obvious in the sections (Pl. XII. figs. 56 & 57) as irregular, often shrivelled-looking strands and masses (f.s.) situated in the spaces between the cells which they frequently only partially fill. They are composed of a homogeneous colloid-like substance, which stains deeply with haematoxylin. Sometimes this substance appears uniform throughout (Pl. XII. fig. 56), more frequently it contains small vacuolar spaces, and these may be so numerous as to give it the appearance of a network or tangle of fine strands (Pl. XII. fig. 57). The secretion occurs not only in the intercellular spaces. but also in the spaces that are present between the cells and the membrana propria on the outer side and the very thin zona on the inner side. But it is by no means uniformly present throughout the extent of the follicular epithelium; some areas contain little or none, in others it is present in fair abundance, and in yet others of limited extent, so much of it may be present that its intercellular portions join together to form an irregular layer lying between the cells and the zona, so that in these particular areas it has attained its definitive position (of. P1. XIII. fig. 62). On the other hand, it may accumulate at the opposite surface and so produce a localised bulging into the enclosing theca.

Platypus Oocyte (oz. 19. 8. 01).

The full-grown twin oocytes in this Platypus ovary measured in diameter 4-3 ><4-1 mm. and 4-4 ><4:-2 mm. respectively, and are the largest Platypus oocytes we have encountered. Unfortunately, the sectional plane intersects the egg-axis almost at right angles, so that the disc and nucleus are not available for description, but the follicular epithelium and the theca interna, both well preserved, are deserving of notice as illustrating the condition of these structures in the fullgrown oocyte of Platypus.

As a matter of fact, the structure of the follicular wall in these oocytes has already been described and figured by Hill and Gatenby (1926, pp. 719-722, text-fig. 2, and pl. ii. fig. 3). Except in regard to the follicular secretion, which is not specifically mentioned by these authors, we have little to add to their detailed account.

The follicular epithelium (Pl. XII. figs. 58-60, f.e.) over most of its extent varies between 0030 and 0034 mm. in thickness, but may be as thin as 0-026 mm. over localised areas and, exceptionally, as thick as 0-05 mm. where the theca interna is absent. As described by Hill and Gatenby, it is composed of large, plump, glandular-looking cells, varying in shape and arranged mostly two cells deep. The cells are delimited by thin but quite definite exoplasmic membranes, below which the cytoplasm is often markedly vacuolated. Irregular vacuolar spaces are of common occurrence at the inner ends of the cells where they rest on the zona. The nuclei are large, deeply staining, and spherical to ovoidal in form, and exhibit a marked tendency to place themselves with their long axes tangential to the egg-surface. The nuclear reticulum is richly developed and supports numerous small granules of chromatin, which are specially abundant just below the nuclear membrane. In it occur nucleoli of two types, highly characteristic ring-like plasmosomes, with a deeply stained periphery and a clear centre, which vary in number (up to six or more) and also in size, and one or two nucleoli (karyosomes) of irregular outline which stain deeply throughout.

Here, again, there is abundant evidence of secretory activity on the part of the follicular epithelium (Pl. XII. figs. 58, 59, 60), though the degree of activity varies somewhat from place to place. In the main, the secretory process appears to be similar to that in E'ch'idna, but there are slight differences in detail. Intracellular secretory aggregations, comparable to those in Echidmz, occur here also and in precisely the same position, adjacent to the nucleus (P1. XII. fig. 58, f.s.), but the secretory bodies, in the form of short irregular strands and irregular flaky masses, are distinctly coarser than those in Eckidna, and are usually clumped together to form a more or less compact mass, though not infrequently they are loosely grouped and extend for some distance into the adjoining cytoplasm as irregularly thickened strands. They never present in section the horseshoe-shaped or ringlike arrangement of those of Echiolna.

In the next phase of the secretory process, the aggregated secretory bodies break up or separate from each other and become dispersed irregularly throughout the cytoplasm, frequently coming to lie close below the cell-membrane. These dispersed granules are again coarser than the corresponding granules in Eckidna, and are never grouped to form a finely granular peripheral zone as in the latter.

In the succeeding phase, the definitive secretion makes its appearance in the epithelium. It takes the form typically of more or less spherical globules (Pl. XII. fig. 59, f.s.), but it also occurs as irregular masses or strands, possibly formed by the confluence of globules (Pl. XII. fig. 60), all of which are composed of a homogeneous colloid-like substance which stains intensely with haematoxylin and which, in the case of the larger masses, may contain vacuoles (Pl. XII. fig. 60). The globules vary in size from quite minute bodies up to spheres 0008 mm. or more in diameter. They are situated mostly in vacuolar spaces at the outer and inner surfaces of the epithelium, but they also lie, as do the strands, in the thickness of the latter, in intercellular spaces, especially at the angles where the cells meet, and they may also occur in intra-cellular vacuoles. Curiously enough, the cell-membranes are sometimes thickened and appear as if they had been replaced by secretion.

We possess no later oocytes of Platypus in which to trace the further history of this follicular secretion, but in Echidna we are fortunate enough to possess a number of oocytes at the phase of maturation, and we are able to show that, greatly increased in amount, it forms a continuous layer, situated between the zona and the inner surfaces of the follicular cells (Pl. XIII. figs. 62 & 68, and Pl. XIV. fig. 70) which completely encircles the oocyte. This layer, as we have already mentioned (ante, p. 454), was originally described by Caldwell (1887) and termed by him the “ proalbumen.” In our opinion it is the homologue of the liquor folliculi of the Graafian follicle of other Mammals (vide pp. 529-530).

It is significant, in View of the secretory activity of the follicular cells, that the membrana propria lying between the follicular epithelium and the theca interna is richly supplied with capillaries.

The theca interna itself is remarkably well developed, though discontinuous, and attains a thickness in places of 0-031 mm. (Pl. XII. fig. 58, th.t.). The theca. interna cells, very small compared with the follicular cells, are polyhedral in form and their cytoplasm is coarsely alveolar, frequently indeed markedly vacuolated, and sometimes to such a11 extent that the nucleus appears to be surrounded by a clear space, which on closer examination is seen to be crossed by delicate radiating threads of cytoplasm. Their nuclei, spherical to oval, possess a well-marked reticulum with fine chromatin granules and one or more small nucleoli. Hill and Gatenby (1926, pp. 756-760) have already called attention to the obviously glandular character of the theca interna cells, in connection with their Work on the formation of the corpus luteum in Monotremes.

Summary and Discussion

1. Growth of the Oocyte.

We recognise three phases iii the growth of the ovarian oocyte in the Monotreme :——

Phase 1 .

This phase includes the smallest “ resting ’ oocytes with a diameter of about 0-06 mm. as well as larger oocytes up to 0-15 mm. or rather more in diameter. They are characterised by the excentric position of the nucleus and the differentiation of the cytoplasm into a rather broad peripheral zone containing fatdroplets and a central zone, rich in mitochondria and enclosing in the smallest oocytes the centrosphere or “ yolk-body.” The follicular epithelium is flattened to cubical, and the zona has made its appearance in the larger oocytes belonging to the phase.

Phase 2.

This second phase comprises oocytes ranging in diameter from about 0-2 to about 0-5 mm. They are characterized by the presence of a peripheral cytoplasmic zone, a cortical fatty zone, and an extensive central or medullary zone relatively fat-free. The follicular epithelium is cubical and the zona is well established. Towards the end of this phase, yolk-sphere primordia make their appearance in the cortical fatty zone, Which, consequent on the correlated utilisation of the fat—droplets, becomes transformed into a cortical yolk-zone, whilst fluid—filled vacuoles are beginning to appear in the medullary cytoplasm.

Phase 3.

This phase includes oocytes ranging in diameter from about 0-6 to a maximum of 4-3 mm. in Platypus and of 3-9 mm. in Echtdna (:the stage of the full—grown oocyte). This is the phase of intensive yolk-production and most active growth. Oocytes of this phase are characterised by the possession of a germinal disc or its primordium and of definitive yolk-spheres. 6 0

We recognise two sub-phases: Sub-phase A includes oocytes 0-6-0-7 mm. in diameter with a germinal disc primordium, a peripheral cytoplasmic zone, a cortical yolk-zone with definitive yolk-spheres, and a central or medullary zone distinguishable into a vacuolated outer zone, in the vacuoles of which yolksphere primordia eventually appear, and a central non-vacuolated core.

Subphase B comprises oocytes ranging from a diameter of about 0-8 mm. upwards to the full-grown condition. In this sub-phase yolk-formation reaches its acme. The latebra is already recognisable at the commencement of this sub-phase. It represents a persistent portion of the central (medullary) cytoplasm in which coarse yolk-spheres are never developed. Its body is largely formed by the now-vacuolated central cytoplasmic core, whilst its neck is derived from the narrow axial cord of vacuolated medullary cytoplasm which extends down from below the nucleus to join the body, and which becomes localised and defined by the formation all around it of coarse yolk-spheres in the surrounding medullary cytoplasm. The cortical yolk-zone loses its individuality at the commencement of the sub-phase and becomes merged with the later-developed medullary yolk to form a broad “ yolk-zone,” whilst, in addition, a “latebral yolk-zone” is established at the periphery of the latebral body and neck. The latebra, with its enclosing latebral yolk-zone, is regarded as the seat of production of the small yolk-spheres which are added to the yolk-zone and so in this way the latter is enabled to increase and to form by far the greater bulk of the yolk-mass of the full-grown oocyte.

We accordingly distinguish two stages in yolk-formation in the Monotreme oocyte, Viz., a first stage, occurring in the early oocytes of phase 3, in which yolkformation proceeds in the main from without inwards, yolk-spheres first appearing in the cortical zone and then later in the vacuoles of the medullary zone, i. e., yolkformation proceeds in the centripetal direction, and a second stage, involving oocytes from about 1 mm. in diameter upwards in which yolk-production proceeds from within outwards, the latebral yolk-zone acting as the centre for the formation of small yolk-spheres which pass outwards into the yolk-zone, 2'. e., yolk-formation mainly proceeds in the centrifugal direction.

During the early part of this sub-phase the fine-grained yolk of the yolk-bed is laid down in the thin layer of vacuolated cytoplasm which immediately underlies the nucleus and germinal disc-primordium, and which is directly continuous below with the more coarsely vacuolated cytoplasm of the latebral neck.

The growth-phases of the Monotreme oocyte, briefly characterised above and described in detail in the body of this paper, are based primarily, though not exclusively, on our study of the process of yolk-formation, and it is a matter of interest that the phases recognised by us for the Monotreme oocyte show a general correspondence with those formulated by van Durme (1914) in her well-known paper on the vitellogenesis of the oocyte of the Bird, and which have been accepted by Marza and Marza (1935) as being most in accordance with the “ known facts of histophysiology.”

Van Durme’s conclusions are summarised in her own paper (pp. 176-180), so that here we need only institute a brief comparison of the three phases she recognises with our own.

Her first phase corresponds fairly closely with our phase 1, but overlaps with our phase 2, and includes like ours the smallest oocytes, characterised by the excentrically situated nucleus, the presence of a centrosphere (“ corps vitellin ”) with a surrounding mitochondrial zone (“ couche vitellogene ”) and of fatdroplets, at first distributed around the latter, but as the oocyte grows coming to form a fatty nuclear cap (“ capuchon granulo-graisseux nucléaire ”) and a cortical granulo-fatty layer, the former unrepresented in the Monotreme oocyte and the latter the equivalent of the cortical fatty zone of our phase 2. Marza and Marza state that, as the oocyte grows, the fat-droplets decrease in size, so that the granulo-fatty zone is only “dimly visible in ovules of 1-2 mm. diameter,” as Konopacka (1933) also found to be the case, and as we also describe as happening in our phase 2 when the primordia of the yolk-spheres are beginning to appear. In both the Bird and the Monotreme the centrosphere early disintegrates, van Durme describing it as dividing into three or four parts, all traces of which have disappeared in oocytes of 0-20 mm. diameter, whilst the mitochondria of the mitochondrial zone spread out and become fairly uniformly distributed throughout the cytoplasm of the oocyte. But this condition, according to van Durme, is of short duration in the Fowl, for in the slightly more advanced oocytes it is possible to distinguish three zones in the cytoplasm, characterised by their mitochondrial content, viz., a cortical mitochondrial zone, the equivalent of the superficial cytoplasmic zone of our phase 2, an endoplasmic zone adjacent to the nucleus, and a sub-cortical exoplasmic zone. The latter two zones are not differentiated cytologically in the Monotreme, though the endoplasmic zone seems to correspond to the axial sub-nuclear medullary cytoplasm of our subphase 3, destined to furnish the yolk-bed and latebra, and ‘the exoplasmic zone, to the central (medullary) cytoplasm, excluding the central core.

Van Durme’s second phase includes oocytes which exhibit features characteristic of those belonging to our phases 2 and 3. Distinctive of the early oocytes belonging to her stage A of this phase is the appearance of clear fluid-filled “ vitelline vesicles ” which first arise in, and especially internally to, the cortical granulo-fatty zone, and from there gradually extend inwards towards the centre of the ovule. In oocytes of 2 mm. diameter, Marza and Marza state that these vesicles “ constitute a layer which takes up the Whole centre of the ovule.” Konopacka (1933) figures them in the oocyte of 1-2 mm. diameter, and describes them as clear droplets composed of liquid protein. They clearly correspond to the vacuoles present in Monotreme oocytes belonging to sub-phase A of phase 3, and Loyez (1906) also found them in the Reptilian oocyte at the corresponding stage, describing them as alveoli or vacuoles. Stage B of this same phase in the Fowl is characterised by the appearance of yolk-spheres, first in the exoplasmic zone where coarse spheres are laid down and then in the endoplasmic zone where finer spheres are formed. In the Monotreme, yolk-sphere primordia are first seen in the cortical fatty zone in our phase 2.

Van Durme believes that the first-formed yolk-spheres (primordial yolk-spheres of Marza and Marza) arise in two ways: (a) by the direct transformation of enlarged mitochondria, (b) from the contents of the “ vitelline vesicles.” Characteristic also of this phase in the Bird is the appearance of the primordium of the germinal disc. In the Monotreme we have recorded its presence in oocytes at the beginning of our phase 3.

Van Durme’s third and last phase corresponds fairly closely to sub-phase B of our third phase (ante, p. 509) and includes actively growing oocytes up to the fullgrown condition, in which the yolk-spheres have increased in size (and presumably in number, though she does not expressly say so) and are distinguishable into white and yellow, the latebra and yolk-bed (nucleus of Pander) become established and the germinal disc reaches its final condition prior to the onset of maturation. At the beginning of this phase in the Bird, the cortical mitochondrial zone has almost completely disappeared.

We are not here concerned with the distinction of yolk-spheres into white and yellow, since in the Monotreme they are all of the same type, and there is no structural or developmental justification for terming the small spheres white and the large yellow, as some previous authors have tended to do on analogy with those of the Bird. The Monotreme yolk-spheres would seem to correspond to what are termed white spheres in Birds and yellow spheres, which according to most authors (of. Konopacka, 1933) are derived from the white, are never formed. In this respect the Monotremes would seem to agree with the Reptiles, though we have not been able to find much information in the literature concerning Reptilian yolk. Waldeyer (1906) states that the yolk-elements of the Reptilia are similar to the white yolk (“ Dotter-cytoide ” of His) in Birds. Loyez (1906) quotes Sarasin to the effect that in the Lizard he is not prepared to recognise a distinction of the yolk into yellow and white, but she herself offers no information on the subject. In the egg of the Skink, Mabuia multifasciata, Kohlbrugge (1901) states that there are no white and yellow spheres, only layers of larger and smaller similar spheres, whilst in the eggs of the Gecko, Hoplodactylus maculatus, Miss M. M. Boyd, M.Sc., informs us that only one type of yolk-sphere is present. On the other hand, Riddle (1911) states that in the turtle’s egg there exist “ alternate layers of white and yellow yolk somewhat comparable to those of the Bird.”

As concerns the latebra, van Durme states that the parts of the White yolk constituting the latebra, the nucleus of Pander (yolk-bed), and the latebral neck all take origin from her endoplasmic zone, and she repeatedly affirms that their formation is to be attributed in large part to a special nutritive action emanating from the nucleus, but provides no positive evidence in support of that affirmation. She presumes that the axial white yolk subserves a purely nutritive function in relation to the germinal disc, but neither in her account of vitellogenesis nor in that of any subsequent author is any indication given of the source of origin of the new yolk-spheres, which we must suppose are added to those originally formed, as the oocyte increases in size, unless of course that increase is to be attributed solely to growth in diameter of the spheres in question.

Van Durme states that her peripheral yolk—zone, which furnishes the peripheral layer of white yolk characteristic of the Bird’s egg, is still present at the beginning of the third phase, but there is no suggestion that this layer of white yolk contributes to the underlying yellow yolk, and there is no evidence in later oocytes of the production in situ of new yolk—spheres between the fully formed spheres. Van Durme, when dealing with late oocytes belonging to her third phase, states that the Very large yolk—spheres are separated by a fine interalveolar cytoplasmic network, but we have failed to find such a network between the spheres in the largest ovarian oocyte (4-8 mm. in diameter) of the Sparrow we have been able to examine, and in the Monotreme it is no longer present in oocytes of a diameter of about 2 mm. The point we want to make is that, once this cytoplasmic network has disappeared, yolk-spheres do not arise de novo in amongst those already formed, and so we are left with the cytoplasm of the latebra as the only possible source of origin of new spheres.

So far as we can find, no detailed study of the latebra of the oocyte of the Bird at successive stages in its growth has ever been made. Konopacka (1933) makes passing reference to its structure in an oocyte of 12 mm. diameter, describing it as consisting centrally of lilac-staining granules, which are gradually transforming themselves into a layer of white yolk. Marza and Marza (1935) describe, no doubt quite accurately, the different layers of yolk that compose it in an oocyte of 20 mm. diameter, but their attempt to provide a “ histo-physiological explanation ” of its formation does not seem to us very successful, since for them “ the latebra is the yolk of the second phase of yolk-formation, pushed back deeper and deeper by newly formed layers of yellow yolk.”

We have ourselves examined the latebra in an ovarian oocyte of the Sparrow measuring 4-8 mm. in diameter. At this stage the latebra consists centrally of a vacuolated cytoplasmic core (about 0-72x0-36 mm. in diameter) completely devoid of yolk or other granules, which merges peripherally into a narrow, more coarsely vacuolated zone (about 0-17 mm. in width). In the inner part of this zone there occur minute eosinophil granules intermingled with slightly larger spheres possessing an eosinophil periphery and a basophil core. Traced outwards these latter spheres become transformed into larger uniformly basophil spheres, and these, still further increased in size, acquire at the periphery of the zone enclosing eosinophil mantles. From there they pass out to become incorporated in the narrow ring of white yolk (021 to 0'3 mm. in thickness) which forms the outer part of the latebra] body. At the junction of this ring with the broad zone THE DEVELOPMENT OF THE .\IOl\'0TREI\-IATA. 513

of yellow yolk—spheres which constitutes the main bulk of the oocyte, it can be seen that the basophil white yolk-spheres break down to form numerous small blackstained granules, which become distributed throughout the matrices of what are now large yellow yolk-spheres, precisely in the way Konopacka (1933) has described.

These observations suggest that the latebra of the Bird is fundamentally of the same nature as that of the Monotreme. The vacuolated central core is clearly a comparable formation in the two, whilst the more coarsely vacuolated zone which surrounds the central core in the Sparrow, and in which yolk-formation is in progress, evidently corresponds to the latebral yolk-zone in the Monotreme. We accordingly conclude that the latebra of the Bird has a comparable function to that of the Monotreme, viz., the production of yolk-spheres, which are added in the centrifugal direction to the yolk-mass and so serve to increase its bulk.

After having completed, as we thought, the foregoing section of the discussion relating to the growth of the oocyte, we remembered that we had not confirmed for ourselves the reference of Loyez to Sarasin’s views on the nature of the yolkspheres in Lacerta agilis. On looking up his paper, which is published in Semper’s ‘ Arbeiten,’ Bd. 6, we were agreeably astonished to find that already in 1883 he had advocated for the latebra or “ Dotterheerd ” as he terms it, of the egg of L. agilis precisely the same yolk-producing function as we have independently postulated for the latebra of the Monotreme egg.‘

Loyez refers to Sarasin’s “ Dotterheerd,” but she quite failed to appreciate the significance of his observations, and no subsequent writer who has dealt with the growth of the Sauropsidan egg appears to have been familiar with them. We therefore feel justified in providing a short summary of that section of his paper which dealswith the growth (“ Reifung,” he terms it) of the egg.

Sarasin begins his account of the growth of the egg with a description of the arrangement of the yolk in an oviducal egg at the stage of the first cleavage furrow. He describes the yolk as being arranged in alternating layers composed of coarser and finer yolk-spheres. These layers vary from egg to egg, not only in their degree of distinctness, but also in their size and form, and he suggests they are probably the expression of a periodicity in the growth of the egg, resulting from variations in nutrition, temperature, etc., affecting the mother animal. The layers are arranged concentrically around a curiously shaped mass which in the particular egg he is describing is situated centrally but it is frequently excentric. He was struck by the fact that the contours of the inner layers in particular, rather exactly reproduce that of the central mass, and this suggested to him the idea that the layers might owe their origin to this mass. If, he says, this idea is correct, then the inner mass ought to contain small immature yolk-spheres, and this he shows is the case. The whole central formation consists of fine yolk-granules, arranged in places in a net-like fashion, and these when traced outwards exhibit all transitional stages to large yolk-spheres. The conclusion that yolk is formed in the interior of the egg itself is, he says, confirmed by a-study of the growing oocyte. In oocytes 1-1-5 mm. in diameter he describes the interior as occupied by a protoplasmic network in which uniformly fine granules are situated. The beginning of yolk-formation is seen in oocytes 2-5-3 mm. in diameter. He recognises a rather broad outer zone of finely granular protoplasm, followed by a ring of yolk-spheres which centrally become progressively smaller and pass over without limit into the extremely fine granules which lie imbedded in the inner protoplasmic network which evidently corresponds to our vacuolated core. Towards its periphery the very fine granules increase to form small yolk-spheres and these, as mentioned, pass by way of intermediate-sized spheres into the larger spheres of the yolk-ring. He also describes the existence in the 3 mm. oocyte of a short, thin, conical process composed of fine yolk-granules, which passes down from below the nucleus to join the central core, but it is not always recognisable. It evidently corresponds to the latebral neck. In oocytes 3-5-4 mm. in diameter, yolk-formation has made further progress and already several distinct layers are present (three are shown in his fig. 3, Taf. xii.) which still enclose an extensive protoplasmic network. Comparison of oocytes of this stage with the oviducal egg shows that the central mass of the latter is to be regarded as the remains of the protoplasmic network, now somewhat altered in its constitution. The very fine granules of the early network have grown to form small yolk elements, but their arrangement around fine vacuolar spaces and the presence at the periphery of the mass of numerous spheres transitional between the very small and the large are clear indications of the original relations.

Sarasin states with reference to the protoplasmic-network that in all the eggs he examined, “ ein solcher gewissermassen embryonal gebliebener Thiel existiert,” so that he clearly realised that this network is none other than a central persisting part of the egg-cytoplasm, and embryonal in the sense that it is still capable of giving origin to yolk-spheres.

From his observations he is led to express the belief that the physiological significance of the central cytoplasmic network lies in yolk-production, and accordingly he designates it “ Dotterheerd,” but without thereby affirming that it is the exclusive seat of formation of new yolk. He shows, in fact, that yolkspheres are formed in the peripheral cytoplasmic layer of the oocyte, possibly here also from fine granules arising in the cytoplasm. Lastly, after a study of the structure and relations of the latebra (“ Dotterhohle und Stiel ”) in the ovarian egg of the Parrot, he comes to the conclusion that the “ Dotterheerd ” of the Reptile and the latebra of the Bird are comparable (analogous) formations with a comparable function.

Sarasin’s conclusions are therefore in complete accord with our own so far as concerns the significance of the latebra as a yolk-forming centre, and to this acute observer belongs the credit of having been the first to recognise some fiftyfive years ago that yolk-production is the primary function of the latebra of the Sauropsidan egg.

2. Yolk—Formation.

It appears to be usual for writers on the formation of yolk to begin with the statement that it is a very complicated process, and to that statement we subscribe. For an account of the histo-chemical aspect of yolk-formation in the Bird’s egg and the literature relating thereto the reader is referred to the papers of Konopacka (1933), Marza and Marza (1935), and V. D. Marza (1935). Here we are only indirectly concerned with these histo-chemical problems, since all we can do is to attempt to correlate our purely histological findings with the results arrived at by these workers after the employment of histo-chemical methods.

In its broad outlines yolk—formation in the Monotreme oocyte seems to follow along much the same lines as in the oocytes of Reptiles and Birds. The early oocyte starts out with a rich store of fat in the form of rather coarse droplets distributed throughout the cytoplasm, but leaving free the central region, which is occupied by the centrosphere and its enclosing mitochondrial zone. These droplets soon become located shortly below the periphery of the oocyte, where they form a definite cortical fatty zone, whilst the mitochondria become distributed more or less uniformly throughout the cytoplasm and the centrosphere disappears.

The first yolk-spheres (yolk-sphere primordia) make their appearance in the deep portion of the cortical fatty zone of the Echidna, oocyte, 0-29 X0-27 mm. in diameter, in the form of minute pale-staining spheres, homogeneous in character. Along with them occur specifically staining oval or rounded granules and aggregations of such, suggestive of swollen mitochondria. As the yolk-sphere primordia increase in number, so the fat-droplets undergo reduction, and by the time the oocyte has reached a diameter of about 0-4 mm. they appear as minute shrivelled remnants lying in the cytoplasm between the yolk-sphere primordia. The latter are now present in considerable numbers, and form with the remnants of the fatdroplets a well-marked cortical vitello-fatty zone, situated internally to the superficial mitochondrial zone forming the peripheral layer of the oocyte (Pl. III. fig. 17). The majority of the yolk-sphere primordia, both large and small, appear homo ’ geneous ; the remainder, including many of the smaller more superficially situated primordia, contain inclusions in the form of minute granules.

The origin of these yolk-sphere primordia is discussed, and the View is expressed that they are possibly derived from enlarged mitochondria. Perhaps of importance in this connection is the fact that they do not arise in preformed vacuoles, but appear in the form of minute spheres situated in the cytoplasm, and only later come to lie in vacuolar spaces. The direct derivation of yolk-spheres from mitochondria 516 PROF. T. THOMSON FLYNN AND PROF. J. P. HILL O.\'

has been affirmed by numerous investigators working on a great variety of eggs, but here We need only refer to the observations of van Durme (1914) and Brambell (1925) on the oocyte of the fowl. According to van Durme, the first undoubted yolk-spheres appear in the peripheral layer of the exoplasmic zone and are formed by the direct transformation of mitochondria. These increase in size, undergo a chemical alteration of their substance, and become enclosed each in a clear vacuole. According to Brambell’s description, as the mitochondria enlarge, a vacuolar space forms around each. In the smaller vacuoles a light coagulum is present which becomes denser in the larger vacuoles, and eventually forms a layer which he terms the M-C-yolk (our mantle layer) enclosing the yolk-sphere.

As concerns the later history of these first-formed yolk-sphere primordia in the Monotreme oocyte, it is evident from their staining reactions that they undergo marked changes in their chemical constitution as they increase in size and become enclosed in vacuolar spaces. Pale-staining and fragile-looking to begin with, they grow into dense homogeneous bodies which eventually stain intensely and uniformly with haematoxylin. These we regard as definitive yolk-spheres. They first appear at the close of phase 2, in oocytes just under 0-5 mm. in diameter (Pl. III. fig. 19) and are Well marked in the cortical yolk-zone of oocytes just over 0-6 mm. in diameter, belonging to sub-phase A of phase 3 (Pl. IV. fig. 20, Pl. V. figs. 21 & 22). In Pl. IV. figs. 22 & 22 B some of the spheres are seen to be enclosed in mantle-layers.

The next stage in yolk-formation is‘ heralded by the appearance of clear vacuolar spaces in the outer zone of the medullary cytoplasm. These vacuoles begin to form towards the end of phase 2 (Pl. III. fig. 19), and in sub-phase A of phase 3 (oocytes of about 0'6 mm. in diameter) they completely occupy the entire extent of the outer zone, situated between the cortical yolk-zone on the outer side and the central cytoplasmic core on the inner side (Pl. IV. fig. 20). It is in these vacuoles that the next crop of (medullary) yolk-spheres makes its appearance (Pl. IV. figs. 22 A & 22 B). As already mentioned (p. 510), a corresponding vacuolation of the medullary cytoplasm has been recorded by various observers in the oocyte of the Bird and Reptile.

Konopacka (1933), using a combination of histological and histo-chemical techniques, has provided an illuminating account of the formation of yolk-spheres from the contents of these vacuoles or droplets, as she terms them, in the oocyte of the fowl. According to her description, in the oocyte of 0-8 mm. diameter, with the cortical fatty zone established, homogeneous clear droplets composed of very liquid protein make their appearance in the cytoplasm not far from the centre of the oocyte. Under treatment with ordinary reagents the protein substance of the droplets is dissolved, and the clear spaces left appear as vacuoles. By the stage of the 1-2 mm. oocyte, the droplets occupy all the cytoplasm in~ ternally to the fatty zone, with the exception of a slightly excentrically situated core, and they also eventually appear in the fatty zone. Konopacka regards these droplets as the actual primordia of the yolk—spheres. The “ radial ” cytoplasmic zone has meantime been formed at the periphery of the oocyte, and across this there penetrate from the follicular epithelium granules of protein and the substances necessary for the synthesis of fats. The protein granules aggregate together to form larger spherules, and two or three of these penetrate into each primordium, or a single large one may so penetrate. According to the authoress, these spherules are composed of a more dense proteid substance combined with phosphatides. In later stages the fluid matrix of the clear droplet comes to contain a denser substance which is not dissolved by the ordinary reagents, a certain amount of fat penetrates into it, and its contained spherule increases in size and takes up an excentric position in the matrix. In this way, according to Konopacka, there is formed a white yolk-sphere, composed of an acidophil matrixsubstance derived from the protein liquid of the clear droplet as a basis and an enclosed basophil sphere formed of a protein in combination with a phosphatide. As we have already indicated, the matrix substance corresponds to What we have termed the mantle-layer and to Brambell’s M-C-yolk, whilst the basophil sphere corresponds to What is ordinarily termed the yolk-sphere.

On the basis of Konopacka’s observations we may presume that the fluid substance which occupies the medullary vacuoles of the Monotreme oocyte is likewise of a proteid nature, but in our material it has been completely removed by the reagents employed. The yolk-sphere primordia first appear in the vacuoles when the oocyte has attained a diameter of a little over 0-6 mm. (Pl. IV. figs. 22 A & B). The early primordia in the stage represented by these figures are most abundant in the inner part of the medullary yolk-zone, but they also occur throughout its thickness. Like those of the vitello-fatty zone, they appear as small rounded bodies composed of a homogeneous substance which stains faintly with eosin. They usually occur singly in the vacuoles, which they only partially fill, though occasionally two may be found together in one vacuole. As they mature they acquire a denser consistency and stain rather more deeply. Their transformation into definitive yolk-spheres seems to be effected along two slightly different lines. In some primordia the central portion of the matrix-substance undergoes differentiation to form a dense, more deeply staining rounded body or sphere, whilst the remainder forms a thin, lighter-staining envelope surrounding the latter. Primordia of this type, increasing in size, would seem to be transformed more or less directly into small definitive yolk-spheres, staining deeply and uniformly with haematoxylin. In other primordia, granules make their appearance In the matrix-substance. They stain more deeply with eosin than the latter and vary in number and size. They may be small and fairly numerous or, more commonly, they are larger and less numerous, and not infrequently they vary in size, one or more large granules being accompanied by smaller. As the primordia of the coarsely granular type grow in size, so also their contained granules increase and, losing their acidophil character, stain lightly with haematoxylin. Finally, the matrix-substance seems to disappear, and the granules now staining more deeply with haematoxylin form a more or less loose cluster of small yolk-spheres situated in a vacuolar space. In the case of primordia with fine granules, these latter would seem to become directly incorporated in the matrix of the definitive sphere, which is normally perfectly homogeneous, though we have recorded the occurrence of spheres completely filled by refractive spherules. The mantle-layers which eventually appear around the definitive spheres are presumably derived as in the fowl from the fluid-substance which fills the vacuoles, and which must have undergone some alteration in its chemical constitution, since the mantle-layer is capable of withstanding the action of ordinary reagents.

In connection with the above-described multiple formation of small yolk-spheres from a single granule-containing primordium, it is of interest to note that Loyez (1906, p. 177), in her account of yolk—formation in Anguis and various species of Lizards, describes the formation of small yolk-spheres through what she believed to be a process of budding on the part of the first-formed spheres, the sphere covered with its buds presenting, according to her description, the appearance of a small morula. We are tempted to suggest that she may have been dealing not with a true budding process, but with the formation of small spheres from a granule-containing primordium in the way described above.

Formation of the Latebml Y ollc.

Our observations show that in the oocyte of Echidna a little over 0-6 mm. in diameter (Pl. IV. fig. 22 A) the constituent parts of the latebra are already clearly distinguishable. Its future body is represented by the central cytoplasmic core of the oocyte, the neck by a vacuolated tract of cytoplasm leading down from below the nucleus to join the central core, whilst a ring of coarsely vacuolated cytoplasm forming the periphery of the central core represents the site of the latebral yolk-zone. In the vacuoles of the latter, as Well as in those of the neck, the first yolk-spheres formed are minute basophil spheres, to the number of one or two in each vacuole, and fine spheres of this type are also the first to appear in the vacuoles of the yolk-bed below the germinal disc.

By the time the oocyte in Platypus has reached a diameter of just over 0-8 mm., the latebral yolk-zone is definitely established and is becoming active as the yolk-producing centre of the oocyte, but it does not reach full productivity until a somewhat later stage (oocytes of 1-5-2-O mm. diameter). The most striking and characteristic feature in the formation of the latebral yolk is unquestionably the multiple production of small yolk-spheres from a single primordium, a phenomenon we have recorded as also occurring in the formation of the yolk-spheres of the medullary yolk-zone, but in this latter site it is on a much smaller scale than in the latebral yolk-zone, where it reaches its greatest intensity.

The yolk-sphere primordia in the oocytes of Platypus Which are concerned with this multiple production are highly characteristic——indeed, unique bodies which we have termed yolk-vesicles. In Platypus oocytes ranging from 0-8 to 2 mm. in diameter, they are readily seen in the latebral yolk-zone and in the lower portion of the latebral neck where they are specially numerous (Pl. V. fig. 24, Pl. VI. figs. 26 & 31). They take the form of vesicular spheres, apparently fluid-filled, with very thin pellicle-like walls, on the inner surface of Which are situated numerous spherules at first minute and eosinophil, but later becoming larger and basophil. These spherules on being liberated from the vesicles furnish the fine yolk-spheres of the latebral neck as well as the small yolk—spheres of the latebral yolk-zone. The yolk-vesicles themselves are formed by the enlargement and vacuolation of granulecontaining eosinophil primordia which are of common occurrence throughout the latebral yolk-zone and the basal portion of the latebral neck. Such yolk-vesicles, curiously enough, are not normally found in the latebra of Eckidua, but there occur in the cytoplasmic network of the latebral body, in oocytes about 1 mm. in diameter, large numbers of minute eosinophil spheres similar to those of Platypus, in the matrix-substance of which appear very fine granules, at first eosinophil, but later becoming intensely basophil (Pl. VIII. fig. 37). In later oocytes (about 2-5 mm. in diameter) such spheres, now larger, are specially abundant in the periphery of the central cytoplasmic core of the latebral body, and from there they extend out amongst the small yolk-spheres of the latebral yolk-zone (Pl. VIII. fig. 39). They are readily distinguishable owing to the presence in their matrix-substance of numbers of basophil spherules, really minute yolk-spheres, Which may be of fairly uniform size or one or more may be larger than the others. Since composite yolk-spheres do not normally occur in the yolk-mass, We conclude that these basophil spherules are liberated on the disappearance of the matrix-substance of the parent spheres and furnish the small yolk-spheres of the latebral yolk-zone.

Latebral yolk-formation i11 both Monotremes would thus seem to be eflected, not perhaps exclusively, but in the main, by the multiple production of small yolk-spheres from eosinophil primordia, that production involving in Platypus the formation of the unique vesicular bodies We have termed yolk-vesicles, Whereas in Echiolna these same primordia Would seem to give origin to small yolk-spheres directly, Without passing through a vesicular phase.

3. Germiual Disc and Nucleus.

As Gatenby (1922) has emphasised, the polarity of the Monotreme oocyte is very early established. In the earliest oocytes encountered in the ovary the nucleus already occupies an excentric position close below the surface, and that position does not change, but is maintained and accentuated during the growth of the oocyte, so that if We draw a line passing in the early oocyte through the centre of the nucleus and through the centrosphere, that line will mark out the 4 A 2 egg-axis. A localised area of the peripheral cytoplasm overlying the nucleus is destined to give origin to the germinal disc at the upperpole, whilst the axial tract of cytoplasm underlying the nucleus and the central mitochondrial zone will together participate in forming the latebra. It is therefore clear that it is the nucleus which determines the organisation of the oocyte, but what factor or factors determine the position taken up by the nucleus itself we do not know.

The structure of the nucleus of the early oocyte is described in some detail (p. 460, Pl. I. figs. 3-6), and the conclusion is reached that the chromatin occurs in the form of small granules situated in the strands forming the pseudo-reticulum. A large acidophil nucleolus is constantly present as well as smaller nucleolar granules. Except for an increase in size, no noteworthy change occurs in the nucleus until we reach sub-phase B of phase 3. At the beginning of that phase, in the oocyte of 0-6 mm. diameter, the nucleus is still approximately spherical (Pl. IV. fig. 20). It has now almost reached its definitive peripheral position and lies below and in contact with the primordium of the germinal disc, which first becomes recognisable at this stage. In the oocyte of just over 0-8 mm. diameter (Pl. IV. fig. 23) the nucleus is no longer spherical, but has assumed an ovalish form, its long axis being disposed more or less parallel to the disc and its upper surface tending to be flattened against the same. In the 2 mm. oocyte (Pl. IX. fig. 43) it has acquired a plano-convex lens-shaped form, its flattened upper surface lying in contact with the underside of the disc and its long diameter being about three times greater than its thickness. Although this change in shape only becomes apparent after the appearance of the disc—primordium, it is undoubtedly to be correlated with its presence.

The germinal disc-primordium first appears in the oocyte of 06 mm. diameter (Pl. IV. fig. 20) as a localised thickening of the peripheral cytoplasmic zone overlying the nucleus. It is devoid of a definite peripheral boundary, the discthickening simply merging gradually into the cytoplasmic zone, a condition which persists throughout the growth-period.

VVhen the latebral neck becomes defined in the oocyte of about 0-8 mm. diameter, it is seen to be continuous above with a narrow layer of cytoplasm, less coarsely’ vacuolated than that of the neck, which underlies the nucleus as well as the discprimordium. In this layer fine yolk-spheres are laid down, and so there is formed the fine—grained yolk-bed on which the nucleus at first rests (Pl. V. fig. 24, and P1. VI. fig. 27).

Except that the disc-primordium increases in area and in thickness as the oocyte grows, no essential change is noticeable in it until the oocyte has attained a diameter of about 3-5 mm. At this stage in growth an important advance is seen to have taken place in its str ucture, inasmuch as it is now distinguishable into two layers, viz., (a) a superficial zone of homogeneous, finely granular, “formative” cytoplasm, really the remains of the cytoplasm of the original disc—primordium, and (b) an underlying zone continuous with (a) and formed of vacuolated cytoplasm, rich in fine yolk-spheres, which merges below into the underlying yolk-bed. It differs from the latter in its much more open character and the much smaller size of its contained yolk-spheres.

The nucleus, as the result of the formation of this deep zone, has now come to be situated entirely within the disc.

Except for some increase in area and in thickness, the disc so constituted (Pl. XI. fig. 50) persists without any marked change up to the time of ovulation. Pl. XI. fig. 54 illustrates its condition just prior to maturation and Pl. XIII. fig. 65 its condition just after the separation of the first polar body.

The deep zone of the disc makes its appearance in the interval between the growth-stages represented in P1. IX. fig. 44 and P1. X. fig. 48, so that we have not been able to trace its origin, but it seems clear that it is derived, at least in part, from the cytoplasm of the original disc-primordium, indications of the extension of which to underlie the nucleus are to be seen in earlier oocytes (Pl. IX. figs. 43 & 45). In how far the yolk-bed contributes to it at its first appearance we cannot determine, but there is fairly definite evidence that once established the yolk-bed is utilised in its subsequent increase in thickness (Pl. XI. fig. 50).

The formation of this deep zone, with its fine yolk-spheres, bears witness to the onset of a greatly increased growth-activity in the disc, and is clearly anticipatory of the marked increase in the formative cytoplasm, which is such a striking feature in fertilisation stages (Pl. XV. fig. 77, and P1. XVI. fig. 82).

We have not been able to find any detailed account of the origin of the germinal disc in the Reptilia, Loyez (1906) giving practically no information on the subject, but van Durme (1914, p. 124) provides a description of its formation in the fowl. According to her account, as the nucleus approaches its definitive peripheral position at the upper pole of the oocyte and assumes a plano-convex form, the vacuolated nuclear cap (derived from the fatty nuclear cap of the early oocyte) which overlies it, becomes displaced laterally, its vacuolated cytoplasm being incorporated in the thickened portion of the cortical mitochondrial zone (“ perinuclear vitellus ”) which surrounds the nucleus and which is characterised by the fineness and abundance of it contained yolk-spheres. As to the nuclear cap, van Durme considers that it probably represents “ la toute premiere ébauche du vitellus plastique définitif.” However that may be, it may be concluded that in the fowl the germinal (disc originates as a perinuclear thickening of the cortical mitochondrial zone, which increases at the expense of the localised cytoplasmic mass or nuclear cap originally situated between it and the nucleus.

Whether a nuclear cap of the type described by van Durme is present in the oocytes of Reptiles remains to be determined. Possibly it is represented by the patch of modified cytoplasm described by Loyez (1906) as lying immediately above, and in contact with, the nucleus in the oocyte of Tropidonotus vipermus (her pl. iv. fig. 28) and in the oocyte (about 10 mm. in diameter) of Vipem aspis (her pl. v. fig. 108). There is no trace of such a formation in the Monotreme oocyte. 522 PROF. T. THOMSON FLYNN AND PROF. J. P. HILL ON

Later H istory of the Nucleus.

As the figures included in Pls. IX., X., and XI. demonstrate, the nucleus, during the growth-stages following on the 2 mm. oocyte represented by P1. IX. fig. 43, undergoes a progressive and very marked increase in size and a very striking alteration in its form from plano-convex to saucer-shaped. The nucleus in P1. IX. fig. 43 measures in diameter O-124 X0-13 mm. and in thickness 0-034 mm., the corresponding measurements of that of P1. X. fig. 51 are O-165 X0-14 X0-O25 mm. and of that of Pl. XII. fig. 55, 0-159 ><0-11><0-0258 mm., so that during growth the nucleus increases very definitely in diameter, but decreases somewhat in thickness, its maximum diameter being some five or six times greater than its thickness. Its final form is that of a large, somewhat flattened, saucer-shaped body, the under surface of which is convex and the upper surface concave (Pl. XII. fig. 55). During the early stages of this growth, the nucleus comes to lie so close below the egg-membrane that it is separated from the same only by a quite thin layer of disc-cytoplasm, but as growth proceeds the latter thickens irregularly and minute yolk-spheres are formed in it. As the result, the underlying nuclear membrane, on the upper surface of the nucleus takes on an irregularly wrinkled appearance and develops inbayings of varying size, occupied by disccytoplasm, containing fine yolk-spheres in small numbers. Eventually there is formed a large inbaying or concavity more or less centrally situated and filled by greatly vacuolated disc-cytoplasm containing numbers of fine yolk-spheres (Pl. X. fig. 49, Pl. XI. fig. 52, and P1. XII. fig. 55). In this way the nucleus attains its characteristic form, that of a shallow saucer, the wide rim of which is separated from the egg-membrane by a quite thin layer of disc-cytoplasm, whilst its central concavity is filled by vacuolated disc-cytoplasm as just described. It may be suggested that the originally thin layer of disc-cytoplasm enclosed between the nucleus and the egg-membrane is overstimulated, so to speak, by the relative excess of nutrient material passed into it from the adjacent follicular epithelium and so undergoes local hypertrophy, thus inducing the wrinkling and inbaying of the underlying nuclear membrane.

A corresponding growth of the nucleus to form a large flattened pancake-like body is described in late oocytes of Reptiles and Birds by Loyez (1906) and by van Durme (1914) in the fowl, but it would appear that in these forms its upper surface comes to lie in direct contact with the egg-membrane, no disc-cytoplasm intervening between the two as in the Monotreme, and so it remains approximately flat or becomes at most only slightly concave (Loyez).

During the later stages in the growth of the oocyte, the nucleus not only increases in size and alters in form as just described, but it also exhibits certain striking internal changes. These comprise (a) the appearance of what we have regarded as linin-threads, bearing minute granules of chromatin, and (b) a notable increase in the number and size of the nucleoli.

There appear to be two distinct types of nucleolar bodies in the Monotreme nucleus. Type ((1) includes nucleoli which reach a relatively large size (up to 0-021 mm. diameter), are mostly spherical in form, and in late stages stain intensely and uniformly with haematoxylin, though in earlier stages (Pl. I. fig. 5, Pl. V. fig. 24, and P1. IX. fig. 43) they may contain granular or vesicular inclusions and are at first intensely eosinophil. Occasionally such large nucleoli are irregular in outline, and appear as if they were giving origin to smaller spherules by a process of budding, such as Loyez (1906) describes as occurring in the nucleoli of Reptilian oocytes. Usually one, sometimes two of these large nucleoli are present in the nucleus along with a variable number of quite similar but smaller spherules. Type (b) comprises very much smaller and much more numerous nucleolar bodies or granules as we have termed them, which are more or less closely associated with the pseudo-reticular strands. They vary somewhat in size from nucleus to nucleus (about 0-0014 mm. in the nuclei in P1. X. fig. 48 and P1. XII. fig. 55, up to 0-0022 mm. in P]. X. fig. 51, and up to 0-0036 mm. in P1. XI. fig. 53, Where they are exceptionally large). They appear less dense than the (a) type, and stain in varying degrees of intensity with eosin, and sometimes also with haematoxylin, but never quite so intensely as the (a) type. It is noteworthy that they become more numerous and larger at the time of formation of the deep vacuolated zone of the disc (Pl. X. fig. 48).

Loyez (1906, pp. pp. 160-162) shows that in the Reptilia the nucleoli Vary greatly in number, size, and disposition in the various species she has investigated, being specially abundant in the nucleus of the oocyte of Vipera aspis, and she discusses their origin and fate in considerable detail. Her discussion, however, does not help us towards an interpretation of their significance in the Monotreme and we ourselves have no suggestions to offer.

It may be recalled that in describing the nucleus of the early oocyte (Pl. I. figs. 5 & 6) we had difficulty in locating the chromatin, and finally arrived at the conclusion that the larger granules in the pseudo-reticular strands were probably chromatinic. No further light on this problem is encountered until we reach oocytes in the last stages of phase 3 (Pl. X. fig. 49, Pl. XI. fig. 53, and P1. XII. fig. 55). In the central region of the nucleus in these oocytes there is present a system of very fine threads forming what seems to be a very loose open network, or quite possibly a pseudo-reticulum, and in these threads are situated at intervals very fine granules. We have interpreted them as linin-threads and the granules as chromatinic. When the threads first make their appearance in the stage represented by P1. X. figs. 48 & 49 the pseudo-reticular strands are no longer recognisable as such, and we suggest they are derived from the latter—a suggestion which is supported by the condition of the pseudo-reticulum in the nucleus of the oocyte of Platypus X V (Pl. X. fig. 47), Where the pseudo-reticular strands, in which are situated minute granules, are quite thin and very similar to the linin-threads.

The saucer-shaped nucleus of P1. XII. fig. 55, in which the linin-threads are very clearly seen, is the most advanced nuclear stage we have obtained, and is of importance as indicating the condition of the nucleus at the conclusion of phase 3 and shortly before the onset of the first maturation division. The stage immediately preceding the latter event we have not succeeded in finding.

We have not been able to find in the literature any descriptions of nuclei of oocytes at the end of the growth—period with a comparable organisation to that of the Echidna nuclei described herein.

In Reptiles, Loyez (1906) states that towards the end of the gr0Wth—period structures which she identifies as chromosomes, after several changes of form, finally appear as short filaments aggregated together to form a small group centrally situated in the nucleus, and from these the rodlets of the first polar spindle are derived. In the 2 cm. oocyte of Cistudo, for example, with a peripheral plano-convex nucleus, she figures the chromosomes as filamentous structures with fine granules aligned along them, situated near the centre of the nucleus and surrounded by a broad ring of nucleoli. VVhat appear to be similar filamentous bodies are shown by Munson in the nucleus of the corresponding stage of Olemmys (Pl. VII. fig. 87, cf. also fig. 85). These filaments distantly recall the linin-threads we have described. In the oocyte of the Fowl, shortly before the first maturation division, van Durme also describes and figures (pl. vii. figs. 62 & 63) a similar centrally situated group of small chromatin bodies, which are of very varying form and eventually just before that division condense to form spherules or short thick rodlets.

Amongst the Monodelphian mammals we have had no better success in finding oocytes with nuclei in any way comparable with those of the Monotreme. In the Cat, R. van der Stricht (1911) maintains that the nucleus of the oocyte never exhibits a true reticulate condition, but that the rings of the dictyate stage persist, and at the oestrous period the rings, now Inuchlcontracted and thickened, give origin to chromatin spherules representing the primordia of the bivalents of the first polar spindle. In Gama, Lams (1913) describes the nucleus of the oocyte at the end of growth as containing spherules of chromatin, mostly aggregated together to form a large irregular mass.

Our observations show that in the Monotreme the bivalents (“ chromosomes ” of Loyez) do not retain their individuality during the growth-stages of the oocyte, but must be reformed prior to the onset of the first meiotic division.

4. Follicular Epithelium and Formation of the Follicular Fluid.

Caldwell (1887) showed that in Echidua the follicular epithelium attains its maximum thickness and exhibits its maximum secretory activity when the oocyte is full-grown, and this we are able to confirm (cf. also Hill and Gatenby (1926)). In the earliest oocytes we have examined, the follicular epithelium consists of a single layer of flattened cells 0-003 to 0-005 mm. in thickness (Pl. I. fig. 3). In the full-grown oocyte it has become transformed into a conspicuous layer, which in Eckidmz attains a thickness of up to 0-048 mm. and in Platypus of about 0-034 mm., and is composed of greatly enlarged cells irregularly arranged to form a layer usually two, sometimes three, cells in depth (Pl. XII. figs. 55 & 58). In certain Reptiles (Lacertilia and Ophidia, Loyez (1906)) two types of cells, glandular and non-glandular, enter into the constitution of the follicular epithelium, but such is not the case in Monotremes, the cells being all similar in structure and function.

Text-figure l. Graph illustrating the relationship between the size of the oocyte and the thickness of the follicular epithelium and the zona during the growth and maturation of the oocyte.

When the thickness of the follicular epithelium is plotted against the size of the oocyte, as has been done for the epithelium in both Echidna, and Platypus in the accompanying graph (text-fig. 1), certain interesting facts are brought to light.

The curve shows that in both genera the growth of the epithelium readily falls into three periods as follows :—

(a) In the first period, corresponding to oocytes up to about 1-00 mm. in diameter, there is a rapid and continuous increase in the thickness of the epithelium.

(b) In the second period, corresponding to oocytes of 1-00 to just over 22-00 mm. in diameter, the epithelium, instead of showing an increase in thickness, undergoes a slight decrease. This happens in both genera. Here may be noted the curious and interesting fact that in these two periods the thickness of the follicular epithelium in Platypus is only about half that in Eckidna.

(c) In this final period, which corresponds to oocytes from about 2-00 in diameter to the full-grown condition (43 mm. in Platypus, 3-9 mm. in Echidna), there occurs a rather sudden and marked increase in the growth of the epithelium, at first much more accentuated in Platypus, so that in this genus when the oocyte has a diameter of about 3-00 mm. the epithelium is thicker than that of Eckidna. This reversal of conditions, however, is only temporary. In the case of Echidna active growth continues unabated up to the time of maturation, whilst in Platypus growth slows up to some extent, and so the curve tends to flatten out, the maximum thickness of the epithelium of the full—grown oocyte of Platypus being definitely less than that of Echidna, as we have noted above.

The beginning of sub-phase B of our third phase of oogenesis which comes into the second of these periods (b) is marked by the tendency. in both genera, for the follicular epithelium to enter on a two-layered condition. This is shown by the fairly frequent occurrence of mitotic figures, so arranged that the plane of division is tangential to the surface of the epithelium (Pl. IV. fig. 28). In the later oocytes of phase 3, however, mitoses are absent and the marked increase in the thickness of the epithelium which takes place as the time of maturation is approached is due, not to new cell-formation, but to the great increase in size of the existing cells. This increase is correlated with the onset of a specific secretory activity on the part of the epithelium (described in detail in the text, pp. 505-507, and referred to below) which results in the formation of a layer of colloid-like fluid situated between the inner surfaces of the follicular cells and the zona and completely surrounding the oocyte. This layer, the “ pro-albumen ” of Caldwell, we regard as the homologue of the “ liquor folliculi ” of other mammals.

The growth of the follicular epithelium of the fowl has been considered at some length by Marza and Marza, who publish a graph illustrating their conclusions (1935, diagram 3, p. 164). In the fowl the epithelium increases in thickness during the first phase of yolk-formation and the growth continues on into the second phase, reaching its peak about the middle of this phase at a time when the oocyte has a diameter of 25-30 mm. From then on the follicular epithelium undergoes a steady decrease in thickness.

Correlating this with yolk—formation, the authors state (p. 164) that “ the beginning of the precipitation of yolk into the ovule coincides with the beginning of the diminution of depth in the follicular cells. During the third phase, when 90 per cent. of the yolk which will constitute the deutoplasm of the mature egg is deposited into the ovule, their depth is very reduced (text-figure 20).” If we institute a comparison between our own results as shown graphically in text-fig. l and those obtained by Marza and Marza, we find :

(a) the preliminary steep rise shown to occur in the oocyte of the fowl is also found in Monotremes, but it extends into our third phase of growth, Van Durme’s phase 2 overlapping with our phases 2 and 3,

(b) in the fowl the peak period of epithelial growth is followed by a decline, but in the Monotreme the only decline noticeable is comparatively slight and of short duration, and follows the initial period of active growth,

(0) in the fowl the decline is continuous ; in the Monotreme, in oocytes of a diameter of just over 2-00 mm., the epithelium again begins to increase in thickness, in anticipation, no doubt, of the formation of the follicular fluid, and this increase is maintained up to the time of maturation.

So far as the oocyte is concerned, the function of the follicular epithelium may be regarded as threefold :—

(a) the secretion of the zona pellucida,

(b) the transmission and elaboration of the materials which are utilised in the growth of the oocyte,

(c) the secretion of the follicular fluid. Throughout the life of the oocyte, therefore, the follicular epithelium is a secretory layer of primary importance.

(d) Zona pellucida.

In the Monotreme oocyte, as we have seen, two layers are present between the follicular epithelium and the egg-membrane, viz., an outer homogeneous layer to which we restrict the term zona pellucida and an inner radially striated layer which we have termed the striate layer.

The various views relating to the origin of the zona have been summarized by Mjassojedoff (1923, p. 74), Schroder (1930, p. 342), and Corner (1932, pp. 15681571), and into a detailed consideration of these views there is no need for us to enter. As is well known, some investigators regard the zona as being a product of the surface of the oocyte, others, and to this category the greater number of modern investigators belong, are of the opinion that it is solely a product of the follicular epithelium, and this is the view that we ourselves advocate. Of modern authors, Mjassojedoff is alone in believing that both the follicular epithelium and the oocyte contribute to the formation of the zona. Most investigators come to the conclusion that the primordium of the zona consists of a series of anastomosing or interlacing strands, forming a kind of network, which afterwards becomes a more or less homogeneous layer. 0. Van der Stricht (mammals) and Miss Thing (turtle) regard this network as being formed at first by the terminal bars which close the intercellular spaces of the follicular cells. By the addition of further secretion the openings of the fenestrated membrane formed by the thickening of the terminal bars gradually become reduced until only fine pores are left, through which processes of the follicular cells pass to enter into connection with the cytoplasm of the oocyte. According to Mjassojedoff (1923) the zona consists at first of branching cytoplasmic processes of the follicular cells (“ periovular net ”), which become embedded in a gelatinous ground-substance produced by direct metamorphosis of the cytoplasm of the follicular cells. From this periovular network radiating processes pass to the oocyte and become surrounded by a fluid ground-substance derived from the surface of the oocyte. As evidence of this derivation he describes the presence at this stage of fluid-filled vacuoles in the superficial cytoplasm of the oocyte, and states that the egg-membrane disappears for the time being. This latter portion of the zona he calls the “ radiar Schicht.”

As we have pointed out above, the zona pellucida in the Monotreme is a perfectly homogeneous layer. We have not met with any evidence in this group to support the View put forward by 0. Van der Stricht (1923) and Thing (1918) that the terminal bars contribute to the formation of the zona inits early stages. Nor have we observed in oocytes of Platypus or of Echidua any processes of the follicular cells passing directly through the zona into continuity with the oocyte (as suggested by many authors) or forming a network (as described by Mjassojedoff). Neither have We met with any condition of fenestration or of pore-formation in the zona which would have for its object the passage of such processes.

The graph (text-fig. 1, p. 525) gives some information as to the variation in thickness of the zona during the life of the oocyte. In Echidua oocytes measuring up to about 030 mm. diameter there is a remarkably rapid increase in the thickness of the zona (P1. 11. fig. 13). In Platypus oocytes at this stage, although the zona thickens considerably, the increase is not nearly so striking, no doubt in correlation with the fact that the follicular epithelium is thinner than that of Echidua. In both genera, from now on, the zona begins to get thinner until in oocytes of approximately 2-5 mm. it appears as an extremely tenuous membrane of no measurable thickness. It would therefore appear that the follicular epithelium during the period of its temporary decline in thickness (text-fig. 1) finally ceases to produce the secretion on which the zona depends for its increase in thickness, and so from now on that structure behaves as an elastic membrane, becoming progressively thinner as the oocyte increases in size.

(ii.) Striate Layer.

The presence of a radially striated layer inside the zona (first recognized by Gatenby in the Platypus oocyte under the name of “ cortical fibrillae ”) is confirmed. Its structure is described (pp. 474~475), and it is shown that its “ fibrils ” are certainly not cytoplasmic. As to its mode of origin no final conclusion is reached.

As the growth of the oocyte proceeds, the striate layer becomes greatly reduced, but it does not disappear and at the time of maturation is replaced by the fluidfilled perivitelline space.

It is pointed out that in the possession of this layer the Monotreme oocyte agrees with those of Reptiles and Birds and differs from those of other Mammals.

(iii.) Formation of the Follicular Fluid.

As We have already recorded, Caldwell in his paper of 1887 (p. 472, pl. xxix. fig. 4) described, in the ripe ovarian ovum of Echidmz (3 mm. in diameter, so he states), the presence of “ a dense homogeneous substance ” situated between the zona and the follicular epithelium and formed as a secretion by the latter. He termed it the “ pro-albumen,” on the supposition that it absorbed fluid in the Fallopian tube and became the albumen layer of the uterine egg. Although we have no precise knowledge of the fate of this pro-albumen layer at the time of ovulation, we do know that it presents when seen in section a quite different appearance to that of the albumen layer. Moreover, C. J. Hill (1933), from her study of the histology of the oviduct, has come to the conclusion that the main part of the albumen layer is formed by the granular secretion of the epithelial cells lining the upper two-thirds of the Fallopian tube. It therefore seems justifiable to conclude that the pro-albumen does not furnish the albumen layer of the uterine egg, but is lost at the time of ovulation.

Caldwell’s discovery of this secretion is, nevertheless, of the greatest interest and importance, but owing to the fact that, up to the present, it has remained unconfirmed, and to the further fact that he himself regarded it simply as the precursor of the albumen, no one has taken the trouble to enquire whether it might not have another and quite different significance. In our opinion, it is to be regarded as the homologue of the liquor folliculi of the Graafian follicle of other Mammals. We have accordingly termed it the follicular fluid.

Its existence renders untenable the prevalent View that the Monotreme follicle differs from that of all other Mammals, and agrees with those of Reptiles and Birds in being solid throughout and devoid of any trace of follicular fluid.

In confirmation and extension of Caldwell’s description, we have shown that, in oocytes of Eckiclna undergoing the final phases of maturation, the follicular fluid forms a continuous layer composed of what appears in sections as a dense colloidlike substance or coagulum, often markedly vacuolated, which completely surrounds the oocyte and lies between the inner surfaces of the follicular cells and the zona (Pl. XIII. figs. 62, 65, & 68, and P1. XIV. fig. 70). We have further shown that in full-grown oocytes of both Eckidna and Platypus, prior to maturation, there are present in the cytoplasm of the follicular cells minute flaky bodies arranged more or less definitely in groups (Pl. XII. figs. 55 & 58). These We have regarded as secretory in nature and the precursors of the intercellular secretory material which appears a little later, in the form of irregular strands, droplets, and masses of a colloid-like substance situated between the follicular cells, and in all respects similar to that which forms the continuous layer of follicular fluid in maturation stages (Pl. XII. figs. 56, 57, 59, & 60). We have therefore arrived at the conclusion that the follicular fluid is the final product of the secretory activity of the follicular cells. In our material we have not been able to elucidate the precise nature of the secretory mechanism. We have seen no evidence of an actual shedding of the intracellular secretory bodies into the intercellular vacuolar spaces, and think it more probable that they undergo elaboration Within the cell and that the resulting product is passed in fluid form into the intercellular vacuoles. However this may be, the important point is that the follicular epithelium does secrete a fluid in Which, as Caldwell has well said, “ the whole egg is suspended,” so that the Monotreme, still possessed of a large macrolecithal ovum of really enormous size compared with those of other Mammals, but much reduced as compared with those of Reptiles, shows us the very first stage in the evolution of the fluid-filled Graafian follicle which is so distinctive of the ovaries of other Mammals *. As Brambell (1928) has pointed out, the acquisition by the Didelphia and Monodelphia of a follicle of this type, tensely filled with fluid, is no doubt to be correlated with the reduction their ova have undergone to minute microlethical bodies of microscopical dimensions and the consequent need of a mechanism such as the follicular fluid provides, for facilitating the bursting of the follicle and the transference of the minute ovum to the Fallopian tube.

The mode of formation of the follicular fluid in higher Mammals is a question which has been much discussed (see, amongst others, Schroder (1930, p. 342), Mjassojedoif (1923), Robinson (1918, p. 321), Regaud and Policard (1901) T). In the main, three methods of origin have been suggested: (1) by secretion from the follicular cells, (2) by the breakdown of the same, and (3) by the transudation of fluid from the capillaries in the thecal coat. Some investigators, indeed, attribute to the fluid a double origin. G. Levi (1903 and in litt.) holds that the fluid is formed partly as a secretion by the follicular cells, partly by the active destruction of these cells. As evidence of the latter process he instances the Call and Exner bodies, and points to the frequency of mitoses in the follicular cells, Which, iI1 his opinion, compensates for the active destruction that is going on, whilst he also points to the observations of Dogliotti (1926), which show that the mitoses become much more numerous when the follicles begin to form the follicular fluid. But other observers (Corner, 1932) are not inclined to attach the same importance to the Call and Exner bodies in view of the variability in their occurrence. Brambell (1928) states they do not occur in the mouse, and they are certainly not present in the Monotreme, and We may add that at no stage in the growth of the follicular epithelium in the latter have We observed any evidence of degeneration of the cells.

As concerns the possibility of a contribution to the follicular fluid through transudation from the thecal blood-vessels, all We can say is that the capillaries of the membrana propria are richly developed at the time of the appearance of the follicular secretion. It is obvious that the metabolic activities of the follicular cells are dependent on materials transmitted to them by Way of the blood-stream, and it is equally obvious that We have no means of telling whether or not such materials are directly added to the follicular fluid.

Certain of the Centetidae (H em-icentetes, Ervlc-ulus), according to the recent observations of Bluntschli and Goetz, and Strauss (Biomorph. 1, p. 281, 1938) lack a fluid-filled antrum folliculi. T C. R. Assoc. Anat. 3 Réunion, Lyon (1901). TABLE I.——Maturatlon Stages. The oocytes below were all fixed ln Bouin’s jlaicl.

Measurements in millimetres.

1)‘. V.

epithelium. 1% 5‘ Follicular i

Oocyte ( 1

(diameter). Th_ k I Celuayer E (iniiluilieri: ‘Diameter «X (thickness). l

l ( secretion). i‘ M ‘

Designation and date of 9 collection.

i Stage.

is !

Thickness . 1

MA Echidna,


3'64 (approx) .

0-026—0'043i Av.0'0-14. 1 p (av. 0'035). ‘ 3


_4-OO><3'3 p (compressed).

MB . Eohidna,

B.20.7.29. .

______m|___ ! E 3'75 0'02l-0'047. 1 ()'038—O'055 1' $066. 00084.

‘ (estimated). to as much pa

1 as 099.




4'50>< 3'43 . . . . (compressed). 1


MD . Echtdmz,


l;0'69. I . . . . e { _ ) ~ Nucleus. I

‘ Thickness

(including i vacuolated 5 zone). ‘

First polar . spindle, length 0-0093, breadthi 0'00-'36. 5

p First polar :

spindle, lcngtll 

1 1 0-0087.

i Second polar § spindle, length 3 0‘0O8, breadth __

0-0058. Remarks.

possibly abnormal. First polar body present, second polar chromo somes formed.

First polar body formed, second polar spindle present.

Fourth Phase. Maturation .

The three previously described oocytes (Echidm A.20.7 .29, C.20.7.29, and Platypus oz.l9.8.0l.) represent the stage of the full-grown oocyte. This has a. diameter of approximately 4 mm. in Platypus (4-2-4-3 mm.) and in Echidmt of just a little less (3-5-3-96 mm.). The nucleus has attained a characteristic saucer-like shape and the linin reticulum with its minute chromatin granules is distinct. The follicular epithelium and the theca interna have attained their maximum thickness, and in the former the follicular secretion is beginning to appear. We now pass to the description of four oocytes which enable us to give for the first time afairly connected account of the process of maturation in Echidmt. The list of stages available to us is given in Table I. (p. 531).

Stage MA.

Echtdna 16.7.29 (Pl. XIII. figs. 61 & 62). The oocyte (approximate diameter 3-64 mm.) is the earliest of our maturation stages and shows the first polar mitotic figure in the metaphase (Pl. XIII. fig. 61). The figure is situated in the superficial granular zone near the centre of the germinal disc, just under the egg-membrane and is disposed with its long axis nearly tangential to the surface. It is extremely small, the spindle measuring only about 0-009 mm. in total length and the equatorial plate (ring ?) of bivalents 0-0056 mm. in breadth. On one side (the right in the figure) the spindle fibres converge to a point occupied by a very minute granule, close to Which is another granule, but We are not prepared to suggest that the former granule is a centriole. On the opposite side the fibres converge to a more truncated extremity and there is no trace of granules. The bivalents of the equatorial plate are so clumped that We have found it impossible to make an accurate count, but the number is certainly not large. A rough estimate makes it 15. They have the form of oval granules of varying size.

D'isc.—In the ovarian oocyte the disc has no constant position in relation to the follicle, and as there is no external indication of its position the direction in Which it is cut is purely a matter of chance. In the present instance it was cut very tangentially, so that its thickness cannot be determined with any accuracy. We estimate its diameter as i0-73 mm. Its structure closely resembles that of the discs in previously described oocytes. It consists of a finely granular superficial cytoplasmic layer (Pl. XIII. fig. 61, fcd.), which passes over below into a finely vacuolated zone (vcd.) containing numbers of very small basophil yolkspheres, which become larger and more numerous in its peripheral region; also THE DEVELOPMENT OF THE MONOTREMATA. 533

present in this vacuolated zone are numerous pale eosinophil spheres (up to 0-003 mm. in diameter) possibly of nucleolar origin (cf. the eosinophil spheres in the nucleus in P1. XI. fig. 53). Pl. XIII. fig. 6], taken at a high magnification to show the details of the first polar spindle, includes only a small portion of the disc, suflicient, however, to show clearly the superficial granular zone and the deeper vacuolated zone above described.

Below the latter zone is the yolk-bed containing rather coarser basophil yolkspheres. Yolk-spheres of the type of the latter occur also in a small localised area of the central region of the disc below the first polar spindle. These may have spread up from the region just mentioned. On the other hand, but this we regard as unlikely, they may be formed by the yolk-spheres which in preceding stages are to be found in the concavity above the saucer-shaped nucleus and which, upon the dissolution of the latter, are free to become disseminated through the central part of the disc.

The Follicular Epithelium (Pl. XIII. fig. 62).—This shows an interesting advance on that of the preceding oocytes, inasmuch as the follicular secretion (f.s.), previously in the form of isolated strands and droplets, has increased in amount and has given origin to a continuous layer of c0lloid~like coagulum, by far the greater part of which lies between the inner ends of the follicular cells and the extremely thin zona. This coagulum appears as a dense, perfectly homogeneous substance which stains deeply with eosin, and in which are present numerous large vacuolar spaces, mostly oval. In its general characters it strikingly recalls the colloid substance of the thyroid vesicles, and on analogy with that substance we may suppose that it was laid down originally as a colloidal fluid and that the characteristically dense and vacuolated texture exhibited by it in section is the result of fixation and dehydration. It varies in thickness from 0-003 to as much as 0-028 mm., but we are unable to lay down any conditions which would determine the sites of the most abundant secretion, and these seem to have no relation whatever to the position of the disc.

In addition to forming a continuous layer at the deep surface of the follicular epithelium, the secretion also appears between the follicular cells, where it forms intercellular partitions suggestive at first sight of thickened exoplasmic membranes. These are in direct continuity with the main mass of secretion. Exceptionally, isolated masses of vacuolated coagulum may be found enclosed between a number of follicular cells remote from the zona. The follicular epithelium (f.e.) (including the follicular secretion) has an average thickness of 0-044 mm. (maximum 0-049 mm.), whilst that of the epithelium itself is about 0-035 mm. (maximum 0-043 mm., minimum 0-026 mm.). .

The follicular cells are arranged one to two cells deep and vary greatly in shape. They do not differ essentially from those of A and C 20.7.29 (Pl. XII. figs. 55 & 57), but their nuclei seem plumper and larger. The cytoplasm of the cells is dense, finely granular, and only occasionally vacuolated at the periphery of the cell. Frequently a denser perinuclear zone is distinguishable from a lighter peripheral zone.

The nuclear reticulum stains deeply and bears fine granules and also several irregular larger nucleoli, intensely stained. Occasionally also a large plasmosome may be present.

The tkeca mtema (Pl. XIII. fig. 62, th.z'.) has a thickness of only 0012 mm. in marked contrast with that of the Platypus oocyte, in which at a much earlier stage (Platypus X V, Pl. X. fig. 46) it has already attained an average thickness of 0-040 mm.

Perivitelline Space (Pl. XIII. fig. 61, pv.s.).—A characteristic feature of these maturation phases is the presence of a cleft-like space between the zona and the egg-membrane in the position occupied by the striate layer, comparable with that figured by Harper (1904) and Van Durme (1914) in the oocyte of the bird of the corresponding stage. This is the perivitelline space, and is so termed by van Durme, whilst Harper simply refers to the presence of a “ perivitelline liquid.” It is clearly seen in our stage MC (Pl. XIII. figs. 65 &; 66, pv.s.) where it is occupied by a granular material. In the present oocyte it is not recognisable in the sections as a space owing to the tangential sectional plane, but what we take to be the material filling it is seen in P1. XIII. fig. 61, just above the delicate egg-membrane, between it and the follicular secretion, as a narrow layer presenting the appearance of an irregular honeycomb. Stage MB.

Echidna B.20.7.29 (Pl. XIII. figs. 63 & 64).

This oocyte (4-0 ><3-30 mm. in diameter) is also at the stage of the metaphase of the first meiotic division. Judging by the appearance and size of the first polar spindle, and the indications of degenerative change in the follicular epithelium, it is not quite normal. That the condition of the follicular epithelium is not due to imperfect fixation is shown by the fact that the ovary, fixed in Bouin’s fluid, is otherwise quite well preserved. Although not quite normal, the oocyte is worthy, we think, of brief description.

The Germinal Dz'sc.—In this oocyte the disc does not occur in the exposed portion of the follicle. It is of normal appearance and is similar to that of the preceding oocyte. It has a diameter of _—_i-_0-72 mm. and a total thickness of about 0-056 mm. The superficial cytoplasmic zone is distinct, as also is the underlying finely vacuolated zone containing numerous minute yolk-spheres. The latter zone passes over below into the yolk-bed containing finer and coarser spheres. Details of the central portion of the disc are given at a high magnification in P1. XIII. fig. 63. The figure shows the first polar spindle (p.sp.l), which is situated in the superficial zone approximately in the centre of the disc. The mitotic figure is still in the metaphase, but has now rotated so that its axis lies almost but not quite at right angles to the surface. It is considerably larger than that of the preceding stage MA, the spindle having a length of 0-0145 mm. and the equatorial plate a breadth of 0-0087 mm. The corresponding measurements in oocyte MA are 0-009 and 0-0056 mm., and those of the second polar spindle in MD (Echialna B.2l.7.30) 0-008 and O-0058 mm. '

The spindle in this stage (MB) is therefore very large as compared with those found in the oocytes just mentioned, but whether the measurements fall Within the range of variation in size of the first polar spindle we have no means of determining. The spindle fibres (Pl. XIII. fig. 63) are thick and very distinct. On the deep side of the spindle they converge to form a typical truncated cone, but on its upper side they splay out fanwise and some of them at least appear to reach the egg-membrane. It is this arrangement of the spindle fibres which further tends to confirm our supposition that the oocyte is not quite normal.

The bivalents of the equatorial plate are less clumped than in stage MA, but though their number is small We have not been able to make a satisfactory count. They vary somewhat in size, and it looks as if they were in process of separating into their constituent chromosomes.

The Follicular Epithelium (Pl. XIII. figs. 63 & 64, f.e.) averages about 0-048 mm. in thickness. Over the disc region it is thicker, measuring 0-048-0-056 mm. As already indicated, the follicular epithelium exhibits evident signs of degenerative change. Over most of its extent the cells are poorly delimited; the cytoplasm presents a hyaline degenerate appearance, and vacuolation, intercellular and intracellular, is excessive. The nuclei vary greatly in size, and many of them are irregular in outline and tend to stain deeply.

The Follicular Secretion (Pl. XIII. fig. 64, f.s.) is present as a discontinuous layer of very varying thickness, situated between the epithelium and the zona and as irregular strands between the cells. It is very poorly developed in comparison with that of the preceding oocyte.

The egg-membrane and zona are both very thin, but distinct, and between the two there is a definite perivitelline space (pv.s.). This, above the disc, has a depth of 0-0014 mm., but over the remainder of the oocyte is so narrow as to be only just recognisable. It contains a material which, as in the previous stage, presents a vacuolated appearance. In succeeding oocytes this perivitelline space reaches a remarkable development.

Stage MC’. Echidna A.l.8.3O (Pl. XIII. figs. 65-68). This oocyte is of special importance, since it represents. the stage just after the separation of the first polar body. The chromosomes destined to form the equa torial plate of the second polar spindle are present in the cytoplasm immediately below the first polar body, there being as yet no trace of the fibres of the second polar spindle. The oocyte has an estimated diameter of 3-75 mm., its lower hemisphere being incomplete.

The germinal disc (Pl. XIII. fig. 65) is situated in the exposed wall of the follicle. It has a diameter of $066 mm., and is thicker and richer in cytoplasm in its central region than is the case in the preceding oocytes.

The superficial cytoplasmic zone of the disc (Pl. XIII. figs. 65 & 66, fcd.) is well marked, but thin, its thickness being about 0-0084 mm. It merges below into the much thicker finely vacuolated zone (vcd.) containing numerous very fine yolkspheres, the combined thickness of the two zones being about 0-05 mm. Though there is no definite limit between the two zones, the junction is easily recognisable owing to the superficial zone being practically devoid of yolk-spheres. It tends, however, to be wavy and irregular, owing to the presence here and there of short irregular cytoplasmic prolongations passing between the superficial and vacuolated zones. In this connection it is interesting to note that there occur, here and there, throughout the extent of the latter, small irregular patches of cytoplasm largely free from yolk-spheres, which seem to be related to the above-mentioned prolongations, whilst in the yolk-bed, almost directly below the first polar body and the chromosome group, is a larger patch of vacuolated cytoplasm (about 0-005 ><0-03 mm. in diameter) containing sparse fine yolk-spheres. The appearances suggest that cytoplasm is passing upward from the deep region of the disc to augment the superficial layer, the process being accompanied by the utilisation of the fine yolk-spheres of the deep zone.

The deep zone of the disc (vcd.) passes over below without limit into the yolkbed, which is rich in fine as well as somewhat coarser yolk-spheres. It continues out to the periphery of the disc, where it merges into the ring of medium-sized yolk-spheres surrounding the latter.

The first polar body has now separated ofl, and is seen lying in the perivitelline space, a little to one side of the centre of the germinal disc (Pl. XIII. figs. 65 & 66, 1012.1). In the figures it is a bizarre-looking structure, in appearance so unlike any other mammalian polar body known to us that it was some time before we actually convinced ourselves that it could be none other than the first polar body. It measures 0-0385 in length, 0-0084 in greatest vertical thickness, and 0-05 mm. in sectional thickness. As seen in sections (Pl. XIII. fig. 66) it appears as an elongated irregular structure presenting a curious undulating outline due to the presence along it of irregular indentations. It is bounded by a definite limiting membrane, and its interior is occupied by greatly vacuolated cytoplasm. In the cytoplasm in one of the sections (Pl. XIII. fig. 66) is a group of four larger and two smaller granules of chromatin.

Below the polar body (Pl. XIII. figs. 65 & 66) there occurs an irregularly wrinkled thickening of the egg-membrane (about 0-02 mm. in length), which no doubt marks the site of the gap through which the polar body passed into the perivitelline space. Directly below this, situated in the superficial cytoplasmic zone of the disc, is the group of chromosomes destined to form the equatorial plate of the second polar mitotic figure (ck7'.2), but there is, as yet, no trace of the spindle fibres. .

The chromosomes are in the form of rounded and oval granules varying somewhat in size, but owing to a certain amount of clumping We have not been able to make an accurate count. The number does not seem to exceed 20.

Follicular Epithelium (Pl. XIII. figs. 65, 67 & 68, f.e.). This resembles generally that of stage MA and calls for no detailed description. Intercellular vacuoles are more numerous than in preceding stages, and, with the increase in the amount of follicular secretion, there is a more marked tendency for the follicular cells and their nuclei to be extended tangentially to the surface of the oocyte. Its thickness varies a good deal. Over the disc it has a thickening of 0-055 mm., including the secretion (f.s.). Excluding the latter it measures from 0-021 to 0-047 mm. Elsewhere its total thickness varies from 0038 to 0-O55 mm., and in places reaches as much as 0-09 mm.

The follicular secretion (f.s.), unlike that of stage MA, is finely granular in character. It varies in thickness from 0-004 to 0-047 mm. and is definitely more abundant than in the preceding oocyte. It forms a complete investment to the oocyte outside the zona. Vacuoles between it and the follicular cells are markedly developed. The zona (21).) is thicker than in preceding oocytes, appearing as a double-contoured membrane about 0-007 mm. thick. It is thickest over the disc, much thinner over the rest of the oocyte.

An interesting advance on preceding oocytes is to be seen in the marked enlargement of the perivitelline space (pv.s.), which, above the disc, has a depth of 0-0098 as compared with 0-0014 mm. in stage MB. It lies between the zona and the much thinner, but very definite egg-membrane. The space is here seen to be filled by a finely granular coagulum, much less dense than the follicular secretion. It narrows somewhat toward the periphery of the disc, but continues some distance beyond its margin as an easily recognisable space before becoming reduced to a quite narrow slit.

There is little doubt that the perivitelline space is formed in the site occupied by the striate layer in earlier oocytes.

Stage MD. Echidna B.2l.7.03 (Pl. XIV. figs. 69 & 70).

This oocyte presents us with a further stage in the maturation process, the second polar spindle being present. The oocyte is therefore valuable as being at the stage

just prior to ovulation. The germinal disc (Pl. XIV. fig. 69) is situated in the exposed wall of the follicle, 538 PROF. T. THoMsoN FLYNX AND PP.Ol~‘. J. P. HILL ox

and has a diameter of about 0-69 mm. It possesses a well-marked, finely granular superficial zone (fc-d.), in which the second polar spindle (p.sp.2-) is situated. This zone passes over below into a conspicuously vacuolated region (vcd.), not so rich in fine yolk-spheres as iii the previous stage, but containing also numerous fine eosinophil spherules. In its central region, the disc has a thickness of 0-05 to 0-06 mm., but is difficult to measure accurately owing to some damage to the yolkbed in sectioning. In this and the preceding oocyte there is a greater concentration of the disc material in the central region than in previous stages. The disc has therefore made further progress towards its final form.

The second polar spindle (P1. XIV. fig. 69, p.sp.2) is situated approximately at the centre of the disc in the superficial cytoplasmic zone. It measures about 0-008 in length Xabout 0-0058 mm. in width. The spindle lies directly below the first polar body (pb.l ), and is disposed slightly obliquely both to the egg-membrane and to the plane of section. It is relatively short and stumpy, with the spindlefibres quite distinct. The chromosomes of the equatorial plate are not compactly arranged. Some are still paired, others have moved towards the poles, indicating the commencement of the anaphase. They are in the form of oval and rounded granules, varying slightly in size. VVe estimate their number to be about 18.

The first polar body (Pl. XIV. fig. 69, 1013.1) lies in the perivitelline space (pv.s.) just over the spindle. It measures 0-030 in length, 0-009 in vertical thickness, and 0-04 mm. in sectional thickness, so that it is comparable in size with that of the preceding stage. It shows the same elongated, irregularly lobed and indented form as in the latter oocyte, and the same structural characters. It should be noted that in the section figured (Pl. XIV. fig. 69) it is not seen at its full length. The chromatin is represented by a small oval granule connected by a thin fibre with a group of three small granules and, in addition, by a larger fusiform body lying below the latter.

The follicular epithelium (Pl. XIV. fig. 70, f.e.) is one to two-cells deep. Over the disc, the epithelium and its secretion (f.s.) together vary in thickness from 0-06 to 0-07 mm., the epithelium varying from 0-025 to 0-039 mm., the secretion from 0-019 to 0-047 mm. Outside the disc the combined epithelium and secretion vary from 0-022 to 0-08 mm. (exceptional), the secretion itself from 0-05 to 0-0056 mm. The zona pellucida and the egg-membrane (Pl. XIV. fig. 69, zp. & em.) are much as in the preceding stage, and there is, as before, awell-marked perivitelline space (pv.s.), whose depth at the first polar body is 0-O11 mm.

Changes during the Period of Jllaturation. Phase of First Polar Body Formation (Stages MA and MB).

Nuclear 0hanges.———The first maturation spindle is formed; it lies at first with its long axis tangential to the surface of the disc, but later it rotates to become almost perpendicular to the disc-surface. The spindle is formed approximately at the centre of the disc and is very small, with a length of 0-009 to 0-014 mm. (but it is possible that the latter measurement is abnormal).

The Gerrninal Disc.——At the time of formation of the first polar body the disc has not yet attained its final form. Its periphery is ill-defined and it passes almost insensibly into the thin cytoplasmic zone which lies at the surface of the oocyte just below the egg-membrane. Structurally the disc consists of a superficial, finely granular zone and a deep, highly vacuolated zone containing fine yolkspheres. It may occur at any part of the wall of the follicle.

A perivitelliue space is developed round the oocyte and is becoming particularly obvious above the disc. Here it is seen to be filled, in the fixed oocyte, With a fine vacuolated coagulum. Above the disc the space is not so deep as it is in the second phase of the maturation period.

Follicular E’pithelium.——Both cells and nuclei have increased in size, and as a result of the continued activity of the cells there has appeared between the epithelium and the zona a continuous layer of follicular secretion. This is the “ proalbumen ” of Caldwell (1887). We have homologised it with the follicular fluid of the Graafian follicle of other Mammals (vide p. 529).

Phase of Second Polar Body Formation (Stages MC and MD).

Nuclear Ohanges.——The first polar body is present at or near the centre of the disc. Its measurements are practically the same in both oocytes of this stage (av. 0-038 >< 0-0087 X0-045 mm.). The second maturation spindle is approximately of the same size as the first.

The germinal disc is now beginning to acquire its final form. It has become more concentrated and its superficial zone is being augmented by the addition of cytoplasmic material from the deep vacuolated zone and the yolk-bed.

The perivitelline space has increased above the disc to a depth eight to ten times that of the previous stage and is filled with a finely granular coagulum.

Follicular Epithelium.—The cells and nuclei have still further increased in size. The layer of follicular secretion, though not equally abundant at all parts of the wall of the follicle, is complete.


Previous Investigations.

Only two papers have been published on this phase of Monotreme development.

The first of these is that of Gatenby and Hill (1924), in which these authors gave an account of what they supposed to be an unsegmented ovum of Ornithorhynchus, the sections of which had been cut and stained some twenty years before, but had not been described owing to the difficulty of interpreting the nuclear structures found therein.

We have now re-examined these sections, and after comparing them with our other material are satisfied as already mentioned (ante, p. 494) that in the process of preparing the egg for sectioning, and particularly in the removal of the membranes, the blastodisc had been accidentally torn off. It will therefore be necessary to correct the interpretation given by these authors of the structures found in this egg, as follows :—

(a) The cells regarded by them as polar bodies are probably disc-cells which have been left behind; they are indicated in their pl. X. fig. 1 by the small numerals 3 and 4 and are shown enlarged in figs. 3 and 5.

(b) The sperm-nuclei of these authors are so-called “ periblast ” or “ yolk ” nuclei, the existence of which in the Monotreme egg was at that time unknown. They are indicated by the numerals l and 2 in their pl. X. fig. 1 and are shown enlarged in figs. 2 and 4.

The other work referred to is that of Flynn (1930), who had at his disposal two stages of Eckidna, one at the stage of second polar body—formation, the other a fertilisation-stage showing conjugation of the two pronuclei. He came to the conclusion, and we are satisfied that this holds good also for the egg of Ornithorkynckus, that the egg of Echidna is not polyspermic.

Flynn’s two stages (FA and FE in Table II.) have been incorporated with the material for the present communication and of them new figures and descriptions have been given.

The Uterine Ovum, Albumen, and Shell-Membrane

Before proceeding to the description of our fertilisation stages, we may interpolate here some remarks on the uterine ovum and the secondary envelopes that are laid down around it, during its passage through the Fallopian tube.

During the breeding season, when the ovarian follicles are nearing maturity, the ovary is found to be completely enveloped by the membranous funnel (infundibulum) of the Fallopian tube, and between this and the surface of the ovary there is present in considerable abundance a thick viscous fluid, which, according to the unpublished observations of Dr. C. J. Hill, is secreted by nonciliated glandular cells situated in the ciliated epithelium which lines the funnel. This secretion doubtless facilitates the passage of the ovum into the tube proper As the ovum passes along the latter it is fertilised, and it becomes enclosed first in a coat of albumen and then outside that by the very thin shell-membrane which forms the basal layer of the shell. Its passage through the tube must be fairly rapid, for we have never succeeded in finding a normal ovum in the tube. We did obtain two eggs(E’chidna, FC & B.8.8.30) from the junctional region between the tube and uterus, but the latter proved to be abnormal and had evidently been retarded in its passage downwards. Whether fertilisation is effected before or after the albumen coat is laid down, we cannot say, but it is not uncommon to find numbers of sperms imbedded in the latter. As we have already recorded, the Monotreme ovum is normally monospermic, and we have also recorded the fact of the occurrence of sperms in the uterine lumen as well as in the uterine glands of Platypus XV, with oocytes not yet full-grown.

All our fertilisation stages were obtained from the uterus, and there cleavage begins. The ova in process of cleavage, which Caldwell (1887, pl. xxx. fig. 1, & pl. xxxi. fig. 3) records as having been obtained from the Fallopian tube, are clearly undersized and abnormal.

Egg-emJel0pes.—At the time of ovulation, the zona is an extremely thin membrane, and it apparently persists as such, but it becomes so closely applied to the deep surface of the albumen coat that its presence is not always detectable in sections of the fixed egg. '

The albumen coat, which is laid down in the Fallopian tube, outside the zona, appears in the fresh uterine egg as a quite thin, slightly cloudy, translucent layer, the thickness of which appears to vary from about 0-3 to 0-4 mm. Dr. C. J. Hill, who has made a further study of the secretory processes in the Monotreme oviduct, has been good enough to place at our disposal her unpublished results, and these, with those already published (1933), indicate that the albumen coat is formed of two distinct secretions. The first of these (a) is a dense, rather coarsely granular secretion, which is formed by the glandular cells in the epithelium lining the upper two-thirds of the Fallopian tube. This secretion is in process of being shed before ovulation takes place, and is produced in considerable quantity. It is regarded as furnishing the main part of the albumen coat, which appears in sections of the fixed egg as an irregularly laminated, dense layer closely adherent to the zona, the two together forming the “ zona-albumen layer,” as we have termed it. The second secretion (b) is much finer than (a), and when shed appears iI1 sections of the tube as a delicate, flocculent coagulum, quite different in character from secretion (a), and evidently of a more fluid character. It is secreted by the glandular cells in the epithelium lining the lower third of the tube and the adjoining upper portion of the uterus, and is formed in no great abundance. It appears to be shed at the same time as the secretion of the tubal glands, which Dr. Hill believes is concerned with the formation of the basal layer of the shell. This second secretion, Dr. Hill suggests, furnishes a more fluid portion of the albumen coat, which occupies the very narrow space between the dense albumen layer and the shell-membrane. These conclusions of Dr. Hill are supported by our own. The dense portion of the albumen coat, which forms, with the zona, the zonaalbumen layer, is readily seen in sections of the fixed egg (of. P1. XIV. fig. 71, Pl. XV. fig. 77, and P1. XVII. fig. 89 (zpa.)). It persists up to quite late stages and is composed of a dense, homogeneous substance, sometimes presenting an irregularly laminated appearance owing to the presence in it of minute clefts.

Caldwell (1887) identified the zona-albumen layer as the vitelline membrane (see his pl. xxx. figs. 2 & 3, and pl. xxxi. figs. 1, 2, & 4, mn.), and the granular coagulum, filling the relatively wide space, in the figures, between the latter and the shell as the albumen. If we are correct in our interpretation of the zonaalbumen layer, then Caldwell’s albumen would correspond only to the outer, more fluid part of the albumen coat. Caldwell’s representation of this layer in the above-mentioned figures is probably somewhat schematic; at all events, in our sections of eggs with the shell in situ we have never encountered such a dense coagulum as is depicted in his figures. In the Echidna egg illustrated in P1. XIV. fig. 71, for example, the space between the zona-albumen layer (210%) and the shell-membrane (sh.m.), here artificially enlarged, contains only “ traces of a coagulum, together with occasional fine fibrils, isolated or sometimes grouped to form thin laminae on the inner surface of the shell ” (Hill, 1933). On the other hand, in the 8-celled egg of Platypus, shown in P1. XVII. fig. 91, which was fixed in a picric fluid, the space in question, considerably enlarged as the result of the action of the fixative, contained small, irregular masses of a granular material dispersed through it. These are not shown in the above figure, but are clearly visible in pl. i. fig. 1 of Wilson & Hill’s paper (1907), and are doubtless the remains of the more fluid portion of the albumen coat. Again, in Echidna eggs VVH 1 and VVH 15, fixed in Smith’s fluid, which causes the shell to distend, a thin layer of clear jelly-like material was observed on the surface of the zona-albumen layer, after the removal of the shell. With a fine brush this material could be removed as translucent flakes to which foreign particles readily adhered. Possibly this material is also a remnant of the outer layer of the albumen coat.

On the basis of Dr. Hill’s observations and our own, we conclude that the albumen coat of the Monotreme egg is formed like that of the Bird of two substances: (a,) a denser material constituting the main bulk of the albumen and appearing in the egg after fixation as a definite, more or less laminated layer, to which the zona is so closely adherent that it is not always recognisable as a distinct membrane, the two together forming the zona-albumen layer; and (b) a more fluid material which occupies the normally very narrow space between the inner dense layer and the shell, and which in the fixed egg is poorly preserved, only slight remnants of it, varying in character with the fixative employed, being left.

The outer envelope of the early uterine ovum is constituted by the very thin, transparent shell-membrane, destined to form the basal layer of the shell. For an account of the formation and structure of the latter the reader is referred to the papers of Hill and Hill (1933). 4D2


in Collection and date of collection.


FA Echidnal, l 24.7.29.


FC Echiclna,


FD Echidna, VVI-I 26, 26.7.33.

T FE . . . . Echidna, B.30.7.29.


~ A.21.8.29.

TABLE II.—Fem'lisat13on Stages.

Ovum (measurements).

After fixation. Fresh. }[-—--—- -:——---v--—— Diameter.

With Without

shell. shell. 4'00 3'95 0'52


4'60 1 Shell not


4'50 0'52 X 049

'3 > O E 9-’ r-4

059 X053

(Measurements in millimetres.)

Disc (measurements).

i Thickness. jFixative.

0'O45 (super- ! Bouin. ficial cytoplasmic zone =0'016).

0'06 (cytoplasmic =0-O36).

0'07 (cytoplas- ’ Bouin. mic zone =0-036).

0'077 (cyto- S1nith’s plasmic zone fluid. =0'052). ‘

O'10(cytop1as- Bouin.

H110 Z0110



Two pronuclei present, sep:u'a.tecl by

0'09 mm.

"P1-onuelei overlapping.

i . . . .

Pronuclei in apposition. Pronuclei in conjugation (Flym1’s

Egg No. 2).

Second polar body extruded and pronucleus (Fl_vnn’s Egg No. 1).

establislied ‘

Fertilisation Stages

Of these we possess six, all of Eckidmz aculeata var. setosa, covering the period from the extrusion of the second polar body and the establishment of the female pronucleus to the fusion of the two pronuclei. These are listed in the accompanying table (Table II.).


Echidna 24.7.29 (Pl. XIV. figs. 71, 72, & 73).

This egg is one of the stages described by Flynn (Flynn’s Egg No. 1, 1930, p. 123, figs. 1 & 3). Its measurements, before and after fixation, are given in Table II.

The living egg at this stage is enclosed in a very thin transparent shell-membrane (Pl. XIV. fig. 71, sh.m.) whose thickness it is almost impossible to measure accurately (0-0013-0-0018 mm.). Through this shell and the semi—transparent zonaalbumen layer (2pa.) the position of the disc in the living egg could be faintly made out, but owing to the fact that the yolk in the living egg of Echidna (at any rate in the variety setosa) is white or greyish-white (Flynn, 1930), the disc offered no great contrast to its surroundings. In fixed and preserved eggs the disc can usually be seen quite easily, especially in those eggs in which a wide separation between the shell-membrane and the zona-albumen layer has occurred, as the result of the use of certain fixatives (see Pl. XVII. fig. 91, showing the 8-celled stage of Platypus).

The zona-albumen layer (Pl. XIV. fig. 71, zpa.) is irregularly laminated and varies in thickness from O-017 to 0-03 mm. The sections of this stage were cut in Tasmania, and no figure was made of the disc in surface view. Flynn (1930) described it as being circular, and this is borne out by examination of the sections. This is a feature of some interest since, in respect of its circular form, this disc differs from later fertilisation discs. Its diameter is 0-52 mm. (Flynn, 0-55). As compared with that of the mature oocyte, the disc shows greater concentration of the cytoplasm and a sharper peripheral delimitation, so that its diameter can now be fairly accurately measured. The superficial cytoplasmic zone (Pl. XIV. fig. 71, fed.) is thin centrally (about 0-016 mm.) but becomes thicker mid-way between the centre and periphery of the disc, where there is an irregular annular thickening. Here, we suggest, cytoplasm is being added to the superficial zone from below. The superficial zone passes over below into the deep vacuolated zone (vcd.). In the central region of the disc it is richly vacuolated, and contains numbers of fine yolk-spheres. Its thickness is about 0-03 mm., so that the total thickness of the disc in the central region is about 0-046 mm. Traced outwards, the deep zone becomes richer in fine yolk and its vacuolation becomes less marked. It thins out towards the periphery of the disc where it ends in the marginal zone. The THE DEVELOPMENT OF THE MOl\‘()Tl{E1\I.-XTA. 545

disc composed of these two zones is continuous below with the saucer-shaped, fine-grained yolk-bed (yb.), which continues out below the disc to the surface, where it forms a narrow marginal zone encircling the latter. Approximately at its centre, and directly above the region of the latebral neck, the yolk-bed exhibits a noteworthy differentiation. Here it is formed of a more or less localised mass of almost pure cytoplasmic reticulum which is very similar to the mass in the corresponding position in fig. 50 (Pl. XI.). The strands of the cytoplasmic network are thick and enclose obvious vacuoles containing minute yolk-spheres, but in no great abundance. We suggest that we have here going on the same process of active utilisation of the cytoplasm and the fine yolk of the yolk-bed in the upbuilding of the disc that we have already encountered in the ovarian oocyte (cf. p. 501 and P1. XI. fig. 50).

Nuclear Structures.

Fig. 72 (Pl. XIV.) shows the central portion of the disc much magnified. Here just below the egg-membrane is situated the very minute oval female pronucleus (gm. 9 ). It is enclosed by a distinct nuclear membrane and has stained intensely, but in its interior just below the membrane there are indications of still darker granules. It is present in one section and measures 0-0042 X0-0025 ><0-O05 mm. Just above the female pronucleus the disc is very slightly elevated, an indication that the second polar body (pb.2) has only recently been extruded.

The first and second polar bodies (Pl. XIV. figs. 71, 72 & 73, pb.l, pb.2) are present, both situated in the perivitelline space (pv.s.). This space contains a coagulum disposed in the fixed egg as an irregular thin layer on the upper side of the disc and on the inner side of the zona respectively, with delicate and sparse connecting strands running between the two. This coagulum surrounds both polar bodies (Pl. XIV. figs. 72 & 73).

The second polar body (Pl. XIV. figs. 71 & 72, pb.2) is situated in this space at the centre of the disc and appears as an irregularly rounded structure lying in contact with the egg-membrane, almost directly above the female pronucleus, and invested by the thin layer of coagulum which covers the surface of the disc. It is enclosed by a definite limiting membrane, and in the finely granular, almost clear, cytoplasm occupying its interior is a horseshoe-shaped body composed of a matrix in which are situated four or five darkly staining granules of chromatin. It measures about O-008x 0'0l0 ><O°0l mm. The first polar body(Pl. XIV. fig. 73, pb.1) is situated not quite in the centre of the disc, being separated from the second polar body by a distance of 0-025 mm. It measures about 0017 ><O-012 ><O°0l mm., and is thus much larger than the second polar body. It appears as a shrivelled body partially embedded in the vacuolated layer of coagulum situated below the zona-albumen layer. It is limited by a definite membrane, and in the clear cytoplasm inside are two darkly stained pear-shaped granules of chromatin connected by a thin filament.

Although we have included this ovum in our fertilisation stages, we have failed, as did Flynn (1930, p. 126), to find any trace of the male pronucleus, and this in spite of the fact that sperms are to be found embedded in the albumen layer.

It is accordingly of great interest to note that van Durme (1914, p. 162), from her study of the material of the Pigeon and Swallow (see her pl. xiv. figs. ll & 12) has reached the conclusion that the second polar body may be extruded in the absence of fertilisation. This is certainly not the case in Monodelphia, and apparently not the case in Marsupials (Hill, 1910, p. 22, 1918, pp. 104-5; Hartman, 1916, pp. l7—18, 1919, pp. 35-6).

The question is one of considerable interest, and clearly needs further investigation both in the Monotremata and the Sauropsida, especially as Harper (1904) in his work on the Pigeon insists that the second polar body is not formed until fertilisation has been effected.

The important features shown by this stage may be summarised as follows :—

(CL) The increasing concentration of the germinal disc and the retention of its radial symmetry.

((2) The presence of two polar bodies of unequal size and their position near the centre of the disc.

(c) The absence of the male pronucleus.

Stage FB. Echidna E.8.8.30 (Pl. XIV. figs. 74, 75, & 76).

This is the earliest of our pronuclear stages, and for this reason is of interest. Unfortunately, during preservation (Bouin’s solution) the shell-membrane contracted down on to the disc, so that it was impossible to remove it Without the risk of damage to the latter. For this reason it was impossible to see the external form of the disc or to arrange its orientation for cutting. The sections of this stage were made at a time when we were still in the experimental stage as regards our technique. Nevertheless, none of the sections is missing and, although they are not perfect, the preservation is very good, and we are able to make out with sufficient clearness the most important details.

The Disc.———It is not easy to be sure of the exact size and contour of the disc. Our sections show that it is elliptical in shape, but much longer and narrower than might be expected (its measurements being O-64><0-32 mm.). This is the more surprising since in the next stage, FC, the shape of the disc, although elongated in the direction of one axis, is very nearly circular (O-52 ><0-49 mm.).

The unexpectedly elongated elliptical shape of the disc of this stage may be due to individual variation; on the other hand it may be a result of temporary alterations in the shape of the disc immediately consequent on fertilisation, a phenomenon which is greatly stressed by Harper (1904) in the case of the Pigeon.

Sections show that an important advance has taken place in the structure of the disc (Pl. XIV. fig. 74). Its cytoplasmic zone (fcd.) has attained the plano~ convex form characteristic of all fertilisation stages from now on. The central portion of the cytoplasmic zone, very thin (0016 mm.) in the preceding stage, has now increased greatly in thickness. no doubt as the result of the addition of material from below, its depth (0036 mm.) being now more than twice what it was in stage FA.

The cytoplasmic zone is finely granular, with minute yolk-spheres irregularly distributed through it, and becoming much more numerous in its peripheral region. Below the cytoplasmic zone is the relatively thin vacuolated zone (vcd.), rich in minute yolk-spheres, and well seen in P1. XIV. fig. 76 below the pronucleus (pn. 6 ). The two zones have a combined depth of about 0-06 mm.

Nuclear Structures. Two pronuclei are present (Pl. XIV. fig. 74, pn.3 , gm. <3 ). Although they are shown in the same drawing, the two nuclei are in different sections (three sections apart). The direct distance between the two is about 0-09 mm. One of the nuclei is situated practically at the centre of the disc. It is shown highly magnified in P1. XIV. fig. 75. It is the larger of the two and measures O-O22 ><0-0154 X0-O2 mm. We regard it as the female pronucleus. It is enclosed in a delicate, slightly wavy nuclear membrane, and has a fine close~meshed nuclear reticulum supporting minute chromatin granules, and with a few larger granules in the meshes. The other pronucleus (Pl. XIV. fig. 76), regarded as the male, is situated rather more deeply in the disc, and is slightly smaller than the female, its measurements being 0-O21 X0-0150 ><O-02 mm. Its internal structure is similar, but the granules of chromatin are not quite so coarse.

At either end of the female pronucleus is a clear space, larger on the right side than on the left, possibly indicative of some disturbance or alteration in the perinuclear cytoplasm. This same condition, in even more marked degree, is also visible around the male pronucleus (Pl. XIV. fig. 76).

Stage F0.

Echidna C.l5.8.30 (Pl. XV. figs. 77 & 78). In this egg the male and female pronuclei are fully differentiated, though

they have not yet attained their full size, and partially overlap each other in one and the same section. The disc has made definite progress toward the attainment of its final structure.

The egg was found in the right uterus close to its junction with the Fallopian tube. Unfortunately, here again, owing to contraction during preservation, the egg-membranes have become adherent, and lie close against the surface of the germinal disc. The perivitelline space is thus obliterated, and we have been unable to find the polar bodies. The shell, according to Hill (l933,’p. 445), has a thickness of 00045 mm. and the zona-albumen layer, of 0-008 mm.

The disc (Pl. XV. fig. 77) is very similar in its general constitution to that of stage FA (Pl. XIV. fig. 71), but its cytoplasmic zone, like that of the preceding egg, is much thicker. Including the marginal zone, it measures 0-52 X0-49 mm. in diameter, and so is just beginning to assume the elliptical form which characterises the discs of the two following stages. It measures about 0-07 mm. in thickness. The sections are transverse to its longer diameter.

As in the preceding egg, the cytoplasmic zone (Pl. XV. fig. 77, fcd.) appears plano-convex in section and centrally, at the level of the pronuclei, has the same thickness (0036 mm.) as in that egg. It thins out towards its periphery and becomes richer in fine yolk-spheres before merging into the marginal zone. The underlying vacuolated zone (vcd.), mid-way between the centre and the periphery of the disc, has a thickness of about 0034 mm., and is rather densely laden with fine yolk-spheres. Traced centrally, this relatively thin zone is seen to pass into a very thick, richly vacuolated mass of cytoplasm containing numerous very fine yolk-spheres, which lies below and to the left (in the figure) of the centre of the disc occupied by the pronuclei, This mass attains a thickness of about 0-073 mm., and is nearly twice as wide. Its deep half, which is situated in the yolk-bed, is more coarsely vacuolated than its upper half (which is part of the vacuolated zone), and is clearly of the same nature as the vacuolated mass which occupies a corresponding position in P1. XIV. fig. 71, so that here again We have evidence of the utilisation of the yolk-bed in the growth of the disc.

At the periphery of the cytoplasmic zone the vacuolated zone and the yolk-bed reach the surface and together form a narrow but distinct marginal zone (mg.z.) containing fine-grained yolk, which surrounds the disc.

The Promcclei (Pl. XV. fig. 78).——These are found in the same section, partially overlapping, one being nearer the surface of the disc than the other. They differ somewhat in form and size and also in staining capacity.

The more superficial of the two is elliptical in form, is somewhat longer and narrower, and stains more lightly than the deeper nucleus. It measures 0-018 X0-0098><0-02 mm., as against 0-0175><-011><0-02 mm. for the deeper nucleus. The latter is ovalish in form, with one end more pointed than the other. It is more deeply stained than its companion, and overlaps the middle part of the lower half of the same. Both nuclei lie with their long axes disposed tangentially to the surface of the disc. The nuclear membrane in each case is distinct, as is the nuclear reticulum, which exhibits well-marked nodal thickenings. We regard the more superficial pronucleus as the female, the deeper as the male. If our conception of the extent of the disc, as stated above, is correct, then the two pronuclei‘ are to be found as near as possible at its centre, 2'. e. at the meeting place of the longitudinal and transverse axes.

Worthy of special notice is the fact that, over approximately one-half of the disc, and extending from the region of the pronuclei to the periphery, fine yolkgranules are present in the superficial part of the cytoplasmic zone, though in no great abundance (Pl. XV. fig. 77). The figure shows the region of the disc where the granules begin to make their appearance (also much magnified in P1. XV. fig. 78). They can be followed in subsequent sections till they merge at one end of the long axis into the fine-yolked marginal zone mentioned above. They have either been formed in situ or have been transported from the marginal zone by cytoplasmic movement. The latter seems a possible explanation in view of Harper’s findings as to cytoplasmic movement in the newly fertilised disc of the Pigeon (1904, p. 364). However formed, their presence is the first definite indication of the commencing replacement of the original radial symmetry of the disc by a secondary bilateral symmetry, the latter being fully established in the disc of our next stage.

Stage FD. Echiclna VVH 26, collected 26.7.33 (Pl. XV. figs. 79, 80, & 81).

In this stage the two pronuclei are in contact preparatory to fusion. This is one of the eggs collected by Dr. V. V. Hickman, and was preserved in Smith’s fluid. Removal of the shell was not difiicult, and the ovum was left as a small sphere, 434 mm. in diameter, enclosed in the delicate zona-albumen, through which the disc could be plainly seen. Over the disc this layer is 0-03 mm. thick. In Pl. XV. fig. 79 we provide a surface view of the upper pole of this egg, from which it can be seen that the germinal disc is no longer circular, but has assumed an elliptical outline. In the disc in surface view we can distinguish three zones, as follows :—

(a) An elliptical central area, broader toward the upper side of the figure and narrower toward the lower side.

(b) A narrow white zone enclosing (a), the outer margin of which is more definitely limited on the upper side in the figure than it is laterally and below, but which is wider towards the lower side of the figure than on its upper side.

(0) A darker, slightly mottled peripheral zone, surrounding (b) and appearing more distinctly limited and slightly narrower on the upper side in the figure than on the lower side.

Study of the sections which are accurately longitudinal (Pl. XV. fig. 80) shows that these regions correspond to the following :—region (a) to the thicker central portion of the cytoplasmic zone of the disc and the underlying vacuolated zone ; region (b) to the thinner peripheral portion of the cytoplasmic zone of the disc ; region (c) to the marginal zone, composed of a slightly vacuolated cytoplasmic basis containing fine-grained yolk and formed jointly by the vacuolated zone of the disc and the yolk-bed, where they reach the surface at the periphery of the cytoplasmic zone. In P]. XV. fig. 80 only a small portion of the marginal zone (mg.2.) is included, rather more on the right side (in the figure) than on the left.

In the central area (area (a)) a dark spot can be seen in P1. XV. fig. 79, situated in the median axis of the area, but not quite in its centre. This marks the site of the two pronuclei, and in this region, as can be seen from P1. XV. fig. 80, the disc is distinctly thickened.

Our interpretation of the various regions of the disc at this stage corresponds fairly closely with the description given by Harper for the disc of the Pigeon (1904, p. 360) and illustrated in his pl. i. fig. 6. He shows, however, only two regions, the outer of which is due to the “ abrupt thinning out of the disc.” It is to be noted that both Harper and Miss Blount (1909) describe and figure the disc of the Pigeon as being from the first elliptical in shape.

It is not easy in the sections to determine accurately the limits of the disc, but we judge that it is present in approximately 53 sections, its transverse diameter being, therefore, about 0-53 mm. Its longitudinal diameter, exclusive of the marginal zone (which is not shown to its full extent in P1. XV. fig. 80) is 0-59 mm. It is therefore clear, even without the evidence of fig. 79 (Pl. XV.), that the disc is no longer radially symmetrical as in the earlier stages, but is now bilaterally symmetrical about a main axis.

Furthermore, it is evident from our description of the surface view of the disc that there has already appeared in it a definite axial differentiation or polarity, inasmuch as it exhibits at its two ends well-marked differences afiecting in particular its cytoplasmic zone. These differences are noted below, and certain of them are clearly brought out in P1. XV. fig. 80, representing a median longitudinal section of the disc.

The disc has the same constitution as in the preceding stage, but the cytoplasmic zone (Pl. XV. fig. 80, fad.) now shows a definite increase in thickness, measuring at the level of the pronuclei 0-052 mm., whilst the vacuolated zone (vcd.), though still well marked, has decreased in thickness, the total thickness of the disc (0-07 7 mm.) being about the same as in the preceding stage. Evidently the cytoplasmic zone has increased at the expense of the vacuolated zone. In the central region of the disc, the latter zone varies in thickness from 0-0258 mm. to 0-034 mm.

On the left side of fig. 80 (Pl. XV.) it can be seen that the cytoplasmic zone rapidly thins out and terminates rather abruptly just below the minute polar body (pb.) situated just above its surface. This extremity evidently corresponds to the more sharply delimited margin of the cytoplasmic zone (i. e. of the zone (b) of the disc as seen in surface-view). At the opposite end of the section, 2'. e. the right side of the figure, the cytoplasmic zone thins more gradually, being continued on for a short distance as a quite thin superficial layer. This end would therefore correspond to the less sharply delimited margin of the cytoplasmic zone in surface-view.

Further, it is to be noted that the width of the marginal zone varies at either end of the disc. At the end marked by the polar body its width is about 0-04 mm., while at the opposite end it measures about 0-07 mm. These measurements are approximate, since the marginal zone is not very distinctly delimited at its outer edge. It will be remembered that in the preceding egg the cytoplasmic zone of the disc was superficially “ infiltrated ” at one end by fine yolk-spheres. The evidence indicates (see especially our description of the next following stage) that this “ infiltration ” (if it may be so termed) takes place from the less sharply delimited margin of the cytoplasmic disc. In the present egg a like infiltration is present, but it occurs at both ends of the disc (Pl. XV. fig. 80) and is slight, though perhaps rather more extensive at the less sharply delimited margin.

The Pronuclei - The two pronuclei (Pl. XV. figs. 80 & 81) no longer occupy the centre of the disc but, as measurements show, are situated slightly nearer the less sharply limited margin. They are therefore approaching the position they occupy in the succeeding disc (Stage FE).

The two pronuclei now appear to be accurately superimposed, the one on the other (PI. XV. fig. 81), but over the area of contact it is possible to make out that the nuclear membrane of each is still intact and that, indeed, there is a very slight overlap in this region. This overlap is not indicated in P1. XV. fig. 81.

They are now definitely larger than in the preceding egg, the more superficial one measuring 0-023 X0-014 mm., the deeper 0-O22><0-014 mm., but they show no difference in staining capacity or in structure. The nuclear reticulum is distinct and rather coarse, and has fine chromatin granules distributed through it.

Polar B0dies.—Only one of these could be found (Pl. XV. fig. 80, pb.). It is present in two sections, but the following section, which might have contained the other body, is, unfortunately, damaged in the region of the disc. The interesting point about this one polar body is that it is not situated centrally over the disc in the region of the pronuclei, as might be expected, but is found in the median plane at that end of the cytoplasmic zone which is more distinctly limited. Here. it lies in a shallow depression just at the junction of the cytoplasmic zone with the marginal zone. The perivitelline space, which was doubtless filled by fluid, is still present, and through it the polar body must have migrated along the surface of the disc from the centre where it was extruded to the periphery, where it is now found.

It is a small elongated body with an irregular wavy contour measuring 0-03 x 0-008 >< 0-02 mm., and from its size is probably the first polar body. It possesses a very distinct and definite enclosing membrane, and in its cytoplasm a few minute chromatin granules are present.

Stage FE’.

Echidna B.30.7.29 (Pl. XVI. figs. 82, 83, & 84).

This is Flynn’s Egg N0. 2 (Flynn, 1930, p. 126, figs. 2, 4, & 5).

This stage follows closely on the previous one, the pronuclei having now fused, though they are still distinguishable.

The thickness of the shell and zona-albumen, according to Hill (1933, p. 445), is respectively 0-0018 and 0-0054 mm.

The disc is elliptical in shape, but distinctly smaller than that of FD (VVH 26). Its superficial dimensions are :—Cytoplasmic zone 0-43 ><0-31 mm. ; including marginal zone, 0-49 ><0-37 mm. These measurements must be regarded as approximate only, as it is not easy to define the limits of the marginal zone in sections. The smaller superficial dimensions of the disc may be due either to individual variation or to contraction. The maximum thickness of the cytoplasmic zone (Pl. XVI. fig. 82, fcd.) is 0-050 mm., which is about the same as in the preceding stage, while the vacuolated zone (vcd.) seems to be a little thicker than that of VVH 26, the disc having a total thickness of approximately 0-10 mm. In our earliest cleavage stage (CA), in which the first cleavage-spindle is present, the disc is still smaller in extent, but its thickness is greater. It therefore appears as if a contraction of the disc, associated with an increase in thickness, is taking place in preparation for cleavage. The disc (Pl. XVI. fig. 82) has been cut longitudinally, and the pronuclei and the two polar bodies occur in the same section. Structurally the disc is similar to that of preceding eggs, and exhibits the same bilateral symmetry and the same axial differentiation. At the end marked by the two polar bodies (pl). 1 & 2) the cytoplasmic portion of the disc (fcd.) ends rather abruptly, so that this margin in surface View would appear sharply delimited. At the opposite end the finely granular cytoplasm merges almost imperceptibly into the marginal zone (mg.z), so that the limit between the two is only determinable with difficulty. Moreover, from this end a thin layer of fine and rather coarser yolk-spheres extends in the superficial cytoplasm of the disc almost up to the level of the pronuclei, so that in section the two margins of the formative disc are readily distinguishable from each other. The cytoplasmic portion of the disc passes over below into the usual vacuolated zone containing fine yolkgranules. Round the periphery, the latter zone forms, with the yolk-bed, the marginal zone which measures about 0-1 mm. in breadth at the yolk-rich end, while at the other end it is narrower, with a width of about 0-06 mm.

The pronuclei (Pl. XVI. figs. 82 & 83) are now no longer approximately central, but are situated definitely nearer the more yolk-rich margin of the disc, a little more than one-third the total diameter of the disc from that margin. They have the same relations to each other as in the preceding stage, that is, they are superimposed the one on the other, but now, round the periphery of the former surfaces of apposition, the nuclear membranes of the two are in direct continuity, while between the two it is no longer possible to recognise a definite continuous limiting membrane or membranes (Pl. XVI. fig. 83). We therefore conclude that the two pronuclei have now become fused to form the single membranate cleavage nucleus, in which the nuclear reticulum of each component still remains separate and distinct.

The two pronuclei (Pl. XVI. fig. 83), if we may still for convenience speak of them as such, differ in size and in staining properties. The more superficial one measures 0-025 ><0-0091 ><0-O15 mm. and stains more lightly than the deeper, which is distinctly smaller (0-020><0-0084X0-015 mm.). Though in both the nuclear membrane shows some evidence of wrinkling, shrinkage is more apparent in the deeper nucleus. Round its periphery, and to a slighter extent round that of the more superficial pronucleus, there is present in the cytoplasm an irregular narrow contraction space. The nuclear reticulum of the larger pronucleus is of a looser, more open character than that of the smaller, and its linin threads are rather finer. In both they support numerous minute chromatin granules, whilst in the more superficial pronucleus there is also present a minute lightlystaining spherical nucleolus.

Polar B0dies.—Both polar bodies are present (Pl. XVI. figs. 83 & 84). They lie close together in the perivitelline space immediately over the margin of the less yolk—rich end of the disc. They differ in size ; the more peripheral body is the larger, and is probably the first (pb.1). It measures 0-0168 X0-0084 mm., while the smaller (pb.2) measures 0-0126 ><0-007 mm. The larger is irregularly oblong in section and the smaller is irregularly lobed. Each contains a minute mass of chromatin granules, and in the cytoplasm of the larger one there are present eosinophil spherules of varying size, suggestive of yolk-granules—an interesting point of detail.

A Stage FF. Eckidna A.2l.8.29.

This egg was fixed whole in the fluid of Bles, and is in a poor state of preservation. Hill (1933, p. 445) gives the thickness of the shell as about 0-0045 mm., and makes the remark that it is “ evidently a fertilisation stage, but the cleavage (?) nucleus is poorly preserved.” It is possible that it represents the final stage in the fusion of the pronuclei before they proceed to the formation of the first cleavage-spindle.

Summary of Maturation and Fertilisation Stages

(1) GermmalD1Isc.

(a) Until maturation has been completed the disc is circular and its symmetry is radial.

(b) Fertilisation has the effect of producing a definite bilateral symmetry in the disc, as evidenced by the following features :—

(i.) The disc becomes elliptical in shape.

(ii.) At one end of the long axis, the disc becomes superficially infiltrated by fine yolk-spheres. '

(iii.) The pronuclei When in conjugation lie in the long axis, but nearer that end of the disc which is superficially charged with yolk-spheres.

(iv.) The polar bodies move from their original central position to the margin of the disc which is less rich in yolk-spheres and so come to lie at one end of the long axis.

(0) In its final form the unsegmented disc consists of a superficial zone of granular cytoplasm and an underlying vacuolated cytoplasmic zone containing fine yolk-spheres. The combined thickness of these two zones in the final form of the disc (in the cleavage stage, CA) is approximately 0-13 mm.

Underlying the disc, and in continuity with it, is the yolk-bed, composed of a cytoplasmic basis densely infiltrated With fine yolk-spheres intermingled With coarser spheres. It reaches the surface round the periphery of the disc, and there combines with the vacuolated zone of the latter to form the marginal zone. The yolk-bed is directly continuous centrally with the latebral neck.

(2) Nuclear Structures.

(a) The conjugating pronuclei, as stated above, take up their position on the long axis nearer that end of the disc Which is superficially charged with yolk-spheres.

(b) The pronuclei fuse before proceeding to the formation of the first cleavage spindle.

(c) At the time of fusion there is a difference in size and staining reactions in the two pronuclei, the deeper being smaller and staining more intensely than the more superficial one. It is regarded as the male pronucleus.

(3) Polar Bodies.

(a) The first maturation spindle is very minute and is formed at or near the centre of the disc.

(b) The first polar body is given ofl in the ovary.

(c) The second polar body is extruded in the oviduct, and in egg FA extrusion appears to have been effected in the absence of fertilisation, but Whether this is the rule We have not been able to determine.

(cl) The second polar body is smaller than the first, and is also formed at the centre of the disc.

(e) The polar bodies migrate through the perivitelline space and take up a position at the margin of the less yolk-rich portion of the disc, as noted above. (f) The first polar body remains undivided.

(4) General. Polyspermy does not normally occur in Monotremes.

Early Cleavage


The earliest papers dealing with the development of the Monotreme ovum are those of Caldwell (1884, 1887). This investigator, with an abundance of material, was able to establish the oviparity of the group and the fact that cleavage is meroblastic (1884). Of his work all we need take note of here is his account of cleavage, of which he gave a description up to the completion of the four-celled stage. He showed that the first cleavage-furrow divides the germinal disc into a larger and a smaller area, and that the second furrow is laid down at right angles to the first, so that the four-celled stage is composed of two larger and two smaller blastomeres, connected with each other and with the underlying yolk. These important observations we are able to confirm. Although his description of the cleavage-process does not extend beyond the four-celled stage, he figures sections of two later stages (pl. xxx. fig. 2, and pl. xxxi. fig. 4). Fig. 2 represents the marginal region of the blastodisc of an Echidna egg of 6 mm. diameter, and fig. 4 a section of the disc of a H mm. egg. Both eggs appear to be at the stage when the blastodisc is about two cells thick. Caldwell identified the superficial cells as constituting the epiblast and the deep as hypoblast, an erroneous interpretation, as Semon (1894) has pointed out and as our own material demonstrates.

Semon (1894) added certain facts of importance to our knowledge of the early development of the Monotremes, but there are wide gaps between the few early cleavage-stages he possessed, and consequently his account of the cleavage-process is very incomplete and, unfortunately, many of his figures are somewhat schematic and lacking in essential detail. His description of the four-celled stage is less accurate than that of Caldwell, since he states that the blastomeres are all approximately equal in size (Taf. ix. fig. 30). His next stage is a disc composed of twenty—four blastomeres, arranged in the form of a circular plate, one cell thick (Taf. ix. fig. 31). Following on this, he figures a blastodisc of Eckidna (Taf. ix. fig. 32), plano-convex in section and up to about six cells in thickness in its central region. This stage is succeeded by a blastodisc of Platypus (Taf. ix. fig. 34), which is beginning to spread peripherally over the yolk, and in so doing is becoming thinner. Spreading continues and results in the conversion of the blastodisc into a unilaminar membrane, which gradually extends round so as to enclose the yolk-mass (Taf. ix. figs. 37 & 38). Though he failed to elucidate correctly the mode of origin of the endoderm, these discoveries of Semon relating to the spreading of the blastodisc form a most important contribution to our knowledge of the early development of the Monotremata.

In 1907 appeared Wilson and Hill’s monograph on the ‘ Development of Omithorkynchus.’ Of the stages investigated by these authors three only are of value to us at the moment. These are (1) the eight-celled stage (Wilson and Hill’s Specimen A and AA) ; (2) the later segmentation stage (Specimen NN) ; (3) an early stage of germinal—layer formation (Specimen 0). This material is now in the possession of one of us (H.) and has been re-examined, and the results incorporated in this and the ensuing parts of this research. The second of these stages (Specimen NN) appears to have lost its surface layer of cells, and as we have a fairly abundant series of a comparable stage, we have decided not to include it in our description.

No papers have appeared dealing with cleavage in Monotremes since the publication of Wilson and Hill’s monograph, but Hill in 1910 (p. 86) gave a summary of the then existing knowledge of Monotreme development and its bearing on the early ontogeny of the Mammalia.

Description of Stages. For list of early cleavage stages see the accompanying table (Table III.)

Stage CA.

Echidna VVH 29, collected 26.7.33 (Pl. XVI. figs. 85 & 86).

In this eg, as with others preserved in Smith’s fluid, the shell Was easily removed, so that a surface view of the disc could be obtained. This is reproduced as fig. 85 (Pl. XVI). The disc appears as a White elliptical area, which passes over at its periphery into a relatively broad darker ring, the marginal zone, there being no definite boundary between the two.

The sectional plane cuts the disc parallel with its long axis (Pl. XVI. fig. 86) and, as in preceding eggs, there is a well-marked axial differentiation, one evidence of which is the presence of a distinct superficial layer of yolk-spheres extending from the marginal zone at one end (left side in the figure) to about the centre of the cytoplasmic zone (fcd.). In this egg the latter does not end quite so abruptly at the opposite margin as in previous stages.

Precise measurements are not easily made but, as near as We can judge, the cytoplasmic disc has a long diameter of about 0-43 mm. and a short diameter of 0-36 mm. It is therefore not very different in superficial extent from the disc of stage FE, but it attains a greater thickness. The cytoplasmic zone has a maximum thickness of about -07 2 mm. It merges rather gradually into the underlying vacuolated zone (vcd.). The latter is rather more compact and more densely laden with yolk—spheres immediately below the cytoplasmic zone than it is deeper down. The total depth of the disc, including the vacuolated zone, is approximately 0-13 mm., that is, it is definitely thicker than in preceding eggs.

It is worthy of note that the disc attains its maximum thickness at about the level of the first cleavage figure, the half of the disc which is superficially rich in yolk-spheres being definitely thicker than the other half. Stage.


Designation 3

in Collection

and date of *


TABLE III.——Ea,rly Oleowage Stages.

Measurements (in mm.).



With shell.

After fixation.

o i l

Without shell.


v x




Echidnct VVH 29, 26.7.33.


470 X 460


Smith’s fluidf

First cleavage-spindle.




Eohidna, VVH 1 , 29.6.33.

E >




Smith’s fluid.‘

Platypus A and AA, 23.7.01.

Echidna VVH 22, 21.7.33.



5'30 '




Echidna VVH 38, 28.7.33.

Eckidna A,


Eokidna V1I.’3l, 3.8.31.





5'00 .


0 4' 70 X 450

Picro-nitric solution.

4-celled stage.

"8-celled stage, Wilson and

Hill’s stage A and AA.


Smith’s fluid.

1‘ 8-celled stage.

16-celled stage.

550 ! 4-o0><3-s0


Smith’s fluid.

Smith’s fluid.

31-celled stage.

32-celled stage.

Sparse fine yolk-spheres, apparently derived from the dense accumulation in the vacuolated zone, are disseminated through the cytoplasmic zone, and are found most abundantly in that half in which superficial yolk-spheres are less abundant. Below the central region of the disc the yolk-bed is very distinctly vacuolated, and contains numerous small spheres varying in size down to quite minute granules, together with sparse larger spheres. Peripherally it merges with the vacuolated zone, and the two together form the marginal zone, which is Well marked but poorly delimited on its outer side. In this egg its yolk-spheres comprise a considerable proportion of medium—sized spheres intermingled with smaller, and the same holds true for the superficial yolk of the cytoplasmic zone.

The first cleavage mitotic figure is now present (Pl. XVI. fig. 86), and is situated in the thicker half of the disc, that in which superficial yolk is present. The figure is in the telophase and consists of two minute groups of clumped chromosomes situated in adjoining sections and connected by a thin strand of spindle fibres. In fig. 86 (Pl. XVI.) the mitotic figure has been reconstructed. The axis of the spindle lies in the long axis of the disc, so that the first cleavage-plane will be transverse to that axis and will divide the disc into two unequal cell-areas.

From Text—figure 2.

Diagram of the germinal disc, after the establishment of bilateral symmetry. The disc is oval and surrounded by the marginal zone. The polar bodies (P.B.) are situated at its relatively yolk-free end; the opposite end is rich in superficially situated fine yolk-spheres. The position of the first cleavage plane is indicated by the line I——T, that of the second by a line joining II——II.

the structure of the disc it might be expected that one of these would be smaller and thicker and rich in superficially situated yolk-spheres, whilst the other would be of greater superficial extent but thinner, and largely devoid of superficial yolkspheres.

It may be stated here that although the other conditions as postulated, hold good, the expectation of a difierence in thickness in the two cell-areas is not borne out by the early cleavage (four~celled) stage We possess. If the configuration of the disc in the stage we are now describing is the normal one, then the disc-material must become rearranged at the beginning of cleavage if the blastomeres are to be of more or less equal thickness, as they are in our four-celled stage.

Although the series is complete, we have been unable to find the polar bodies. It is possible that they have degenerated earlier than usual.

We have failed to secure an egg in the two-celled stage, and we now proceed to the description of a stage exhibiting four cell—areas.

Stage CB.

Echidna VVH I, collected 29.6.33 (Pl. XVII. figs. 87, 88, & 89, Pl. XVI. fig. 90, and text-fig. 3).

This egg is at the four-celled stage, or rather the stage of four cell-areas, since the prospective blastomeres are not delimited below as is the case in all early cleavage stages. When, for convenience, we designate them as cells or blastomeres, that fact should be borne in mind.

A surface-View of the blastodisc is given in P1. XVII. fig. 87, whilst text-fig. 3 provides a plan of the arrangement of the blastomeres, and also shows the positions of the first and second cleavage planes, as determined by a study of the sections of the blastodisc. From the figures which are siinilarly orientated, it will be seen that the blastodisc forms an area which is roughly circular in outline and not elliptical as in the unsegmented disc (text-fig. 2). Its diameter in the intact egg is about 0-45 mm. Surrounding the blastodisc (Pl. XVII. fig. 87) is a narrow, rather ill-defined lightish ring, varying in width and in distinctness, and outside that again is a more prominent, much Wider and darker ring. These two rings together form the marginal zone, which has a Width in the intact egg of from 0-20 to 0-26 mm. The inner ring in section is seen to be of a looser, more vacuolated character than the outer, and to contain only very fine yolk-granules. It is formed by the outcrop, at the surface, of the vacuolated zone, which underlies and is continuous with the blastomeres (Pl. XVII. fig. 89), whilst the outer ring represents the part of the marginal zone which is formed by the outcrop of the yolk-bed. Its contained yolk is more compactly arranged and, for the most part, distinctly coarser than that of the inner ring (Pl. XVII. fig. 89, and P1. XVI. fig. 90, mg/.z.).

The blastomeres are separated from each other by well-marked cleavagefurrows (Pl. XVII. figs. 87 & 89), which stop short of the marginal zone. Their peripheral margins, though quite well defined, are not limited by definite furrows, but merely by a very shallow circular groove, marking the junction of the blastomeres with the marginal zone. Here the cytoplasm of the blastomeres is directly continuous with that of the marginal zone, just as it is with the cytoplasm of the vacuolated zone.

If fig. 87 (Pl. XVII.) and text-fig. 3 be examined it will become evident that the four blastomeres can be grouped into two pairs differing in size, viz., a pair of larger blastomeres numbered 1 and 2 in teXt—fig. 3 and a pair of smaller, numbered 3 and 4, the components of each pair again differing slightly in size, 1 being slightly larger than 2 and 4 larger than 3. If, now, we examine the sections, we find ( 1) that blastomeres 3 and 4 are rich in superficially situated yolk-spheres, whilst such spheres are largely absent from 1 and 2, and (2) that the two polar bodies lie in contact with the antero-medial border of blastomere 1 and very nearly in the second cleavage plane (text-fig. 3, P.B.). These facts point to the conclusion that the two larger blastomeres are derived from the larger yolk-free portion of the unsegmented disc, at the margin of which, in the median plane, the two polar bodies are situated, whilst the two smaller blastomeres are derived from the remaining smaller portion of the disc, which is rich in superficially situated yolkspheres (of. text-fig. 2).

Text-figure 3.

Diagram of the four-celled stage, Echidnw VVH l. I—I, first cleavage plane; II—II, second cleavage plane.

Fig. 89 (Pl. XVII.) illustratesa section through the blastodisc along the plane AB in teXt—fig. 3. The three blastomeres cut in this section are 1 (with nucleus), 2, and 4 (ca. 1, 2, 4). In blastomere 4 (on the right) a superficially situated layer of fine yolk-spheres is present, whilst blastomeres 1 and 2 are practically free of such yolk. In fig. 90 (Pl. XVI.) is illustrated a section along the plane CD in text-fig. 3, the blastomeres cut through being 1 (ca. 1), of which only a Very small portion is seen, and 2 (ca. 2). The two polar bodies (10b.l & 2) are visible at the outer margin of blastomere 1, overlying its junction with the marginal zone (mg.z.), and they are also shown in text-fig. 3, PB. Blastomeres 1 and 2 are again seen to be devoid of superficially situated yolk.

In all the blastomeres the nuclei are preparing for division ; in those of 1 and 4 the nuclear membrane is present, enclosing a spireme thread; in blastomere 2, the nuclear membrane is in process of disappearing and the spireme is dismembered; while in blastomere 3, the nuclear membrane is absent and chromosomes have appeared.

It will be observed in fig. 89 (Pl. XVII.) that the cleavage furrows extend down to the vacuolated zone, and that the finely granular cytoplasm of the blastomeres passes over without limit into the greatly vacuolated cytoplasm of the latter zone (vcd.), which is laden with extremely fine yolk-granules. That the vacuolated zone is destined to enter into the constitution of the blastomeres is evident from the structure of the latter in later cleavage stages (see Pl. XVIII. fig. 101). The highly vacuolated character of the zone, and the presence in its cytoplasm of minute yolk-granules, possibly the remnants of larger spheres, suggest that it is an active metabolic centre in which the yolk-spheres are broken down into fluid products, which are utilized by the developing blastomeres. The yolk-bed (g/b.) is also vacuolated, but much more irregularly and in a very much less degree than is the vacuolated zone. Particularly noticeable, however, is a group of vacuolar spaces, one of them specially large, situated immediately below the centre of the vacuolated zone and directly above the junction of the yolk-bed with the latebral neck(IH.}CVII.fig.88)

Polar Bodies.—These are to be seen in surface view (Pl. XVII. fig. 87, and textfig. 3, PB) as two small granules situated in the sectional plane CD, in contact with the antero-medial border of blastomere 1. They lie very nearly in the second cleavage plane, which is coincident with the median plane of the unsegmented disc, and so have retained, practically unaltered, the position they occupied in stage FD (Pl. XV. fig. 80). They measure 0-024 X0-013 and 0-020 X0-011 mm. in diameter respectively. The larger of the two, probably the first, is the more peripheral, and has an irregular outline with a definite enclosing membrane (Pl. XVI. fig. 90, 106.1 & 2). Its cytoplasm is homogeneous centrally, vacuolated peripherally, and possesses at its centre what appears to be a minute membranate nucleus (0-006 X0-004 mm.), enclosing on one side an elongated mass of clumped chromatin granules.

The smaller polar body (pb.1 & 2) possesses a very thin limiting membrane enclosing alveolar cytoplasm. In the latter, on one side, there are present spherules of varying size, each with a light centre and an eosinophil periphery (incipient yolkspheres ?). The nuclear constituents are represented by two or three fine strands in which are situated chromatin granules.

Fig. 88 (Pl. XVII.), which represents the next succeeding section to that illustrated in fig. 89 (Pl. XVII.), shows very clearly in vertical section, the relation of the blastodisc to the yolk-bed (3/6.), the continuity of the yolk of the latebral neck (l.n.) with the latter, and the body of the latebra (l.b.) with its enclosing latebral yolkzone (ly.z.). The minute structure of the latebra we have already described (ante, p. 494).

From the structure of the disc, the final disposition of the pronuclei in previous stages, and that of the first cleavage spindle in stage CA (-Eckidnla VVH 29), we conclude, in agreement with Caldwell (1887, p. 476), that the first cleavage plane is transverse to the long axis of the disc and nearer the yolk-rich end, thus dividing the disc into two unequal portions, viz., a smaller blastomere superficially rich in fine yolk-granules, and a larger blastomere devoid of the latter (see text-fig. 2). In the stage under consideration we interpret the Z -shaped furrow separating blastomeres 1 and 2 from blastomeres 3 and 4 as marking the first cleavage plane, and the continuous zigzag furrow separating blastomeres l and 3 from 2 and 4 as indicating the second cleavage plane (text-fig. 3, I & II ;.

To summarise, the first cleavage plane is transverse to the long axis of the disc, and divides the latter into a smaller and a larger cell-area. The second cleavage plane is at right angles to the first and coincides more or less accurately with the long axis, the smaller blastomere of the two-celled stage becoming subdivided into two small blastomeres of slightly unequal size, the larger into two blastomeres, also slightly unequal. Cleavage accordingly is of the unequal meroblastic type.

Stage 00.

Platypus A and AA, being twin eggs collected by Mr. John John in Queensland on August 23, 1901 (P1. XVII. figs. 91 & 92, and P1. XVIII. fig. 93).

These eggs form part of the material on the examination of which Wilson and Hill based their monograph on the Development of Omithorhynckus (1907). They constitute “ Specimens A and AA ” of these authors. Neither of the eggs had been cut into sections, but text—figures are given (1907, p. 37, text-figs. 1 & 2) showing the arrangement of the cell-masses in both eggs, and a photograph of the complete egg of specimen A is reproduced, in which the blastodisc can be faintly seen through the shell-membrane (Pl. I. fig. 1). The latter is seen to be separated from the yolk-mass by a wide space (largely artefact), partly occupied by the coagulated remains of the albumen. We reproduce another figure of this egg complete in its membranes (Pl. XVII. fig. 91), but with the coagulum omitted.

Although neither of these eggs had been sectioned, one of them (AA) had been double-embedded in celloidin and paraffin wax, and had remained in the block until it was sectioned for the purposes of the present research—a period of over thirty years! Although somewhat shrivelled, it yielded an excellent series of sections, one of which is shown in Pl. XVIII. fig. 93.

Specimen A had been kept for the same period preserved in spirit, and of it a preparation was made of the blastodisc. This is shown in fig. 92 (Pl. XVII.) and text-fig. 4. From these figures, as well as those of Wilson and Hill (1907, text-figs. l & 2), it is evident that the blastodisc at this stage takes the form of an elongated ellipse (0-572 ><0-416 mm.), and that the blastomeres are arranged as two linear aggregates of four cells each, on either side of the long axis of the disc. The blastomeres at one end of the long axis are appreciably smaller than those at the other, so that the axial polarity present in the four-celled stage of Echidna is still apparent in the eight—celled stage of Platypus. The axial differentiation of the blastomeres, and their disposition in two linear aggregates of four, can only be accounted for by the third series of cleavage planes having developed on either side of, and more or less parallel to, the first cleavage plane. This is shown in the accompanying text-fig. 4.

Of the eight blastomeres it is evident that the four smaller, situated at one end of the long axis, are derived from blastomeres 3 and 4 of the four-celled stage, the remaining group of larger blastomeres at the other end of the axis being the result of the division of blastomeres l and 2. Stage A shows a very regular and typical arrangement of the furrows, but irregularities of cleavage may arise even at this

Text-figure 4.

Diagram of eight-celled stage, Platypus A. I—I, first cleavage plane ; II—-II, second cleavage plane ; III—-III, third cleavage planes.

early stage, such as can be seen in the disc of specimen AA (see Wilson and Hill, text-fig. 2). A similar disturbance of the bilateral symmetry occurs in our eightcelled stage of Eckidna (stage CD, Pl. XVIII. fig. 94). 4

A picture of a section through the disc of specimen AA is given in P1. XVIII. fig. 93. The sections are obliquely transverse. The two blastomeres figured are the bottom right in Wilson and I-Iill’s text-fig. 2 (whose nucleus is cut) and the second from the bottom on the left-hand side.

As in the four-celled stage, the blastomeres are flattened masses of cytoplasm fully open to the yolk below and at the periphery.

There is no great difference between the two ends of the disc as regards the superficial yolk-content of the blastomeres, and this is also the case in the eight-celled stage of Echidna, to be examined next.

Stage OD.

Echidna VVI-I 22, collected 21.7.33 (Pl. XVIII. figs. 94, 95, 96, & 97 and text-fig. 5).

This egg was fixed in the fluid of Bouin-Hollande. The cytological detail is excellent, but the fixative has caused the yolk-mass to swell and to disintegrate into its component yolk-spheres. This swelling has also affected the blastodisc, which is abnormally enlarged, and its cleavage planes much exaggerated. The measurements of the disc are therefore excessive (approx. 1-00 ><0-60 mm.).

Text-figure 5.

Diagram of eight-celled stage, Echidna VVH 22.

The blastomeres (Pl. XVIII. fig. 94) have the same arrangement as in the preceding stage of Platypus. There is, however, no great disparity apparent in the sizes of the cells at either end of the long axis, although on the Whole the upper series of blastomeres in the figure would appear to be the larger, and these contain appreciably more superficial yolk.

In none of the eight-celled stages either of Platypus or of Echidna have We seen the polar bodies. The disc of this stage was sectioned transversely to its long axis. Pl. XVIII. fig. 95 passes through the second pair of blastomeres from the top approximately along the plane AB of text~fig. 5, but is a combination of two adjoining sections. The blastomeres are similar to those of previous stages.

In all the eight blastomeres the nucleus is dividing, being in the metaphase of mitosis (Pl. XVIII. figs. 96 & 97). In all cases the long axis of the spindle is transverse to the long axis of the disc. The fourth series of cleavage-planes, resulting in the sixteen-celled stage, will therefore be approximately parallel to the second plane. In this way the division of a regularly arranged eight-celled stage, such as specimen A of Platypus (stage CC), will result in the formation of a sixteen-celled stage in which there will be four central cells and twelve marginal cells.

In the blastodisc of Echidna now being considered, owing to an irregularity in cleavage, the blastomere second from the top on the right-hand side of P1. XVIII. fig. 94 is already centrally situated and on division will form two central cells. Division of the blastomeres of this disc will therefore result in a sixteen-celled stage consisting of five central cells and eleven marginal cells. A further complication arises from the fact that in some blastomeres the spindles are not perfectly central (cf. the two sides of Pl. XVIII. fig. 95), so that in these the division will be unequal.

Stage OE’.

Echidmt VVH. 38, collected 28.7.33 (Pl. XIX. figs. 98 & 99, and P1. XVIII. figs. 100 & 101, and text-fig. 6).

This is an ovum in which the blastodisc consists of sixteen blastomeres containing thirty-two nuclei. Division of the original sixteen nuclei has taken place, the synchronisation being almost perfect, while the blastomeres have not yet divided, notwithstanding that superficial cleavage furrows are present in practically all of them (Pl. XIX. figs. 98 & 99).

The blastomeres with their nuclei are shown in text-fig. 6. The dimensions of this blastodisc, measured in the intact egg were 0-72 ><0-62 mm., but according to the sections are approximately 0-62 ><O-57 mm. As in corresponding stages of reptiles and birds, the blastodisc consists of two sets of blastomeres, namely a peripheral series of larger “ marginal ” blastomeres, open to the yolk peripherally and below, and an inner group of smaller “ central ” blastomeres, surrounded by the former and delimited on all sides except below where they are still open to the yolk (cf. Blount, 1909, p. 46).

The blastodisc consists of eleven marginal cells and five central cells. It has become approximately circular, so that the longitudinal axis of previous stages is not easily determined. It is therefore almost impossible from external examination alone to recognise the axis of bilateral symmetry. To find this axis we have to take into consideration the structure of the blastomeres and the position of the polar bodies.

In Pl. XVIII. fig. 100, of a section along plane AB of text-fig. 6, the large marginal blastomere at the extreme left is that in the upper right-hand corner of the textfigure (adjacent to the letter B). Two sections farther on, the polar bodies are found in the cleavage-furrow which separates this blastomere from the marginal blastomere immediately above and to the left of it (text-fig. 6, P.B.). Sections show also that the blastomeres at the upper side of the figure are much less rich in superficial yolk than are those at the lower side. We conclude, therefore,

Text-figure 6. Diagram of 16-celled stage, Echidna VVH 38. P.B., position of polar bodies.

that the plane of bilateral symmetry corresponding to the longitudinal axis of the original elliptical disc is one running vertically through the centre of the blastodisc, as shown in text-fig. 6, and corresponding more or less closely with the second cleavage-plane (II). Cleavage-planes I, III, and IV can now be determined, as shown in the text-figure, but the fifth cleavage-planes show no constancy of arrangement, since the planes of division of the central cells are quite variable and the marginal cells may divide to form two new marginal cells or into a marginal cell and a central cell.

Sections through the blastodisc, along the planes AB and CD respectively of text-fig. 6, are given in P1. XVIII. figs. 100 & 101. In the latter figure two central blastomeres only are shown (c.c.). Comparison of the blastomeres of this stage as seen_ in section with those of the four-celled stage (Pl. XVII. fig. 89) reveals important advances. The cleavage-furrows are now much deeper and extend right down into the site of the former vacuolated zone. The blastomeres, as the result, are now much thicker than in previous stages. They are largely composed of homogeneous cytoplasm, whilst their basal portions are formed to a varying extent of vacuolated cytoplasm, rich in very fine yolk-granules, and evidently derived from the vacuolated zone. Owing to this incorporation, the latter zone has practically disappeared as such and the small yolk-spheres of the yolk—bed pass over directly, without any line of demarcation, into the fine, mostly lighter-staining granules in the vacuolated cytoplasm of the blastomeres. The marginal cells (Pl. XVIII. fig. 100, ms.) possess only a thin layer of vacuolated cytoplasm, which separates them from the yolk-bed and, as in the four-celled stage, they are in continuity marginally with the cytoplasmic network of the marginal zone.

The yolk—bed itself presents a dense appearance owing to its rich content of yolk-spheres, comprising small deeply staining basophil spheres, as well as minute lighter-staining granules. Distinct vacuoles occur in it at intervals throughout its extent, but its central region, at and just above its junction with the fine-grained yolk of the latebral neck, shows an intense vacuolation (Pl. XVIII. fig. 101, yb.v.), whilst the small yolk-spheres are largely replaced by minute yolk-granules. We conclude, then, that the blastomeres have increased in thickness at the expense of the vacuolated zone, and that the yolk-spheres of the yolk-bed are broken down and the products utilised in that growth.

The two polar bodies are present in the position indicated above and shown in text-fig. 6 (P.B.). They are situated a little to the right of the hypothetical axis of the blastodisc. They lie in close contact and measure 0-O22 X0-O14 and 0-022 ><O-021 mm. respectively. They are wrinkled membranate structures and their cytoplasm contains numerous small definitely limited vesicular bodies and a number of chromatin granules.

Of the eleven marginal cells, five will form each a central and marginal cell, the other six producing each two marginal cells. The five central cells will divide to form ten smaller centrals. The result will be 32 blastomeres—17 marginal and 15 central. Of the central cells, those newly cut off from marginal cells are larger than those produced by the division of pre-existing central cells.

In this way irregularities arise in the sizes of the central cells (cf. Pl. XIX. figs. 103 & 104, of the 31- and 32—celled stages). It is therefore established that the differences in the sizes of the various blastomeres at the 32-celled stage are not due to lack of synchronisation, such as an increase in the rate of division in any special region, but to the inherently unequal nature of the cleavage itself.

Stages CF and CG. 31- and 32-celled stages respectively.

Echidna, A. 31.7.30, and Eckidna VII. ’3l, collected 3.8.31 (Pl. XIX. figs. 102, 103, 104, 105, and P1. XX. figs. 106 & 107, all of CF, and P1. XX. figs. 108, 109,

& 110, of CG.).

Surface views of the two blastodiscs are given in P1. XIX. figs. 102 & 103 (CF, 31 cells) and P1. XX. fig. 108 (CG, 32-cells).

In both blastodiscs the arrangement of the cells is similar to that of the sixteen-celled stage. Each has an outer ring of larger marginal cells, enclosing an area occupied by smaller central blastomeres. The latter are of two average sizes, the larger being those which have the more recently been cut off from the marginal cells. The dimensions of the two blastodiscs are :—CF (31 cells) 0-78 X0-72 mm., CG (32 cells) 0-79 X0-68 mm., the central cell-area measuring 0-45 ><0-34 mm. and 0-53 X0-43 mm. respectively. In CF the blastodisc consists of 14 marginal and 17 central cells, while in CG there are 15 marginals and 17 centrals.

Axial polarity is not now recognisable with any certainty. Neither surfaceviews nor sections give us any evidence of value in this regard.

Polar bodies are present in both CF and CG. In the former (Pl. XIX. fig. 103) the single polar body occurs in the cleavage-furrow situated below the very large marginal blastomere at the extreme top right-hand side of the figure. The furrow separates this blastomere from the two large centrals lying below it. The polar body is found in the angle between the three.

In CG the (again) single polar body is found, singularly enough, in the inner field of blastomeres. It can just be seen in P1. XX. fig. 108 (pb.) as a minute whitish sphere, just to the outside of one of the largest of the central blastomeres.

In neither case does the position of the polar body help us in determining the plane of symmetry.

Pl. XIX. fig. 104 is a low-power view of a vertical section of the upper hemisphere of the 31-celled stage (CF), showing the blastodisc, the disposition of the yolk, and the latebra in its full extent. The blastodisc appears to lie in a shallow saucershaped depression at the upper pole, but actually only its central cells are so situated. Surrounding the ovum and covering the blastodisc is the zona-albumen layer, left behind after removal of the shell-membrane. This section is shown at a higher magnification in P1. XX. fig. 106. The central blastomeres (see also Pl. XIX. fig. 105) generally resemble those of the 16-celled stage, but are more rounded in form, so that when cut through their peripheral regions, they appear isolated from the yolk-bed. In many of them their vacuolated basal portions are so densely infiltrated by fine yolk-spheres and granules that the vacuolation is largely obscured. Basally they are in direct continuity with the yolk-bed. The nuclei in the central as well as in the marginal blastomeres are either in the prophase or are replaced by mitotic figures in the metaphase. THE DEVELOPMENT OF THE MONOTREMATA. 569

The marginal blastomeres (Pl. XIX. fig. 105 and P1. XX. figs. 106, 107, m.c.) are now flattened and, as in previous stages, are continuous below with the peripheral portion of the yolk-bed, whilst they are continuous at their outer margins with the marginal zone. Their peripheral cytoplasm, above and below, is now infiltrated by fine as well as by sparse coarser yolk-spheres (Pl. XX. fig. 107, m.c.) a process which was only just beginning in the 16-celled stage.

The marginal zone is much less marked than in previous stages. It is narrow and contains medium-sized, in addition to quite small spheres.

The central region of the yolk-bed immediately above its junction with the latebral neck is here again very richly vacuolated, appearing in the sections as a. conspicuous light area of irregular form, and clearly visible in P1. XIX. figs. 104, 105, and especially in P1. XX. fig. 106. It is composed of a coarse cytoplasmic reticulum containing sparse fine yolk-spheres and enclosing vacuolar spaces, evidently fluid-filled during life. The occurrence of this vacuolated area and the presence of fine yolk-spheres in the basal portions of the blastomeres again bear witness to the active utilisation of the yolk-spheres of the yolk-bed in the growth of the blastodisc.

The yolk-bed resembles that of the preceding stage, but the yolk in the peripheral portion of the bed is much coarser-grained, medium-sized spheres being intermingled With the fine.

Egg CG is perhaps not so typical as CF. The central blastomeres are smaller and more widely separated (Pl. XX. figs. 109 & 110). Their basal vacuolated portions are less yolk-laden and the granules are finer. Moreover, such granules are also present as a quite thin layer in their peripheral cytoplasm.

Below most of the area occupied by the central cells, there is present a thin clear vacuolated layer containing sparse fine yolk-spheres, with which the basal portions of the cells are continuous. This layer probably corresponds to the central vacuolated area of the yolk-bed in egg CF.

The marginal cells are here less flattened, but are again infiltrated peripherally by fine yolk-spheres, as well as by a few coarser spheres at their outer ends.

The marginal zone is very unequally developed on the two sides of figs. 109 & 110 (P1. XX.), being much better marked and more normal on the right side (in the figures) than on the left.

The yolk-bed again is thinner than that of CF, and contains numbers of coarse spheres in addition to fine.


Platypus J J . Irregular 8-celled stage with accessory cleavage (Pl. XXI. figs. 111, 112, & 113).

In spirit the entire egg measured 6-0 mm., after removal of the membranes 4-5 mm. 570 PROF. T. THOMSON FLYNN .\.\'1> PROF. J. P. 1111.1. ON

In surface-view the blastodisc presents a number of cell~masses of varying size (Pl. XXI. fig. 111). There are three groups :— '

(aa) Eight large masses, having in surface-view and in sections (left side of fig. 112 (P1. XXI.)) the appearance of true blastomeres, and containing what appear to be normal nuclei. They are arranged more or less symmetrically in pairs on either side of an axis, which in P1. XXI. fig. 111 is vertical.

(b) An aggregate of seven smaller masses situated on the left side of the larger blastomeres in Pl. XXI. fig. 111. Three of them are shown on the right side of fig. 112 (P1. XXI.). Some contain several nuclei, one possessing as many as five.

(c) A group of faintly outlined masses, situated below the second group in P1. XXI. fig. 111 and still smaller than those of the latter. There are six altogether, and here again some possess two or three nuclei. A section through four of them is shown in P1. XXI. fig. 113. They are much flattened and lie quite superficially in the yolk. Their nuclei are small, flattened, and wrinkled, and poor in chromatin.

The stage is obviously abnormal, and it may be that this egg is polyspermic, the penetration of more than one sperm having given rise to an eight-celled stage with accessory cleavages.

Echidmz VVH 15, collected 14.7.33, fixed in Smith’s fluid.

M easurements.—Fresh 4-5 mm. in diameter, in formalin 4-9 mm. After removal of membranes 4-34 mm.

This is an abnormal early cleavage. It is shown in surface-View in P1. XXI. fig. 114, and a section is figured in P1. XXI. fig. 115.

In Pl. XXI. fig. 114 the blastodisc is seen to be made up of one large mass, partially surrounded by four smaller masses. Sections show that each of the latter contains one nucleus. The large mass, however, possesses two, one at normal level, the other situated rather deeply, in an abnormally thickened portion of the mass. The cell—aggregate therefore possesses six nuclei. None of the cleavageplanes reaches the vacuolated zone, so that the separation of the masses is incomplete.

Two polar bodies of unequal size are present in an unusual position, since they lie on the upper surface of the large mass, approximately at its centre. Each consists of more or less clear cytoplasm containing a minute rounded mass of chromatin. The egg in section shows premature liquefaction of the yolk, and the

yolk-bed is not of normal appearance. The vacuolated zone is excessively developed below the large mass (Pl. XXI. fig. 115).

Summary of Early Cleavage

Cleavage is not initiated till the fertilised egg arrives in the uterus. We have not observed the two-celled stage, but the evidence of later stages shows that the first cleavage-furrow is transverse and divides the germinal disc into two unequal cell-areas, a smaller charged with superficial yolk-granules and a larger from which superficial yolk is practically absent.

The second cleavage—furrow is coincident with the long axis of the elliptical disc, and is thus at right angles to the first. The result is that the four-celled stage consists of two pairs of blastomeres, a smaller pair rich in superficial yolk and a larger pair largely deficient in such, the members of each pair differing slightly in size.

The third cleavage-furrows appear on either side of the first furrow and more or less parallel to it, the result being an eight-celled stage consisting of two linear aggregates each of four cells, symmetrically arranged on each side of the original long axis of the disc, 2'. e., of the second cleavage—furrow.

The fourth series of furrows resulting in the sixteen-celled stage arise on either side of and parallel to the second furrow. The 32-celled stage results from the fifth series of divisions, but these exhibit no constancy in their arrangement. The blastodisc in the 16- and 32-celled stages consists of an outer ring of large marginal blastomeres enclosing an inner field of central blastomeres. The latter are smaller, but of two average sizes. The synchronisation of division up to the 32-celled stage is practically complete, and the difierence in size of the various blastomeres is not attributable to any variation in the rate of division.

As cleavage proceeds, the blastodisc increases in area at the expense of the marginal zone and becomes more or less circular in shape. At the four-celled stage its diameter is about 0-45 mm., at the 32-celled stage 0-80 X0-70 mm. (approx.).

Pola,m'ty.——The axial polarity present in the unsegmented disc, evidenced by the shape of the disc, the distribution of superficial yolk, and the position of the polar bodies, is easily recognisable in the four-celled and eight-celled stages.

It is recognisable with difficulty in the sixteen-celled stage, and in the 32-celled stage has become entirely obscured.

Polar Bodies.——These have been observed in all our early cleavage stages except the 8-celled stage. In the 4-celled blastodisc, the polar bodies are situated at the yolk—free end of the blastodisc. As the blastodisc increases in area, the polar bodies lose their peripheral position, so that in extreme cases, as in our 32-celled stage, the single remaining polar body, now degenerate, is found towards one end of the disc in the central cell-area.

The polar bodies have not been observed after the 32-celled stage.

Resume and Discussion

We have been able to give in the preceding pages for the first time a connected account of the processes of maturation, fertilisation, and early cleavage in the

Monotreme ovum.

What knowledge we possess of maturation and fertilisation in this group, We owe only to Flynn’s recently published paper (1930) dealing’ with one maturation and one fertilisation stage in Echidmz.

It may be stated at the outset that in essential details these processes in the Monotremes resemble those of Sauropsida so far as our knowledge of the latter extends.

The Monotreme oocyte is in a condition of preparation for maturation, when its diameter is approximately 3-50 to 4 mm. The nucleus breaks down, its contents are dispersed through the germinal disc, and the first polar spindle appears. This is extremely small (0-0093 X0-0056 mm.). Minute as it is, it is much larger than the corresponding spindle in the case of the pigeon, whose measurements, recorded by Van Durme (1914, p. 155), arc: length 0-005-0-006 mm., breadth 0-006—0-007 mm.

As concerns reptiles, the only record of the dimensions of the first polar spindle that we can find is one made by M. Loyez (1905, p. 91) for Anguis fmgilis, in which the spindle is larger than in the Monotreme (0-023 X0-012 mm.).

With regard to the second polar spindle, recorded observations on the Sauropsida are even more rare. We can find no record at all of the dimensions of this spindle in reptiles, but Van Durme (1914, p. 154) gives us the information that in birds the second spindle is slightly longer and thinner than the first, so that here again the Monotreme spindle is larger (0008 x 0-0058 mm.).

In the Monotreme as in the Sauropsida, the spindles when finally orientated are arranged with their long axes almost perpendicular to the surface of the oocyte. Whether any rotation of the second spindle takes place we are unable to say, but, in the case of the first, when first formed itismore or less tangential to the surface and later undergoes rotation into its final position.

The Polar B0d'ies.—Two polar bodies are normally developed in the Monotreme, although in some of the cleavage-stages only one is present, owing, no doubt, to the more rapid degeneration of one of the two.

The first polar body is given off in the ovary. It is the larger of the two. By the time the ovum has reached the uterus, the second polar body has been formed. Harper (1904, p. 381) has stated that in the Pigeon the second polar body is not formed unless the ovum has been previously fertilised. On this point Bartelmez is also very emphatic (1912, p. 295). He states that the second polar mitotic figure remains in the metaphase if fertilisation does not take place.

On the other hand, Van Durme contests Harper’s statement and gives two figures (1914, pl. iv. figs. 11 & 12), one of the pigeon and another of the swallow, in both of which the second polar body had been formed without any trace of sperms being found in the disc.

Our own contribution to this question comes from the examination of a single egg of Echidna, stage FA (mIdep.546). In this uterine ovum the second polar body had just been extruded and t11e female pronucleus established, but the most detailed search has failed to reveal the presence in the disc of any included sperm or male pronucleus, and this in spite of the fact that sperms are present in the albumen layer. Flynn (1930, p. 126) had previously recorded his inability to find the male pronucleus in this same egg. _

We have seen the polar bodies only in Echidna. They are membranate bodies, of lobed or amoeboid appearance, with sparse chromatin granules in the cytoplasm. They always lie free in the fluid-filled perivitelline space, and are never found buried in the superficial cytoplasm of the disc. In their appearance and structure they resemble very closely those figured by Van Durme for the Pigeon and the Swallow (1914, pl. iv. figs. ll & 13) and by Harper (1904, fig. 21) for the Pigeon.

The polar bodies are formed at or close to the centre of the disc. Their behaviour subsequent to their expulsion from the disc is so singular as to merit special mention. Immediately after fertilisation the small amoeboid bodies become active and migrate through the fluid of the perivitelline space to one end of the long axis of the now elliptical disc, where they take up a position together in the region of the marginal zone. Our observations indicate that this position is a constant one. It is always at that end of the elliptical disc which is free from superficial yolk-granules. We can find no previous record in any Amniote of a comparable migration of the polar bodies.

They are present in cleavage stages up to the 32-celled stage. By this time they are quite degenerate, in fact one has already entirely disappeared, and the other is much shrivelled, with numbers of minute vesicles in its cytoplasm.

Once having attained their final position, the polar bodies apparently lose any power of further migration, and in the 16- and 32-celled stages, owing to the growth in size of the blastodisc, are found within the margin, even within the area of the central cells.

Fertil2'satz'o'n.—The passage of the ovum down the Fallopian tube must be quite rapid, since none of our fertilisation stages (FA—FF) was found actually in the tube itself *. Caldwell states (1887, p. 472) that in the case of two eggs of Ornithorhynchus found in the dilated end of the Fallopian tube both had begun to segment, and one had already acquired eight segmentation nuclei. In his explanation to the figures of this stage Caldwell gives the diameter of one of the eggs as 2-6 mm. (pl. XXX. fig. 1 and pl. xxxi. fig. 3). This measurement is so much less than what we know to be the normal diameter of the ovum of Platypus at the time of ovulation (maximum 42-43 mm.), that there can be little doubt that Caldwell was dealing here with abnormal ova such as We have found in Echidmz, and possibly fragmenting. On this point no information of value can be gained from the study of Caldwell’s figure (pl. xxxi. fig. 3) of a section through thedisc of this ovum.

In our records, it is noted that egg FC was found in the thickened junctional region between the tube and uterus, and, although the precise position of the other fertilisation stages is not stated, it is highly probable that they lay either in this same region or in the upper part of the uterus.

Fertilisation is well known to have fundamental effects on the shape and structure of the germinal disc in many meroblastic ova, and to this the Monotreme ovum is no exception. Bartelmez (1912, p. 276) asserts that in the ovum of the Pigeon bilaterality exists from the stage of the earliest primordial ovum onwards. However this may be in the Pigeon, and it is a statement diflicult of proof, in Echidna it is only after fertilisation that a definite bilateral symmetry becomes externally apparent. It is first manifested by a change in the form of the disc, which, originally circular, becomes elliptical. This change of shape as a result of fertilisation seems to be fairly general in Sauropsida (Harper, 1904, fig. 16 ; Bartelmez, 1912, fig. 43, both for the Pigeon ; Nicolas, 1904, fig. xvii. ; Oppel, 1892, Taf. ix. figs. 1 & 6, both for Anguis fmgilis). On the other hand, Patterson (1910, fig. 1, p. 124) depicts the germinal disc of the fowl as being circular.

In Echvidlna the long axis of the disc is the axis of bilateral symmetry. The constitution of the disc alters in such a way that at one end of the long axis it becomes superficially charged with fine yolk-granules, while at the other end it remains relatively free from such granules. The polar bodies take up a position at the yolk-free end of the axis, and within the disc the two pronuclei meet and fuse on this axis—not in the centre of the disc, as might be expected, but appreciably nearer to the yolk-laden end.

Analogous changes are known to occur in the structure of the disc of the Pigeon, where Harper (1904, p. 365) describes the disc as being distinguishable into a more “ granular ” and a more “ hyaline ” pole (1904, p. 365). These changes in the fertilised disc of the Pigeon are described in greater detail by Bartelmez (1912, p. 299), who draws attention to the difference in the development of the “ periblast ring ” (our marginal zone) at the two ends of the disc, and to the rearrangement of the granular substance of the disc which becomes “moved from the anterior side of the segmental disc and forms a crescent round its posterior margin ” (see his figs. 40 & 41). In comparable Monotreme ova the width of the marginal zone differs at the two ends of the disc.

What appears to be a remarkable resemblance to the condition occurring in Echidna is described by Oppel (1892, Taf. ix. figs. l, 2, 5, & 6) in the fertilised ovum of Anguis fmgilis. His figs. 1 & 6 depict an elliptical disc of very similar form to that of Echidna (see our fig. 79 (Pl. XV.) and text—fig. 2). He shows, also, that the two pronuclei, as in Eckidna, lie in the long axis of the disc, 2'. e., the pronuclear axis is coincident with the axis of bilateral symmetry. Again, if We refer to his fig. 2, which represents a longitudinal section through the disc, we can see at the left side of the figure what appears to be an infiltration of the surface-layer of the disc by fine-grained yolk, this being much less pronounced at the opposite end. To complete the resemblance, the pronuclei are situated nearer that end of the disc which apparently contains more superficial yolk. It is evident then that the Monotreme shows a striking resemblance to the Sauropsida as typified by Anguis in the structural characters of the disc at the time of fertilisation.

As regards the details of fertilisation, however, we would emphasise one important difference between Monotremes and Sauropsida. In the latter it would appear that polyspermyis the rule ; in the former all the evidence shows that they are monospermic. In none of our fertilisation or early cleavage stages is there any evidence of the presence of supernumerary sperms, with the possible exception of the obviously abnormal early cleavage stage of Platypus above described (Platypus JJ), and this in spite of the fact that sperms are usually to be found in the albumen layer of these eggs.

The two pronuclei situated in the plane of the long axis of the disc, nearer the end which contains more superficial yolk, have a subsequent history similar to that recorded by Harper for the Pigeon, and illustrated by him in several figures (1904, p. 364, figs. 10, ll, & 12). Similar figures for Anguis fmgilis are given by Oppel (1892, Taf. ix. figs. 5, 7, & 13, and Taf. X. fig. 15).

In Echidna, as the pronuclei approach one another, differences in size and staining reaction become apparent. One is usually a little smaller, stains more intensely, and is more deeply situated than the other. This we regard as the male pronucleus. Harper shows a similar arrangement in the Pigeon (1904, fig. 10) and gives the same interpretation, whilst Oppel (1892, Taf. ix.) records comparable stages in Anguis fmgilis in which the male pronucleus is also smaller than the female.

In Echidna the pronuclei come into relation in such a way that the female becomes superimposed on the male. Their apposed surfaces become flattened, and the orientation of the two pronuclei is such that this flattened surface is parallel to the disc-surface. Over the surface of contact the nuclear membrane eventually disappears, and in this way the single cleavage nucleus takes its origin.

The pronuclei take up their final position in the long axis of the disc at a spot which is a little more than one-third the length of the disc from its yolk—rich end. At the opposite end are situated the two polar bodies, now at rest, the larger one (presumably the first) being the more peripherally situated.

The disc is now prepared for the onset of cleavage. The only observer who appears to have seen the first furrow in the Monotreme egg is Caldwell, who states (1887, p. 476) that “ the first furrow marks out the germinal disc iI1to a larger and a smaller area ” *. He further goes on to say that “ the second furrow appears at right angles to the first, and divides the germinal disc into four regions, two larger and two smaller” (pl. xxxi. fig. 2, n.1 & n.2). This figure represents a vertical section through the disc of an Echidna ovum of 4-5 m1n., at the stage of four segmentation nuclei. On the other hand, Semen states that all the cells of the four-celled stage are equal in size, and he pictures them thus in his figures of surface views and of sections (Semon, 1894.-, p. 68, Taf. viii. figs. 1 & 10, and Taf. ix. fig. 30).

It should be noted, however, that this is a general statement applying to cleavage in both the Monotremes and Marsupials, and, so far as the latter are concerned, is based on an erroneous inter pretation of the structure of the ovum of Phascolarctus (of. Hill, 1910, pp. 26-28).

Our observations support Caldwell. The position of the pronuclei in later fertilisation stages, and especially the position and general relations of the first cleavage spindle in our stage CA (Eckidna VVH 29), together with the structural conditions found in early cleavage stages, leave us in no doubt whatever that the first cleavage furrow is transverse to the long axis of the disc, and divides the latter into two unequal areas, the smaller of which is impregnated superficially with fine yolk-granules, whilst the larger is practically devoid of such. Thus the first cleavage furrow in Monotremes divides the disc quantitatively as well as to some extent qualitatively.

The relationship of the first furrow to the long axis of the disc does not appear to be always the same in Sauropsida as it is in Monotremes. Nicolas (1904, p. 613) has reviewed in some detail the observations of Kupffer and Benecke, Sarasin, Oppel, Todaro, and Will on the early cleavage of Reptiles, and gives also certain findings of his own. It would appear that the first furrow in Platydactylus mauritanicus and in Angmls fmgilis is excentric, and is parallel to the long diameter of the elliptical disc. The second furrow crosses it at right angles (Nicolas, 1904, pl. xvii. figs. 1 & 2). In the Pigeon, according to Harper (1904, p. 370), “ the first furrow crosses the disc along its short diameter,” and, like Eckidna, the two blastomeres differ in structure, the smaller being granular,” the larger “ hyaloplasmic.” In the absence of detailed investigations into the structure of the reptilian germinal disc at the time of fertilisation and early cleavage it is impossible to correlate the recorded observations relating to the direction of the first cleavage furrow.

The third series of furrows in Reptiles, according to Nicolas (1904, p. 617, pl. xvii. figs. 3-7), are parallel to the second furrow and perpendicular to the first. In this way an eight-celled stage results which in a typical condition, such as is illustrated in his fig. 3, closely resembles the eight-celled stage of the Monotreme, but is arrived at in a different way. We are not informed as to whether there is any constant difference in the sizes or yolk-content of the blastomeres at either end of the long axis of the reptilian blastodisc at this stage.

So far as Birds are concerned, the only statements on this question come from Patterson (1910) for the Fowl and Harper (1904) for the Pigeon.

According to Patterson, the germinal disc of the Fowl is circular at the time of the first division (see his fig. 1, p. 24). The second furrow is at right angles to the first, and the furrows of the third series are parallel to the first. From what we can understand of Harper’s description of early cleavage in the Pigeon (p. 370), the third series of furrows are more or less parallel to the second (pl. iv. fig. 43), although Harper says that considerable variation occurs in the position of the furrows.

Van Durme (1914, pl. iv. fig. 20) gives a figure of the eight-celled stage of the Swallow which closely resembles that of the Monotreme, but she provides no details of the order of appearance of the cleavage furrows.

The net result is that in all these cases where the cleavage is regular, an eightcelled stage is reached, which is composed of two linear series, each of four cells, arranged on either side of an axis of bilateral symmetry. Whether this axis is homologous in all cases we are not able to say, nor is it possible to determine, in the case of the Monotreme, the relationship of the long axis of the unsegmented disc to the polarity of the embryo.

Monotremes resemble mammals in general in the clearness and regularity with which the processes of early cleavage are carried on. Up to the 32-celled stage the synchronisation of cell-division is practically perfect. The 16-celled and 32-celled stages, however, strikingly resemble those of the Sauropsida in that they consist of an outer ring of large “ marginal ” cells enclosing an inner field of smaller “ central ” cells. Figures similar to those of our relevant stages of Eckidna are given by Nicolas (1904, pl. xvii. figs. 8 & 9), Blount ( 1909, figs. 39, 40, & 48), Patterson (1910, figs. 20 & 21), and van Durme (1914, pl. iv. figs. 22 & 23).

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Arch. Biol. 44-. THE DEVELOPMENT or THE .\1ONO'I‘REMA’1‘A. 579

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alb. Albumen. pl). Polar body. bv. Capillary in membrana propria. . pb.1. First polar body. c.c. Central cell—area. 1 pb.2. Second polar body. cxn. Cleavage nucleus. 19.0.2. Peripheral cytoplasmic zone. c/m2. Group of chromosomes for second l gm. 5‘. Male pronucleus. polar mitotic figure. 5 pn. E2. Female pronucleus. cmz. Central medullary zone. p.s'p.l. First polar spindle. cs. Centrosphere. * p.sp.2. Second polar spindle. c.y.z. Cortical yolk-zone. .‘ pus. Perivitelline space. em. Egg~n1en1brane. l sh.m. Shell-membrane. fccl. Superficial formative zone of ger— s.l. Striate layer. minal disc. th. Theca folliculi. f.e. Follicular epithelium. . th.e. Theca externa. f.s. Follicular secretion (fluid). ‘ tho’. Theca interna. f.s’. Precursor of follicular secretion. 3 vcd. Deep Vacuolated zone of germinal ft. Fat—droplet. ; disc. gd.p. Primordium of germinal disc. vf.z. Vitello-fatty zone. l.b. Body of latebra. yb. Yolk-bed. l.n. Neck of latebra. _ ys. Yolk—sphere. ly.z. Latebral yolk-zone. ' yap. Yolk—sphere primordium. m.c. Marginal cell—area. Yolk—Vesicle. mg.z. Marginal zone. g/.2. Yolk-zone. mp. Membrana propria. ‘ zp. Zona pellucida. mt. Mitochondria. ‘ zpa. Zona-albumen layer. 7n.t.z. Central mitochondrial zone. . zps. Secretion destined to form zona.

m.z. Medullary zone. PLATE I.

41 582

Fig. 1. Fig. 2.

Fig. 3.

Fig. 4. Fig. 5.

Fig. 6.


Platypus XV. Left ovary, 16-25 X10-5 mm., with three large follicles, the middle one measuring about 4-5 mm. in diameter. ( >< about 6.)

Echiolna, B.ll.8.30. Left ovary, 11-5><8-5 mm. in diameter, showing a single large follicle 4-7 5 X4-5 mm. in diameter ( X about 6-5.)

Echidna XIX. Section of oocyte, 0-10 ><0-08 mm. in diameter, fixed in F.W.A. 08., centrosphere; f.e., follicular epithelium; ft., fatdroplet ; mt.z., central mitochondrial zone. ( X 800.)

Echidna, 24.7.29. Section of oocyte, 0-083 ><0-079 mm. in diameter,

fixed in Bouin. ( X800.) Eckidna. Nucleus of oocyte, 0-104><0°088 mm. in diameter, fixed in Corr.-Osmic. Text, p. 460. ( X 1500.)

Echidna, 24.7.29. Nucleus of oocyte, fixed in Bouin. ( X 1500.)


Fig. 7. Echidna. Section of oocyte O-128><O-124 mm., fixed in Corr.-Osmic. Text, p. 462. (><600.)

Fig. 8. Eckidna XVIII. Periphery of oocyte, O-103 ><0'O78 mm. in diameter, fixed in .F.W.A. em., egg-membrane; f.e., follicular epithelium; ft.. fat-droplet ; zp.s., precursor of zona. ( X1166.)

Fig. 9. Eckidna XVIII. Periphery of oocyte, 0-13 ><0'O94 mm., in diameter. 212., zona pellucida. ( X1166.)

Fig. 10. Eohidna XVIII. Periphery of oocyte, 0°179><O-148 mm. in diameter. s.l., striate layer. (><ll66.)

Fig. 11. Echidna XVIII. Periphery of oocyte, 0-17‘/<0-16 mm. in diameter. (><1l66.)

Fig. 12. Echidna XVIII Section of oocyte, O'29><O-27 mm. in diameter. m.z., medullary zone; p.c.z., peripheral cytoplasmic zone; vf.z., Vitello-fatty zone. ( X260.)

Fig. 13. Echidna XVIII. Periphery of the preceding oocyte. mt., mitochondria in follicular epithelium (f.e.) ; ys.p., yolk-sphere primordium. ( x 1166.) 3.1.




Fig. Fig. Fig.









Eckidna XVIII. Section of oocyte, 0-296X0-284, mm. in diameter. vf.z., vitello-fatty zone ; 3/3.1)., yo1k—sphere primordium. ( X225.)

Eckidmt XVIII. Periphery of oocyte, 0-31 X0-25 mm. in diameter. f.e., follicular epithelium, with mitochondria (mt.); p.c.z., peripheral cytoplasmic zone; 5.1., striate layer; vf.z., vitello-fatty zone; 210.,

zona pellucida. ( X 1125.)

Echidna XVIII. Section of oocyte, 0-368 X0-328 mm. in diameter. ys.p., yolk-sphere primordium. ( X200.)

Echidmz XVIII. Periphery of oocyte, 0-448X0-36 mm. in diameter. (><600.)

Platypus VI, 13.9.05. Section of oocyte 0-56 X0-50 mm. in diameter. c.y.z., cortical yolk-zone ; m.z., medullary zone. ( X142.)

Platypus XV. Section of oocyte, 0-53 X0-43 mm. in diameter. (X 142.) n]7'r/.//.2’. .1“/. ’/KI’/1;T/5/j %///


Fig. 20. Platypus VI, 13.9.05. Section of oocyte, 0-62 ><0~6l mm. in diameter. cmz., central medullary zone ; c.y.z., cortical yolk-zone ; gd.p., primordium of germinal disc; p.c.z., peripheral cytoplasmic zone; vmzu, vacuolated medullary zone. ( X 117.)

Figs. 21 & 22. See Pl. V.

Fig. 22 A. Echidna, 24.7.29. Section of oocyte, 0-65 ><0-60 mm. in diameter. cmz., central medullary zone; l.n., neck of latebra; p.c.z., peripheral cytoplasmic zone ; g.z., yolk-zone.

Fig. 22 B. Echidna, 24.7.29. Periphery of oocyte, O'64><0'56 mm. in diameter. lg/.z., primordium of latebral yolk-zone ; ys.p., yolk-sphere primordium.


Fig. 23. Platypus VIII, Section of oocyte, 0-86 ><0-81 mm. in diameter. gd.p., primordium of germinal disc; l.b., body of latebra; l.n., neck of latebra; lg/.z., latebral yolk-zone ; y.z., yolk-zone. (><90.)

Fig. 28. Eohidna XVIII. Periphery of oocyte, 1-0><0-95 mm. in diameter. f.e., follicular epithelium, containing minute fat—droplets; ft., fatdroplets in the fine strands of the cytoplasmic network between the yolk-spheres; p.c.z., peripheral cytoplasmic zone containing minute yolk-spheres: s.Z., striate layer; th.i., theca interna; zp., zona.

( X975.)


Fig. 2]. Platypus VI, 13.9.05. Periphery of oocyte depicted in fig. 20. s.l., striate layer ; ys.p., yolk-sphere primordium. ( X 1320.)

Fig. 22. Platypus VI. Periphery of oocyte 0-66 ><0-61 mm. in diameter.

Fig. 24. Platypus VIII. Segment of oocyte depicted in fig. 23, showing the primordium of the germinal disc (gd.p.), the nucleus, the primordium of the yolk-bed (yb.p.), the latebral neck (l.n.). yv., yolk-vesicle. ( X 375.)

Fig. 25. Platypus VIII. Central region of oocyte, 1-10><1-O0 mm. in diameter, showing the vacuolated cytoplasm of the latebral body (l.b.), the latebral

yolk-zone (lg/.z.), the latebral neck (l.'n,.). yv., yolk-vesicle. (X 150.) I/2-/mg. 1( r /. Va-. 7/~zi1/.4/H”/I. './’/. If


Fig. 26. Platypus VIII. Same oocyte as in fig. 25, showing the junetional region between the latebral body (l.b.) and the latebral neck (l.n.). Note the numerous yolk-vesicles (yv.). ly.z., latebral yolk-zone. (><300.)

Fig. 27. Platypus A.13.9.03. Upper polar region of oocyte, diameter 1-09X 0-96 mm., showing the germinal disc primordium (gd.p,), the nucleus and the primordium of the yo_lk~bed (yb.p.). l.n., latebral neck; ly.z., latebral yolk-zone. ( X265.)

Fig. 28. See Pl. IV.

Fig. 29. Platypus X. Horizontal section of oocyte, diameter 1-35 ><l-14 mm. l.b., latebral body; ly.z., latebral yolk-zone; y.z., yolk-zone. * (X50.)

Fig. 30. Platypus VIII. Central region of oocyte, diameter 1-90Xl-80 mm., showing the latebral body (l.b.), latebral neck (l.n.), and latebral yolkzone (ly.z.), yv., yolk-vesicle. ( X206.)

Fig. 31. Platypus VIII. Junctional region between latebral body and latebral

neck of preceding oocyte, under high magnification, showing the numerous yo1k—vesieles (yu). ( X350.) -"/nu/.,: .’Z( r/. -Va-. VV’k.,[A . H’/I. I/’/. I7

PLATE VII. Fig. 32.

Fig. 33. Fig. 34.

Fig. 35.

Fig. 36.


Platypus VIII. Central region of oocyte, diameter 2-0 X 1-8 mm., showing junctional region between the latebral body (l.b.) and latebral neck (l.n.). ly.z., latebral yolk-zone ; yu, yolk-vesicle. ( X190.)

Platypus VIII. Central region of oocyte, diameter about 2-70 ><2-50 mm. ( X230.)

Platypus XV. Section of oocyte, diameter 3-20 ><3‘l0 mm. See text, p. 490. (><20.)

Platypus, y..19.8.0l. Horizontal section through oocyte, diameter 4-46 X4-21 mm., showing the latebral body (l.b.), the latebral yolkzone (ly.z.), and the adjoining portion of the yollczone. ( X 170.)

Platypus X. Horizontal section of oocyte, diameter 2-18 X2-00 mm., showing a ring of small yolk-spheres (s.ys.) between the outer and inner portions of the yolk-zone. See text, p. 491. ( ><37-5.) 7/2-a/z.J.;L «K 2%. '2’z:%{,«%K}{71.C/’/. I ’/


Fig. 37. Echidna, XVIII. Oocyte, diameter 1-10 X0-96 mm. Portion of the body of the latebra, in which are situated eosinophil yolk-sphere primordia, many of them containing eosinophil or basophil granules. See text, p. 492. (Xl620.)

Fig. 38. Eckidna, C.20.7.29. Central region of oocyte, diameter 2-60 X2-20 mm. l.b., body of latebra. ( X206.)

Fig. 39. Eckidna, C.20.7 .29. Portion of the peripheral region of the latebra of the preceding oocyte, under high magnification, to show the multiple production of yolk-spheres from eosinophil primordia. See text, p. 492. (X900.)

F ig. 40. Eckidna. Portion of central region of an oocyte, diameter O-72 X 0-62 mm. from an atretic follicle. cmz., central medullary zone ; ft., fat-droplet ; 3112., yolk-Vesicle. ( X500.) =7/7///.1. 445 r/. Va: 7//.. H /I. V./’/. V///.


4L 598

Fig. 41.

Fig. 42.

Fig. 43.

Fig. 44.

Fig. 45.


Echidna, 0.20.7 .29. Central region of oocyte, diameter 4:-00 ><3-40 mm., in horizontal section. l.b., body of latebra; ly.z., latebral yolk-zone. ( X 206.)

Eckidna, VVH 38, 16-celled stage. Portion of the body of the latebra and the base of the latebral neck. See text, p. 495. ( X680.)

Echiclna, C.20.7.29. Upper polar region of oocyte, diameter about 2 mm., showing the nucleus, the germinal disc primordium (gd.p.), the follicular epithelium (f.e.), the theca (th.), and the yolk-bed (yb.). ( X 320.)

Eckidna, B.3.8.29. Upper polar region of oocyte, diameter 26 ><2-7 mm. ( X 320.)

Echidna, B.3.8.29. Nucleus and surrounding region of preceding oocyte. ( X 865.) T/nmx;. ,1} r-/. Yr}-. ;/Ell"/I5./’/.%/_ If


Fig. 46. Platypus XV. Upper polar region of oocyte, diameter 3-2 X3-1 mm. mp, membrana propria; th.e. & 13., theca externa and interna. ( X276.)

Fig. 47. Platypus XV. Nucleus and surrounding region of preceding oocyte. (><766.)

Fig. 48. Eckidna, B.19.7.30. Upper polar region of oocyte, diameter 4~l X3-1 mm. fcd., superficial formative zone of germinal disc ; vcd., deep vacuolated zone of same ; yb., yolk-bed. ( X320.)

Fig. 49. Echiolna, B.19.7.30. Upper polar region of preceding oocyte, showing the theca, follicular epithelium, nucleus, and adjoining portion of the germinal disc. (><838.)

Fig. 50. See Pl. XI.

Fig. 51. Echidmz, 2.8.28. Upper polar region of oocyte, diameter 3-7 X3-4 mm.

( X 320.)


Fig. 50. Eckidna, D.15.8.29. Oocyte, diameter 3-9><3-6 mm. Section of upper polar region, showing germinal disc (fcd. & vcd.) and nucleus. f.e., follicular epithelium; l.n., neck of latebra; tk.e., tk.z'., theca externa, interna; yb.,yolk-bed. (><244.)

Fig. 52. Echidna, B.11.8.30. Section through upper polar region of oocyte, diameter 4-1 X 3-4 mm., showing the follicular epithelium (f.e.), germinal disc (fcd. & vccl.), and the nucleus containing nucleolar granules and linin—threads. (><365.)

Fig. 53. Echidna, B.ll.8.30. Follicular epithelium, nucleus, and surrounding portion of germinal disc of preceding oocyte. The nucleolar granules and linin-threads in the nucleus are Well seen. ( X 613.)

Fig. 54. Echidna, A.20.7.29. Section through upper polar region of oocyte, diam. 4-30 ><3-63 mm. Reference letters as in fig. 50. ( X290.) -f/27///.1. 1;, /. 1%}: )‘?rT‘/[. H71.


Fig. 55. Echidna, A.20.’7 .29. Follicular epithelium ( f.e.), nucleus, and surrounding portion of the germinal disc of the preceding oocyte (fig. 54), under higher magnification. Note the precursor of the follicular secretion (f.s’.) in the follicular cells and the linin—threads (l.t.) in the nucleus. (><550.)

Fig. 56. Echidna. C.20.7.29. Oocyte, diameter 4'0><3-4 mm., showing the follicular epithelium (f.e.), With the follicular secretion (f.s.). th.e. & th.i., theca externa and interna. ( X600.)

Fig. 57. Echidna, 0.20.729. Another portion of the follicular epithelium from the same follicle as the preceding. ( X 600.)

Figs. 58, 59, 60. Platypus, oc.l9.8.0l. Full—grown oocyte, diameter 4-3 X4-1 mm. Portions of the follicular epithelium, showing in fig. 58 the intracellular precursor (f.s’.) of the follicular secretion, seen as intercellular globules and masses (f.s.) in figs. 59 & 60. Note the Very thick theca interna

(th.z'.) in fig. 58. 210., zona. (><600.) .:-77"(/~/z.a’.(/ /((1,: %%6Z/K/I”/T; J71


4M 606







Fig. Fig.







67. 68.


Phase of jllatumtion. Stage MA. Echidna, 16.7.29. Oocyte, diameter 364 mm.

Section (tangential) through central region of germinal disc and showing the 1st polar mitotic figure (p.sp.l). em., egg-membrane; fcd. & vcd., superficial and deep zones of disc; f.e., follicular epithelium; f.8., follicular secretion ; 1)?/28., material in perivitelline space. ( X 1440.)

Section through follicular wall and periphery of oocyte, showing the vacuolated follicular secretion (f.s.) situated between the follicular epithelium (f.e.) and the zona (zp.); th.e. & tk.z'., theca externa and

interna. ( X390.)

Stage MB. Eckidna, B.20.7 .29. Oocyte, diameter 4-0 X 3-3 mm.

Section through centre of germinal disc, showing the 1st polar mitotic figure (p.sp.l). ]ov.s., perivitelline space. ( X 1250.) Section through the follicular wall. f.s., follicular secretion.

Stage MC. Eckidna, A.1.8.30. Oocyte, diameter 3-75 mm.

Section through the central portion of the germinal disc, showing the 1st polar body (pb.1) situated in the perivitelline space (pv.s.) and the chromosomes (chr.2) destined to form the equatorial plate of the 2nd polar spindle. em., egg-membrane; f.s., follicular secretion; zp., zona. (><360.)

The central portion of the preceding figure, much more highly magnified. fcd. & vcd., superficial and deep zones of the germinal disc. ( X 1140.) Section through the germinal disc some distance from its centre. ( X430.) Section through the follicular wall and the periphery of the oocyte.


( X430.) /M.


4M 608







Fig. Fig.







75. 76.


Stage MD. Echidna, B.2l.7.30. Oocyte, diameter 4-50 X3-43 mm.

Section through the centre of the germinal disc, showing the first polar body (pb.l) situated in the perivitelline space (p'v.s.) and the 2nd polar mitotic figure (p.sp.2), situated in the superficial zone of the disc (fcd.). ( X 1250.)

Section through the follicular wall. f.s., follicular secretion; 210., zona. ( X430.)

Phase of Fertilisation.

Stage FA. Echidna, 24.7.29. Uterine ovum, diameter (fresh) 4-0 mm.

Section including the entire Width of the germinal disc and passing through its centre. fad. &. vcd., superficial and deep zones of disc; pb.2, 2nd polar body in the perivitelline space (pv.s.); sh.m., shellmembrane ; yb., yolk-bed ; zpa,., zona.-albumen layer. ( X350.)

Portion of central region of preceding figure, under higher magnification, showing the 2nd polar body (1962) enclosed in the coagulum in the perivitelline space (pv.s.) and the female pronucleus (gm. Q), situated superficially in the formative cytoplasm of the germinal disc (fcd.). vcd., deep vacuolated zone of disc. (X 1500.)

Section of the disc passing through the 1st polar body (pb.l). albumen ; em., egg-membrane ; 21)., zona. ( X 1500.)

Stage FB. Ec-hidna, E.8.8.30. Uterine ovum, diameter (fresh) 4-6 mm.


Section of germinal disc, showing the male ( gm. 6‘) and female (pm. $2) pronuclei. (><l30.)

Section showing the female pronucleus (pn. $2). ( X 1200.)

Section showing the male pronucleus (gm. 3). (1230.) ..-77?///.J.§[( r/.%(-. 7/ZTZ/L51”/T». i/’/. . 171'


Fig. 77.

Fig. 79.

Fig. 80.

PLATE XV. Stage FC. Echidna, C.15.8.30. Uterine ovum, diameter (fresh) 4-5 mm.

Section of germinal disc, showing the male and female pronuclei (gm. overlapping each other at the centre of the formative zone of the disc

‘NC O9-r

(fcd.). 'vcd., deep vacuolated zone of the same; mg.z., marginal zone ; sh.m., shell-membrane ; yb., yoik-bed; zpa,., zona-albumen layer. ( X400.)

The male (gm. 5) and female (1117. S‘) pronuclei. ( X 1566.)

Stage FD. Echidna, VVH 26. Uterine ovum, diameter (fresh) 4-75 mm.

Surface-View of germinal disc, now elliptical in outline.

(X about 52.) Median longitudinal section through the germinal disc, showing the

superimposed pronuclei (gm-. 5, S2) situated in the formative cytoplasm (fcd.), nearer the less sharply defined margin of the disc (on the right in the figure) and a polar body (pb.), situated over the more sharply

defined margin (on the left). ( X284.)

See text, p. 549.

. The two pronuclei (pm. 5‘ and jam. 9) and the surrounding portion of the

formative cytoplasm of the disc. ( X 1566.) .:77°r/-/1.9‘. 121.! Va: ’“'//,l‘V/_/If/V/IV. 3/’/. /11:’













. 90.


Stage FE. Echidna, B.30.7.29. Uterine ovum, diameter (after fixation) 4-6 mm.

Median longitudinal section of the germinal disc, showing the conjugating pronuclei (c.n.) situated nearer the less sharply defined margin of the disc, which is superficially infiltrated with fine yolk-spheres (on the left in the figure) and the two polar bodies (125.1 & 2) situated over the opposite, more sharply defined margin (on the right in the figure). mg.z., marginal zone. ( X400.)

The two pronuclei now united, but still clearly distinguishable, and the surrounding formative cytoplasm of the disc. ( X 1765.)

The marginal region of the disc showing the two polar bodies (196.1 & pb.2). mg.z., marginal zone; pv.s., perivitelline space; sh.m., shellmembrane ; 2pa., zona~albumen layer. ( X 1650.)

Early Cleawge Stages. Stage CA. Eckidna, VVH 29.

Surface-View of the germinal disc and the surrounding region. 70.)

Median longitudinal section through the germinal disc, showing the 1st cleavage spindle in the telophase and situated in the thicker half of the disc, which is rich in superficial yolk. fcd., superficial formative zone ; vcd., deep Vacuolated zone of disc ; mg.z., marginal zone ; 3/b., yolk-bed ; zpa., zona-albumen layer. ( X255.) ,

Echidna, VVH l, 4-celled stage. Section of the disc along the plane C—D in text-fig. 3, and passing through cell-areas 1 and 2 (car/.1 & ca.2) and the two polar bodies (pb.l & 2). ( X177.)

( >< about 6:».


Sta-ge CB. Four-celled stage. Echidna, VVH 1.

Fig. 87. Surface-View of blastodisc and surrounding region (of. text—fig. 3). ( X 62.)

Fig. 88. Portion of a Vertical section through the egg, passing through the blastodisc and the latebra. l.b., body of latebra ; l.n., latebral yolk zone of neck; lg/.z., latebral yolk-zone ; yb., yolk-bed ; zpa., zona-albumen layer. (><55.)

Fig. 89. Section of the blastodisc along the plane A—B in text-fig. 3. ca.1, ca.2, ca. 4, cell-areas 1, 2, and 4 of the latter. ( X 150.)

Fig. 90. See preceding Plate.

Stage CC. Eight-celled stage. Platypus, A and AA.

Fig. 91. Platypus, A. Entire egg, complete with shell. (Xabout 1331;.) Fig. 92. Platypus, A. Surface-View of blastodisc. (X58.) './’/NH’/Z

PLATE XVIII. Fig. 93. Platypus, AA. Section of blastodisc. ( X194.)

Stage CD. 8—celled stage. Echidna, VVH. 22.

Fig. 94. Surface-View of blastodisc. See text-fig. 4. ( X45.) Fig. 95. Section along the plane. A—B in text-fig. 5. ( X 160.) Figs. 96 & 97. Mitotic figures from two of the cell—areas of this stage. ( X 1150.)

Stage CE. Sixteen-celled stage. VVH. 38.

Figs. 98 & 99. See Pl. XIX.

Fig. 100. Echidna, VVH. 38. Section through the blastodisc along the plane A—B in text-fig. 6, and intersecting a marginal cell—area on each side and two central cell-areas. ( X170.)

Fig. 101. Section through the blastodisc, along the plane C—D in text-fig. 6, but including only the two central cell-areas. ( X377.) %-”/7?(zm:;Z(Ac.K%w~Y2»(-. ‘Z675/Iii!”/1.?/’/. /I 7////.


Fig. 98. Echidna, VVH 38. The egg, after removal of the shell, showing the blastodisc. (Xl5.) Fig. 99. Surface-View of the blastodisc, more highly magnified. Compare text fig. 6. (X60.)

Stage CF. 31-celled stage. Echidna, A.31.7.30.

Fig. 102. The egg, after removal of the shell, showing the blastodisc. ( X 13-5.)

Fig. 103. Surface-View of blastodisc, more highly magnified. As in fig. 99 the distinction of the cell-areas into marginal and central is very clearly seen. (><62.)

Fig. 104. Vertical section of the egg, passing through the blastodisc and the latebra. l.b., body of latebra; l.'n,., latebral neck; lg/.z., latebral yo1k—zone. (X76.)

Fig. 105. Section through the blastodisc. c.c., central cell-area; m.c., marginal cell-area ; 3/b., yolk-bed ; zpa., zona-albumen layer. ( X160.) AK ” /I, 1/’/. - [X17



Fig. 106. Echédna. Stage CF. Section through the blastodisc. Lettering as in fig. 105. l.n., neck of latebra. (X 160.)

Fig. 107. Portion of margin of blastodisc, showing a marginal cell—area (m..c.) in division. X376.

Stage CG. 32-celled stage. Echidna VII, ’3l.

Fig. 108. Surface-View of blastodisc. pb., polar body. (X62.) Figs. 109 & 110. Sections of the blastodisc showing the central (c.c.) and marginal (m.c.) cell-areas. ( X 170.)


VOL. XXIV.——PART VI. No. 23.—-December, 1939.

40 622

Fig. Fig.


Fig. Fig. Fig.





114. 115. 116.



Irregularities in Cleavage. Platypus JJ.

Surface-View of blastodisc. 24.)

Section through the blastodisc and intersecting two of the large cellareas (on the left) and three of the smaller. ( X200.)

Section through the blastodisc, showing four of the quite small faintly outlined cell-areas. ( X435.)

Eckidna, VVH 15. Irregular early cleavage stage. ( >< about 20.)

Irregular 8-celled-stage, with accessory cleavage.

For description, see text, p. 569. ( >< about

Surface—View of the blastodisc. Text, p. 570.

Section through the blastodisc. ( X 180.)

Echidna, VVH 14. Entire egg in which the shell has become drawn out and assumed an ellipsoidal form. Diameter of egg, fresh 6-0 X4-5 mm., after fixation in Smith’s Fluid and transference to 5 per cent. formalin, 7 X4-34 mm. ( X8.)

Echidna, VVH 3. Vertical section of the egg passing through the upper pole and the latebra. Text, p. 495. (><46.) 77

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