Paper - Studies in the development of the opossum 3 (1918)
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Hartman CG. Studies in the development of the opossum (Didelphys virginiana L.). III. Description of new material on maturation, cleavage, and entoderm formation. IV. The bilaminar blastocyst. (1918) J Morphol. 32(1): 1-144.
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Studies in the Development of the Opossum Didelphys Virginiana L.
III. Description of New Material on Maturation, Cleavage and Entoderm Formation
The Wistar Institute of Anatomy and Biology and the University of Texas
III. Description Of New Material On Maturation, Cleavage, And Entoderm Formation
Introduction
a. Prefatory remarks
The writer's work on the development of the opossum began in 1912-13, when a prehminary study of the problem was made and the approximate breeding season determined for Austin, Texas. Active collecting was done in January and February, 1914, and again in 1915, and the results of the study of the 415 eggs secured from twenty females were published in March, 1916. A considerable number of eggs, including several missing stages, were also collected during 1916, and at this time many more eggs and embryos were sacrificed for a series of physiological experiments on the female opossum. As a result of these experiments I learned a simple and comparatively certain means of diagnosing a female opossum in the earliest stages of pregnancy and in early oestrus. Since it was felt that this experience would greatly facilitate collecting in 1917, plans were made to secure a complete series of eggs, embryos, and pouch young of this species. The more than hoped for success of the effort was due to the active interest of Dr. M. J. Greenman, Director of The Wistar Institute, for it was through the generosity of the Institute that I was enabled to secure and care for the requisite number of animals and also to have the advantage of the able services of Dr. C. H. Heuser, embryologist at the Institute, who, with the assistant of Miss Aimee Vanneman, technician in the School of Zoology, the University of Texas, saw to the proper fixation and after-treatment of the specimens. Entire credit also belongs to Doctor Heuser for the unique series of photographs of living eggs, some of which are herein reproduced. To Dr. J. T, Patterson is due the initiation of the work on this interesting marsupial, and his scientific zeal and keen interest in mammalian embryology have been a constant inspiration to the writer. I am indebted for indispensable assistance in the operations on the animals during the last two years' collecting to a number of premedical students of zoology, notably to Mr. Victor Tucker, who stood ready to help at any hour of the day or night and who performed many of the operations; also to Miss Janoch and Messrs. Goff, Stiefel, and Kaliski.
During the year 1917-18 I have enjoyed the privileges of a fellowship at The Wistar Institute and have been its guest while engaged in a study of the material collected. I am further indebted through the Institute to Dr. C. H. Heuser for making some of my best preparations of serial sections and to Mr. T. H. Bleakney, artist at the Institute, for drawing plate 12 and for shading and finishing the figures drawn for this paper.
The new material collected since the publication of my former paper covers many stages there described, and in addition thereto transitional stages not secured before. Among the latter are litters Nos. 194', 344, 349, 356, 175', 339, and 347, which show the process of entoderm formation in an unbroken series. Since this phase of the problem had to be entirely rewritten, and since I now have new material on the early stages, besides a series of photographs of the living egg in all stages, it has seemed desirable and profitable to give a complete account of the development of the opossum from the beginning. This has been done in the present paper; but the reader is referred to the writer's former publication for certain details.
The original notes and the preparations upon which this work is based, together with alcoholic specimens, will be deposited in the archives of The Wistar Institute, where they will be easy of access, and anyone who wishes to do so may examine the material and test the validity of the conclusions at which I have arrived in this paper.
b. Historical
In my former publication I reviewed in some detail the work of Selenka ('87) on the opossum and that of HiU ('10) on Dasyurus. Mention was also made of Caldwell's discovery ('87) of the shell membrane enveloping the marsupial egg (Phascolarctus), and of a short paper by Professor Minot ('11) on the bilaminar blastocyst of the opossum. Simultaneous with the pubhcation of my article, a paper by Spurgeon and Brooks appeared, giving a description of two litters of opossum eggs in cleavage (2 to 8 cells). I wish at this point to recur briefly only to the work of Selenka, leaving the other articles to be discussed under appropriate headings in the body of the paper.
Salenka's work on the cleavage and blastocyst formation is based on 26 eggs secured from two females. One animal yielded one 2-celled, one 20-celled, and nine unfertilized eggs, all badly shrunken. I suspect that the '2-celled' and the '20-celled' eggs are probably specimens in different stages of fragmentation. The other animal furnished two unfertilized eggs, one4-celled and one 8-celled egg, two blastocysts of 42 and 68 cells, respectively, two slightly older blastocysts with a mass of entodermal cells, and six normal vesicles with thin, partly bilaminar walls. Of his unsegmented ova I think all were unfertilized. Hence the 42-celled and the 68-celled blastocysts which Selenka describes are the youngest of his specimens which approach a normal opossum egg. These two are practically normal except for the shrinkage of the vesicle from the vitelline membrane and for the regular gradation in size of the blastomeres from one pole to the other — a condition entirely accidental and not at all characteristic for this or any other stage in the development of the opossum. His pear-shaped, thick-walled vesicle with spreading entoderm (Selenka, '87, Fig. 1 and 2, Taf. XVIII) is clearly a degenerating specimen, as I judge by comparison with numbers of similar preparations from my collection. Whenever, in any batch of eggs, there are very retarded specimens, these are to be regarded with suspicion. Many such abnormal eggs can be seen in my photographs of living eggs, as, for example in figure 4, plate 1, and figures 3 and 4, plate 11. Selenka's interpretation of certain gaps in the walls of his young blastocysts as the 'blastopore' must be rejected for the reason that these gaps are not to be found in completed blastocysts, of which I have a hundred specimens. Where openings in the blastocyst wall do exist in young specimens, they are easily explained when the method of blastocyst formation is understood.
On the origin of the entoderm in the opossum Selenka is not clear. I must support one of his suggestions, however, for his designation 'Urentodermzelle' as applied to the large cell in his 42- and 68-celled eggs expresses its true function. I previously described the rather constant occurrence of such cells, all in an excellent state of preservation; but in the absence of the succeeding transitional stages, I rejected the view that these are true entoderm mother cells and considered them of 'nomorphological importance.' I am now enabled to give a complete account of the most interesting behavior and the destiny of these cells.
On the time relations in the development of the opossum my data substantiates Selenka's account only in regard to the time between copulation and parturition, which is thirteen days.
But the age.s given for all his early stages are far too low, because the author greatly overestimated the postoestrous period, that is the interval between copulation and ovulation, which he states to be five days. The time of beginning of cleavage he fixes at exactly five times 24 hours," a period which he apparently determines on the basis of one experiment in which he secured what I regard as fragmeiiting eggs in a condition that accords very well with eggs about three days old. Again, his 10-hour vesicle is nearer three days old and his 32-hour vesicle nearer four days; hence the interval of twenty-two hours between these two stages is substantially correct. In a subsequent paper I shall discuss these time relations from the abundant, though by no means simple and harmonious data on hand.
c. Material and technique
1 . Material. The opossum eggs on which the present study is t)ased represents collections made during four seasons. In 1914 eighteen litters or batches of fertile eggs were secured; in 1915, seventeen litters; in 1916, fifteen litters, and in 1917, 37 litters — a total of 87 litters. These refer, of course, only to stages coming within the field of this paper, for besides these many litters of older stages were collected; and unfertilized eggs were removed ad nauseam. The 87 fertile litters, which include eggs through the bilaminar stage, contained 1009 eggs, of which 641, or nearly two-thirds, are normal. Thus, about one-third of the eggs secured from pregnant females are unfertilized or abnormal, chiefly the former. My previous estimate of one-sixth is therefore too low. The average number per litter is 11.5, the extremes are 1 (No. 94) and 22 (No. 346'), not taking into consideration No. 117', which numbered 43 eggs by virtue of the compensatory hypertrophy of the ovary. Table 1 summarizes the number of eggs mentioned under "History of the Animals" in the next section.
2. Animals used; reference to illustrations. In the following summary a brief protocol is presented of each animal furnishing €ggs used in the present study. The data for Nos. 21 to 144
TABLE 1 Sirmmary of eggs
a
K a
H
H <
H
3
^ «
§ss
b.
NUMBER OF NORMAL
6. H
s
EGGS
°o:^
D
K B 2
a N £
n " «
J a
s ri °
S°
3
p H Z
ga
^
?
H
A. Previously reported: litters Nos. 21 to 144, 21 different animals
1. From pregnant animals
2. From pseudopregnant animals.
35 2
248
130 37
378 37
B. New material: litters Nos. 173 to 415, 45 different animals
3. From pregnant animals at first operation (left uterus)
22 14
16
16 15
166 (63% normal)
107 (57.5% normal)
137
(78.4% normal)
120
(Estimated)
97
79
39
62 156
263
4. From second operation (right uterus) eggs used for this article
186
5. Later stages mentioned in summary, second operation
176
6. From pregnant animals, proportion es timated
7. From pseudopregnant animals
182 156
Total items 1, 3, 4, and 6
87
641
(63.6% normal)
368
1009
Total mentioned in summary
120
778
600
1378
are abstracted from the writer's previous study (Hartman, '16), to which the reader is referred for further details.
In the system which I have employed for the identification of the specimens each animal receives a number, and the litters of eggs taken from that animal receive the same number. Without further designation, a number may represent all of the eggs secured from both uteri when the animal is merely killed and both uteri removed simultaneously; but when the animal was used for two stages, the simple number represents the first batch of eggs, that is, the contents of the left uterus, removed under anesthesia and aseptic conditions. The litter of eggs removed from the right uterus at a later period is designated by the prime of the number given to the animal. Thus, figure 1 in plate 1 shows the eggs No. 320 taken from the left uterus of animal No. 320 at 9 p.m., Jan. 24; figure 2 shows the eggs yielded by the right uterus of the same animal 5| days later, and these are designated as No. 320'. The same system applies to 299 and 299', 292 and 292', etc. The first litter of eggs is invariably from the left, the second from the right uterus.
For the reader's convenience references are made to the figures illustrating the respective litters of eggs. An asterisk (*) is placed after the figure or plate containing heliotype illustrations of eggs photographed in Ringer's solution in the living state.
Animals usedin the study . No. 21. Killed three days after attempted copulation; mature ovarian eggs (fig. 1, pi. 14).
No. 28. Captured Aug. 23, when seven or eight months old; kept in solitary confinement until Jan. 23, when she was placed with a male; male ahnost killed by female Jan. 26, indicating that oestrus had passed. Killed Jan. 27; large undischarged follicles with ripe eggs (fig. 1, pi. 13).
No. 40. Blastocysts near end of entoderm formation with greatly attenuated non-formative area (figs. 3 and 4, pi. 18).
No. 43. Eggs 0.8 to 1 mm., blastocysts bilaminar throughout (figs. 4 and 4A, pi. 20), except several like those of No. 40 (fig. 1, pi. 18).
No. 46. 2- to 5-celled eggs (text fig. 4, P).
No. 50. Unilaminar vesicles of about 50 to 70 cells with none or with one to several entodermal mother cells (figs. 1 and 3, pi. 7; fig. 11, pi. 13; figs. 2 and 5, pi. 16).
No. 52. From pronuclear to..4-celled stages, but mostly unsegmented eggs (fig. 21, pi. 14).
No. 54. About same as preceding.
No. 55. Numerous bilaminar blastocysts about 1 mm. in diam. (figs. 2, 2A, and 6, pi. 21).
No. 56. Unfertilized tubal ova still devoid of albumen layer (fig. 3, pi. 13; figs. 7 and 14, pi. 14).
No. 58. Undivided, unfeitilized uterine eggs.
No. 76. Tubal ova with small trace of albumen (fig. 2, pi. 13; figs. 8, 11, 13, and 15 to 17, pi. 14).
No. 81. Fertile eggs, all 4-celled (text fig. 4, N).
No. 82. Bilaminar blastocysts like those of No. 50 (figs. 1 and lA, pi. 20).
No. 83. Four 4-celled eggs (figs. 8, 11, and 12, pi. 15) and three young blastocysts^ (figs 12 and 19, pi. IG).
No. 85. Cleavage stages; one each of 6, 7, 9, 10, 12, 14, 15, 17, and 18 cells; three of 8 cells; five of 16 cells (figs. 15 and 17, pi. 15).
No. 88. Of these eggs the collection contains twenty-seven excellent preparations consisting of 50 to 70 cells and ranging up to 103 cells each. Most of the eggs have from one to several entoderm mother cells in their earliest proliferation (figs. 2, 4, and 6, pi. 7; figs. 3, 7 to 11, 18, 21, and 22, pi. 16; fig. 13, pi. 22; compare also page 36, Hartman, '16).
No. 94. A single bilaminar blastocyst about 1 mm. in diameter.
No. 112. Degenerating ova from known second oestrus period.
No. 117'. Forty-three eggs, mostly in cleavage, 2- to 16-celled, from a single uterus, the organs on the opposite having been removed 33 davs before; ovarv hypertrophied; eggs subnormal in size (figs. 9 and 10, pi. 15).
No. 144. Blastocysts more advanced than those of No. 88; attenuation of non-formative area well under way (figs. 1 to 3, pi. 17).
Fig. 1. Three blastocysts and one unfertilized egg of litter No. 175', sketched with the aid of the camera lucida immediately upon immersion in the fixing fluid (aceto-osmic-bichromate). X 8.
No. 173. Received Jan. 17. Left uterus and ovary removed 8 p.m., Jan. 18; about 12 eggs, of which 8 were sectioned: 7 are 4-celled (fig. 5, pi. 3) and one is 3-cellexl (text fig. 4, L; fig. 3, pi. 15).
No. 173'. At 8 p.m., Jan. 19 (interval 24 hours) about 12 just completed blastocysts were secured from right uterus; no entodermal mother cells present (fig. 5, pi. 7). Killed Feb. 9, when the completely hysterectomized, semi-spayed animal was again coming into heat.
No. 175. Received Jan. 17; removed left ovary and uterus; pseudopregnant; the degenerating eggs were not counted or preserved.
No. 175'. Removed male Feb. 9; killed Feb. 14 (interval 28 days after operation); 14 eggs: 6 unfertilized, 8 very attenuated vesicles, entoderm reaching almost to equator (fig. 8, pi. 18; figs. 7 and 7A, pi. 19 and accompanying text fig. 1). The measurements of the eggs of litter 175' are here given as made in salt solution:
^ This is the only instance in all of my records in which the eggs, all removed at the same time from the animal, consisted of two distinct groups or stages, separated by a considerable period of development, in this case about twentyfour hours. There is, of course, a possibility of error on my part due to mixing of labels in this case.
Through shell 0.76 0.74 0.72 0.70 0.70 0.70 0.68 0.64
Through blastocyst.. 0.54 0.42 0.55x0.4 0.50 0.44x0.4 0.4 0.47 0.40
No. 189. Received Jan. 22; operation at 10: 45 a.m., Jan. 23; 10 eggs: 3 unfertilized; 7 bilaminar blastocysts, mostly about 1.2 mm. in diameter; one 0.9 mm. with smaller vesicle probable in dying state (fig. 18, pi. 13; fig. 4, pi. 21).
No. 189'. Killed at 10:30 p.m. same day (interval 12| hours); 12 eggs, five of which measured 1.7, 1.7, 1.8, 1.8, 1.9 mm.; stages just preceding the beginning of mesoderm formation ; no record of unf ertihzed eggs (fig. 20, pi. 13; figs. 4, 4A, 7, 8, 8A, 11, 12A, 12B, pl.22).
No. 191. Jan. 23, 3: 30 p.m., took out left ovary and uterus; 10 eggs: one a. defective 16-celled stage, others just completed blastocysts of about 35 cells; recorded measurements in salt solution average 0.56 mm. through shell membrane and 0.14 to 0.15 mm. through ovum (fig. 10, pi. 13; fig. 1, pi. 16).
No. 191'. Jan. 26, 9 p.m., removed right uterus, leaving ovaiy (interval 3 days, 5| hours); 11 eggs: 4 bilaminar blastocysts, 1.4 mm. in diam. in alcohol, almost 210 albumen; 7 unfertilized eggs. Animal died Feb. 5; no wound infection.
No. 192. Operated Jan. 23, 4:30 p.m.; 12 eggs: 4 unfertihzed; 8 bilaminar blastocysts measuring mostly about 1 mm. in alcohol, but three measure 0.85, 0.90, and 1.20 mm., respectively. In xylol four measurements were 1, 1, 1.01, and 1.06, with formative areas 0.62, 0.67, 0.76, and 0.65 mm., respectively.
No. 192'. Killed Jan. 24, 11: 30 a.m. (interval 19 hours); 15 eggs: 3 unfertilized; 12 vesicles, of which two measure 1.6 and 2 mm., the others about 2.4 mm.; pear-shaped embryonic area with primitive streak.
No. 193. Left uterus and ovary removed Jan. 23, 6 p.m. Number of eggs not recorded; collection contains 10 poorly fixed preparations, mostly of small blastocysts of 25 to 36 cells, one egg, however, in the M-celled stage with two cells in telophase (fig. 8, pi. 3; fig. 9, pi. 13).
No. 193'. Removed remaining uterus Jan. 26, 8:45 p.m. (interval 3 days, 2f houis); number of eggs not recorded; five measured in salt solution 1.15, 1.60, 1.70, 1.70, 1.85 mm.; the first two are bilaminar blastocysts; the last three have primitive streaks in pear-shaped areas; one 1-mm. blastocyst was dead and one of 1.40 mm. has imperfect embryonic area; several unfertilized eggs (figs. 1 and 2, pi. 10; figs. 1, 9, 9A, 9B, 9C, pi. 22). Animal died Jan. 29 of an intestinal disease common in cage animals.
No. 194. Jan. 24, 8:30 p.m., found 7 young degenerating eggs like those shown in figure 6, plate 11, in left uterus which was removed with the left ovary.
No. 194'. Feb. 9, signs of approaching oestrus returned; Feb. 13, 10 A.M., copulation observed; killed Feb. 17, 25 days after first operation; 18 eggs: 9 unfertilized; 9 vesicles with entodemi only at embyronic area, stage intermediate between Nos. 356 and 352 (fig. 5, pi. 12, figs. 13, 14, 15, pi. 17 and accompanying text fig. 2). Measurements in salt solution and in fixing fluid are as follows: In Ringer's solution (average 0.66 mm. and 0.34 mm.).
Through shell 0.73 0.68 0.65 0.65 0.65 0.60 0.65 0.65
Through blastocyst 0.35 0.35 0.40 0.37 0.35 0.30 0.32 0.30
In fixing fluid (average 0.57 mm. and 0.33 mm.).
Hill's fluid Aceio-osm.-biochr
Through shell 0.70 0.55 0.53 0.53 0.60 0.50 0.60 0.55 0.54
Through blastocyst 0.34 0.32 0.33 0.31 0.33 0.34 0.32 0.35 0.34
No. 203. Received Jan. 26. Removed only left uterus, leaving ovary, Jan. 28, 8:40 a.m.; about 12 eggs: one with pronuclei (fig. 20, pi. 14; some 2-celled (text fig. 4, E to J; fig. 7, pi. 13; fig. 1, pi. 15); one 3-celled (text fig. 4, M) ; others 4-celled (fig. 8, pi. 13; fig. 7, pi. 15) ; one recorded measurement of whole egg is 0.44 mm. through shell membrane, 0.15 through ovum.
Fig. 2. Five blastocysts with embryonic areas and one unfertilized egg of litter No. 194', sketched alive in Ringer's solution with the aid of the camera lucida. X 8.
No. 203'. Second operation at 11:45; date not recorded in protocol, but cage recoi-d indicates that the time was 11:45 p.m., Jan. 29; hence the interval was probably 39 hours; removed only right uterus, leaving both ovaries. Several young vesicles of about 50 cells; one measurement in salt solution is 0.5 mm. through shell membrane, 0.16 mm. through ovum. Killed Feb. 16; corpora lutea had almost entirely disappeared, follicles still small, but mammae very thick as in pregnancy.
No. 205. Captured by dogs Jan. 28, the skin being ripped at shoulder; operated Jan. 29, 9:15 p.m.; 10 eggs: 9 young bilaminar blastocysts with much albumen at one pole; entoderm quite or nearly reaching non-formative pole; three measured in salt solution 1.05 mm. three 1 mm., two 0.90 mm., and one 0.75 mm.; one unfertilized egg measured 0.72 mm.; in alcohol after two years the eggs measured about 0.70 mm. (figs. 1, 2, 9, 11, and 12, pi. 19).
No. 205'. Killed Jan. 30, 10:10 a.m. (interval 13 hours); 13 eggs: 3 unfertilized, the remainder vesicles with faint primitive streak in rounded areas or with more advanced primitive streaks in pear-shaped areas. Two of the former measured in alcohol 1.45 and 1.83 mm. with areas 1.1 and 1.2, respectively; one of the latter 2 mm. with area 1.32 X 1 mm.
No. 208. Caught Jan. 29; Jan. 30, 11:30 a.m., removed left uterus
containing 4 eggs: 3 unfertilized, measuring in alcohol 1.1 mm., and
one young bilaminar blastocyst measuring in salt solution 0.85 mm.,
'in alcohol 0.8 mm.; size of vesicle in salt 0.65 x 0.6 x 0.5 mm. (figs. 10,
lOA, and lOB, pi. 19).
No. 208'. Killed Jan. 31, 1:45 p.m. (interval 26i hours); right uterus yielded 8 eggs: one unfertiUzed, one defective vesicle, 1.25 mm. in diameter, and others like No. 205', measuring in salt solution 1.30, 1.45, 1.59, 1.59, 1.94, 2.35 mm.
No. 214 (D. marsupialis) . Received from south Texas, Feb. 1; operated Feb. 2; a dozen or more undivided, unfertihzed eggs, a sHght degeneration apparent only after sectioning.
No. 214'. Feb. 6, right uterus removed, leaving right ovary; 14 large eggs with opaque shell membrane, dense albumen and fragmenting ova (interval 4 days). Killed Feb. 28; after 22 days the completely hysterectomized and semi-spayed animal had again come into heat.
No. 256. Removed three pouch young Feb. 9; 10 days later, numerous small eggs in early stage of degeneration were found in uteri.
No. 285. Caught Jan. 12; injured. Jan. 13, 10:25 p.m., 10 eggs: 2 unfertilized, the remainder small blastocysts partially Uned with ento
Fig. 3. A, B, and C, three eggs of litter No. 285; D, E, and F, three eggs of litter No. 285', sketched alive in Ringer's solution with the aid of the camera lucida. X 8.
derm; eggs measured 0.85 to 0.9 mm. in salt solution; no preparations made of this litter (text fig. 3, A to C).
No. 285'. Killed Jan. 14, 12:30 p.m. (interval 14 hours); 14 eggs: 3 unfertilized; others as illustrated by D, E, and F, text figure 3; entoderaial lining complete (least advanced, figs. 3, 3A, and 3B, pi. 20; most advanced in figs. 7 and 7A, pi. 21).
No. 287. Jan. 15, 8:15 a.m.; 7 or more eggs (the collection contains 7 preparations) undivided, unfertilized, little or no signs of disintegration (fig. 6, pi. 13; fig. 19, pi. 14).
No. 287'. Jan. 18, 6 p.m. (interval nearly 3| days); 13 clear, hyahne eggs, disintegration evident in ovum.
No. 290. Copulation during night of Jan. 11 to 12; motile twoheaded spermatozoa recovered from vagina a.m., Jan. 12; Jan. 17, 8:45 p.m., 5 eggs: one unfertilized; 2 with small thick- walled vesicles at one pole, abnoi-mal (compare No. 290 (3), fig. 2, pi. 6); 2 eggs with normal bilaminar vesicles occupying about one-half of the egg (compare 290 (4), fig. 2, pi. 6; fig. 6, pi. 12).
No. 290'. Killed Jan. 18, 4:30 p.m. (interval 19f hours); 8 eggs: 2 unfertilized ; one retarded blastocyst ; 5 apparent^ normal bilaminar blastocysts a little over 1 mm. in diameter; no preparations made of this litter (figs. 2 and 3, pi. 11*).
No. 292. Caught with male in hollow log Jan. 13; isolated till operation, Jan. 17, 11 p.m; 10 eggs: of the 4 that were sectioned one is a defective 7-celled egg, the others normal blastocysts of 40 to 50 cells with none or only one *entodermal mother cell (fig. 5, pi. 1*; figs. 1 2, 5*, and 6, pi. 6).
No. 292'. Killed Jan. 21, 10:40 p.m. (interval 4 days); 7 vesicles about 3 mm. in diameter, late primitive-streak stage; one unfertihzed egg; one degenerating young bilaminar blastocyst (fig. 6, pi. 1*).
No. 293. Caught Jan. 17; Jan. 18, 8:00 p.m., 13 eggs: of the eight preparations made all are 4-celled except one 2-celled egg; in one case one blastomere, in two cases 2 blastomeres are in mitosis (fig. 1, pi. 2*; figs. 5 and 6, pi. 15).
No. 293'. Killed Jan. 22, 7:30 a.m. (interval 3^ days); 17 eggs: 8 unfertihzed, one defective; 8 bilaminar blastocysts hke those sketched in text figure 3, D, E, F (fig. 2, pi. 2*).
No. 294. Caught Jan. 17; large skin wound on belly; Jan. 18, 8:30 p.m., 15 eggs: 5 unfertilized, 8 with small rounded or irregular blastocysts at one pole, all rather abnormal; 2 apparently normal bilaminar blastocysts Hke F, text fig. 3 (fig. 1, pi. 11*).
No. 294'. Killed Jan. 20, 7 a.m. (interval 34| hours); 14 eggs: 7 unfertihzed; one 1.4 mm. in diameter and 4 small degenerating blastocysts; 2 bilaminar blastocysts about 1.3 mm. in diameter, of which only one is perfectly normal. Thus both litters, 294 and 294', were mostly abnormal (fig. 4, pi. 11*). No preparations were made of this Htter.
No. 298. First copulation Jan. 14, spermatozoa recovered from vagina; at 1 p.m., Jan. 15, the double spermatozoa had mostly divided. Jan. 20, 10: 15 a.m., 14 eggs, of which perhaps 10 are normal blastocysts; five preparations contain 60 to 120 cells each, showing earliest entodermal prohferation (fig. 7, pi. 2*; figs. 7 and 8, pi. 6; figs. 6 and 20, pi. 16).
No. 298'. Killed Jan. 23, 12 m. (interval about 3| days); 6 eggs: 4 vesicles 4.25 and 4.9 mm. in diameter with medullary groove as long as primitive streak; 2 smaller vesicles and 2 unfertihzed eggs (eggs in utero, fig. 8, pi. 2*).
No. 299. Caught Jan. 19; Jan. 20, 8:15 p.m., 12 eggs, of which all of the 7 sectioned are normal 4-celled eggs with small blastomeres (fig. 3, pi. 1*; figs. 6 and 7, pi. 3; figs. 13 and 14, pi. 15).
No. 299'. Killed 11:30 p.m., Jan. 24 (interval 4 days, 3^ hours); 14 eggs: 6 apparently normal, nearly or quite completed bilaminar blastocysts; 3 abnormal blastocysts; 5 unfertihzed eggs (fig. 4, pi. 1*; figs. 1 and 2, pi. 6; fig. 5, pi! 10; figs. 7 and 8, pi. 12; fig. 16, pi. 13).
No. 303. Caught Jan. 19; Jan. 20, pseudopregnant, 7 degeneratmg eg;gs a week old (fig. 7, pi. 11*); killed Feb. 1, the mammary glands still very thick, almost as in pregnancy.
No. 306. Jan. 21, 12 m.; 11 eggs recorded in notes, but only 3 found in collection; two of these are 2-celled with both blastomeres in mitosis (text fig. 4, A to D; fig. 4, pi. 3; fig. 2, pi. 15); one egg is 3-celled (text fig. 4, K; fig. 4, pi. 15).
No. 306'. Killed Jan. 26, 8:30 a.m. (interval 5 days, 20^ hours); 10 eggs: 2 unfertilized; 8 bilaminar blastocysts 0.7 to 0.75 mm. in diameter, of which one has no embryonic area (fig. 7*, pi. 10; fig. 17, pi. 13; figs. 2 and 2A, pi. 20; figs. 1 and lA, pi. 21).
No. 307. Jan. 21, 3:30 p.m.; 11 eggs removed from left Fallopian tube (fig. 7, pi. 1*; figs. 2 to 6, 9, and 10, pi. 14).
No. 307'. Killed Jan. 27, 9:15 a.m. (interval 5f days); 10 eggs, unfertiHzed and fragmenting (fig. 8, pi. 1*).
No. 313. Caught Jan. 19; Jan. 22, 10: 30 p.m., 9 tubal ova, with considerable albumen (figs. 1* and 3, pi. 3; figs. 12 and 18, pi. 14).
No. 313'. Animal died during the night; the 11 eggs taken from remaining oviduct had more albumen than the '313' fitter; eggs poorly fixed (fig. 5, pi. 13).
No. 314. Copulation a.m., Jan. 20; spermatozoa recovered; Jan. 23, 7:30 P.M., 9 eggs (fig. 1*, pi. 5; fig. 1, pi. 6); of the 5 eggs sectioned one is a blastocyst of 26 cells (fig. 2, pi. 5), 2 contain about 30 cells each (fig. 4, pi. 6), and 2 are abnormal (fig. 10, pi. 21).
No. 314'. Killed Jan. 29, 10 a.m. (interval 5 days, 14^ hours); 6 normal embryos with first rudiment of allantois.
No. 318. Jan. 23, 26 eggs in early stage of fragmentation, 13 from each uterus, in which involution had already set in (fig. 8, pi. 11*).
No. 320. Received about Jan. 20; Jan. 24, 9 p.m., 13 4-celled eggs studied in salt solution; subsequent fixation poor (fig. 1, pi. 1*).
No. 320'. Jan. 30, 9:25 a.m. (interval 5| days); 17 eggs: 4 unfertiHzed; 11 vesicles 2.3 to 2.6 mm. in diameter with well-developed primitive streak; one egg contains two vesicles and two embryos; two vesicles have no embryonic area (fig. 2, pi. 1*).
No. 321'. Jan. 25; fitter of foetuses near term accompanied by the 4 eggs shown in fig. 9, pi. 11*; these eggs aie, therefore, nearly 10 days old.
No. 332. Jan. 26, 21 eggs (9 plus 12), degenerating, unfertiHzed, in middle stage of pseudopregnancy (fig. 10, pi. 11*).
No. 336. Jan. 27, 9 p.m., 14 eggs (one was lost before photographing); the 6 preparations made are young blastocysts of 17, 26, 29, 30, 32, and 32 cells, respectively (all figures on plate 4*; figs. 18 and 19, pi. 15).
No. 336'. Killed Feb. 1, 5:45 p.m. (interval nearly 5 days); six 10-mm. vesicles with small embryos; 2 smaller vesicles.
No. 337. Jan. 28, 10:30 a.m., 8 eggs: a study of them in salt solution seemed to show that one egg was unfertiHzed, one 8-celled and six 16-celled; two eggs sectioned are 15- and 16-celled, respectively (fig. 9, pi. 1*; figs. 3* and 4*, pi. 5; fig. 16, pi. 15).
No. 337'. Feb. 1, 10:30 p.m. (interval 4^ days); 14 eggs: 12 blastocysts, about 2.5 to 4.5 mm., first appearance of medullary groove; 2 defective blastocysts (eggs in utero, fig. 10, pi. 1*).
No. 339. Jan. 28, 3:30 p.m., 8 eggs (fig. 6*, pi. 9): 2 unfertilized; 5 eggs with more or less abnormal, round, thick-walled blastocysts at one pole (fig. 2, pi. 6; fig. 15, pi. 13; figs. 5, 5A, and 6, pi. 19); one quite normal thin-walled blastocyst with entoderm spread to equator (fig. 2, pi. 6; figs. 6 and 6A, pi. 18).
No. 339'. Killed 12:30 p.m., Jan. 29 (interval 21 hours); 9 eggs: 6 bilaminar blastocysts measuring about 0.85 mm. in alcohol; one dead blastocyst 0.75 mm.; 2 unfertiUzed eggs (fig. 3, pi. 21).
No. 342. Received Jan. 27; Jan. 28, 9:30 p.m., 19 eggs; of the 4 sectioned specimens two aie defective and the other two are blastocysts of 26 and 28 cells, respectively (figs. 6* and 7*, pi. 5; fig. 20, pi. 15).
No. 342'. Feb. 4, 8:15 p.m. (interval 7 days); 2 dead and 9 normal embryos, the latter about 7.5 mm., head-rump length.
No. 343. Observed copulation, 4 a.m., Jan. 22; Jan. 29, 2:45 p.m., left uterus yielded 15 eggs: 4 unfertiUzed; one small defective blastocyst; one blastocyst with defective embryonic area; 9 normal bilaminar blastocysts about 1 mm. in diameter, embryonic areas 0.64 to 0.76 mm. (fig. 5, pi. 2*; fig. 5, pi. 21).
No. 343'. Killed 7 hours, 20 minutes later (7f days after copulation); 8 eggs, of which 3 are unfertiUzed, 5 normal 1.8-mm. blastocysts just preceding proliferation of mesoderm; embryonic areas 1 to 1.1mm. in diameter (fig. 6, pi. 2*; fig. 2, pi. 22).
No. 344. Received and operated Jan. 29, 4:45 p.m.; 16 eggs: 15 sectioned; of these 6 are unfertilized and fragmenting; 2 are abnormal blastocysts (fig. 4, pi. 16); 7 are noraial blastocysts showing early differentiation of embryonic and non-embryonic areas; the most advanced contains 124 'ectodermal' and 45 entodermal cells (figs. 5*, 6*, and 7, pi. 8; figs. 14 to 17, pi. 16).
No. 344'. Killed Feb. 1, 8:30 p.m. (interval 3 days, 3f hours); 7 eggs, all normal vesicles 4 mm. or more in diameter, with short medullary groove.
No. 346. Received and operated Jan. 29, 8:45 p.m.; 21 eggs: 8 unfertiUzed; one dead blastocyst; 8 normal 1.5 mm. blastocysts, embryonic areas about 1 mm.; the other eggs retarded and defective (fig. 3, pi. 2*). , , .
No. 346'. Killed next morning at 6:35 o'clock (interval 9f hours); 22 eggs: 11 unfertiUzed; 11 blastocysts ranging up to 2.2 mm. in diameter, all in early primitive-streak stages (fig. 4, pi. 2*; fig. 22, pi. 13).
No. 347. Jan. 29, 9:45 p.m.; 15 eggs: 4 unfertilized; 11 normal blastocysts partly or entirely bilaminar (fig. 5*, pi. 9; figs. 5 and 5A, 7 and 7A, pi. 18; figs. 3, 8, and 8A, pi. 19).
No. 347'. Jan. 30, 10:15 p.m. (interval 12^ hours); 17 eggs: 4 unfertiUzed; 13 bilaminar blastocysts measuring 1.1 to 1.24 mm. in alcohol (fig. 1, pi. 22).
No. 349. Front foot wounded in trap; Jan. 30, 3:45 p.m., 5 eggs: 2 unfertilized; one unilaminar blastocyst (fig. 4, pi. 8); 2 blastocysts with spreading entoderm (fig. 3*, pi. 8; fig. 12, pi. 17).
No. 349'. Killed Feb. 2, 11 p.m. (interval 3 days, 7 hours); 10 eggs: 9 vesicles 8 to 10 mm. in diameter with embryos of about 10 somites; one small vesicle.
No. 351. Jan. 30, 5 p.m., animal was opened: freshly burst follicles on left ovary, but no eggs in left oviduct.
No. 351'. Killed at 7:30 p.m., 2| hours later; 14 eggs with a httle albumen found in right oviduct (fig. 2* pi. 3; fig. 4, pi. 13).
No. 352. Jan. 30, 5 p.m.; 16 eggs: 9 unfertihzed; of the 7 remaining, 3 are Hke eggs No. 40, the others less advanced and perhaps not quite normal (figs. 1* and 2, pi. 9; fig. 14, pi. 13; fig. 2, pi. 18; fig. 8, pi. 21).
No. 352'. Killed Jan. 31, 8 a.m. (interval 15 hours); 20 eggs: 8 unfertihzed, one egg with dead vesicle; one egg with two vesicles; the remainder bilaminar vesicles fill one-haK to three-quarters of the egg, which measured fresh about 0.75 mm. (fig. 4*, pi. 9; fig. 4, pi. 10; fig. 4, pL 19).
No. 353. Jan. 30, 7: 45 p.m., 16 eggs: 5 unf ertihzed ; 11 bilaminar blastocysts measuring in alcohol 1.1 to 1.3 mm. (figs, 3, 3A, 3B, 3C, pi. 22).
No. 353'. Killed Jan. 31, 1 a.m. (interval 5^ hours); 12 eggs: one unfertilized; one dead; 10 blastocysts 2 mm. and less in diameter, showing the proliferation of first few mesodermal cells (fig. 21, pi. 13).
No. 356. Had been in cages some time before first operation, Jan. 30,8:45 p.m.; 15 eggs: 10 normal blastocysts averaging about 0.18 mm. through ovum, with numerous entodermal mother cells at formative pole and considerable attenuation of non-formative pole; one abnormal blastocyst with large blastomere (fig. 14, pi. 22) ; 4 unfertihzed eggs (fig. 1, pi. 6; figs. 1* and 2, pi. 8; fig. 3, pi. 9; fig. 4, pi. 12; fig. 12, pi. 13; figs. 4 to 11, pi. 17).
No. 356'. Killed Feb. 3, 12:30 a.m. (interval 3 days, 3f hours); 6 vesicles about 3 mm. in diameter, with short medullary groove.
No. 360. Jan. 30, 9:30 p.m., 11 eggs: 10 bilaminar blastocysts about 1.5 mm. in diameter; one unfertihzed egg (fig. 6 and stereogram, fig. 8, pi. 10; fig. 6, pi. 22).
No. 360'. Killed Feb. 2, 7:30 p.m. (interval nearly 3 days); 2 abnormal and 18 normal embryos about' 5.75 mm. in length with first rudiment of allantois.
No. 415. Feb. 10, 11 fragmenting eggs in early stage of pseudopregnancy, presented for the false '2-celled' and '4-celled' eggs seen in figure 5, plate 11*. . "
3. Material arranged according to stage of, development. The following tabulation is arranged by stages for ready reference. Within a given stage the litters are also placed in ascending order of development.
1. Ripe ovarian eggs: 21, 28.
2. Tubal ova: 56, 76, 307, 351', 313, 313'.
3. Undivided, unfertilized uterine eggs showing little or no degeneration: 58, 173, 214, 287.
4. Cleavage stages:
a. From one to about four cells: 46, 52, 54, 203, 306, 293, 81, 83, 299, 320.
b. From about 8 to 16 cells: 85, 117', 337, 342.
5. Young unilaminar blastocysts :
a. Containing from 25 to 35 cells, mostly without entodermal mother cells: 336, 173', 191, 193, 203', 314.
b. Older stages up to 100 cells, mostly with entodermal mother cells: 50, 83, 298, 292, 88.
6. Young blastocysts with distinct polar differentiation: 344, 144', 356, 349.
7. Young blastocysts with spreading entoderm : 194', 339, 352, 43, 294, 175', 347.
8. The bilaminar stage: 347, 285, 299', 205, 208, 290, 293', 43, 306', 352', 82, 285', 290', 294', 189, 191', 192, 343, 339', 94, 55, 347', 353, 346, 360, 193', 343', 189', 353' (few mesoderm cells) .
9. Primitive-streak stages: 353', 346', 320', 192', 193', 205', 208', 337', 344', 356', 292', 298'.
10. Embryos: 349', 336', 314', 342', 360', 321'.
11. Unfertilized and degenerating eggs: 112, 415, 175, 194, 256, 287', 307', 214', 318, 303, 297, 332, 321'.
4. Securing the eggs. During the collecting season 1916 and 1917 two stages were secured from each female after the method first employed by Bischoff on the rabbit. As the method has proved of great value to the writer in securing a complete series of stages, it is here discussed in some detail.
The female is placed under anesthesia and one uterus is removed under aseptic conditions; the animal recovers and the eggs are allowed to 'incubate' in the remaining uterus for a calculated period of time. In this way, by utilizing gradually accumulating data on the rate of development, it became possible to secure almost any desired stage and thus fill in the gaps still appearing in the series. Thus, for example, I succeeded in securing from animal No. 353 eggs in which the mesoderm was just beginning to proliferate. Animal No. 343 had previously furnished bilaminar blastocysts (fig. 5, pi. 2) from the left uterus ; she was killed seven hours and twenty minutes later and a Utter of large blastocysts, still in the bilaminar stage, was removed (fig. 6, pi. 2) : the interval allowed had been too short. Animal No. 346 (figs. 3 and 4, pi. 2) had yielded bilaminar blastocysts a little larger than No. 343 and an interval of nine hours and forty-five minutes had proved to be too long, for, when the animal was killed, the primitive streak was already well advanced in the second litter of "eggs. Profiting by these two experiments, when animal No. 353 appeared with large bilaminar blastocysts about the size of those in litter No. 346, a five and one-fourth hour interval proved to be the correct one, for the eggs in litter No. 353' contain the first anlage of the primitive streak, one egg having as few as twenty-five mesodermal cells.
In the operations I have found it most convenient to enter the abdomen through a short slit on one side of the pouch. For the sake of uniformity I select the left side as a matter of routine. The animal is shaved over this area and the incision is made as near the pouch as possible, care being taken not to cut through the pouch, especially in multiparae, which possess dilated pouches. The operated animal is bandaged; but it is impossible to keep the bandage on an animal unless the entire trunk is covered. I use over the bandage a jacket with holes cut for head and legs and tied over the back. As the animal usually sweats with the bandages on, the wound will heal better if they are removed at the end of three or four days.
If, on opening the animal, the uteri are purplish and flaccid, the case is one of pseudopregancy and the organs may be left intact and the animal kept for another oestrus period, which takes place in about thirty days. If ovulation is recent, however, one uterus must be removed to ascertain the state of the eggs. If the appearance of the organs indicates that young stages are to be expected, the uterus is placed in warm Ringer's solution and a slit is made through the musculature and peritoneum from one end to the other, and this must be done by a rapid manipulation of the scissors to precent eversion of the mucosa.
The pressure now being removed, the hypertrophied mucosa is pulled apart, preferably under the binocular microscope, with two pairs of finely pointed forceps, and the lumen exposed. The eggs may be picked out from among the delicate folds of the mucosa by means of a pipette. But this method is unnecessarily tedious; the uterus may instead be simply turned inside out in the Ringer's solution and the eggs picked out from the bottom of the dish. To insure finding all of the eggs, a little Bouin's fluid added to the salt solution, after removal of all the eggs that can be seen, makes any specimens overlooked prominently visible. The uterus should also be shaken out in another dish of Ringer's solution for any eggs that may have been hidden in the uterine folds. To keep the solution clear of blood, it is well, before opening the organ, to slit all the superficial bloodvessels and drain them of blood. I may add that the neck of the uterus should be ligated -with a 'lifting' ligature before it is cut from the body, in order to prevent the loss of eggs through the OS uteri.
Young eggs in cleavage and small blastocysts are mostly found near the caudal end of the uterus, often closely bunched together. Hence one cannot speak of 'implantation' of the opossum egg at any early stage. The 'uterine cups' described by Spurgeon and Brooks ('16) do not mark implantation surfaces, but merely accidental pits produced by pressure into the delicate ©edematous mucosa.
If, on opening the animal, pregnancy seems to be advanced, in order to remove entire vesicles intact, it is best to slit the uterus superficially in many places and to trim off the entire musculature before attempting to remove the vesicles, which are closely applied to the mucosa, but never fused with it. This procedure renders the use of a killing fluid to paralyze the musculature entirely superfluous. With a pair of forceps and a fine brush an entire litter of delicate vesicles may be removed intact. They may be transferred to the fixing fluid in a deep mustard spoon or in a shallow, neckless vial. A collapsed vesicle may again be dilated in the fixing fluid by injection with a fine pipette; in fact it is well to irrigate with the fixing fluid the lumen of every vesicle containing a large embryo.
Eggs are easily washed out of the Fallopian tube by means of a stream of Ringer's solution, as has been done in other mammals.
5. Fixing and stai^iing. I have used the following solutions: Bouin's, Bouin's half strength, increased gradually to full strength; Hill's; Flemming's; Carnoy's; Zenker's; formolZenker; picro-sulphuric ; trichloracetic; Bensley's aceto-osmicbichromate. Hill's fluid is made as follows: Mayer's picronitric, 96 cc; 1 per cent osmic, 2 cc; glacial acetic, 2 cc. I stated in 1916: I have found Hill's mixture to be the perfect fixing liquid for the opossum egg." Further experience with it has led me to give decided preference to Bouin's for all older blastocysts; for younger eggs up to the bilaminar stage I get equally good fixation with both; and I also have made some poor preparations with either. For all stages Bouin's is perhaps the safest solution to use; with it the specimens have the advantage of toughness and they can be safely transported, whereas solutions containing osmic acid render the specimens unduly brittle. The half-strength Bouin is not as good as fullstrength. I have some excellent preparations of material fixed in Flemming's fluid, although collapse of blastocysts is more likely to occur in this fluid than in Bouin's. My poorest fixation was with aceto-osmic-bichromate, although superficially the eggs thus fixed seem well preserved. This fluid has the advantage of bringing out cell membranes clearly. I have no perfect specimens fixed in Zenker or formol-Zenker, both of which shrink the material more than any other and render it very brittle. Several fairly good preparations were made with picro-sulphuric. Trichloracetic has the peculiar property of fusing extruded yolk and cytoplasm of the blastomeres into an almost undifferentiated mass (fig. 13, pi. 15).
Hematoxylin stains, especially Heidenhain's iron-alum hematoxylin, have proved entirely satisfactory, both for sections and for surface mounts. Several eggs fixed in solutions containing osmic acid were stained in acid fuchsin, saffranin, or cochineal to differentiate the nuclei clearly from the black yolk granules, but this refinement of technique is not at all necessary.
To prevent collapse of the blastocysts, which my photographs on plates 1 and 2 show to be perfect spheres, it is important to pass from one medium to another (Bouin's to alcohol, water to alcohol, alcohol to xylol, and especially xylol to paraffin) by slow gradations, I use 5 per cent differences, accurately made up in stock solutions. The eggs are placed in small vials and each higher percentage is "added gradually. Vesicles from about 1.5 mm. on up may easily be cut in half equatorially with fine scissors; but such hemispheres, if of approximately equal size, should not be placed in the same vial, because they are likely to telescope in such a way that they are hard to separate without injury to them.
The material was imbeded in paraffin and most of the newer material was sectioned by Huber's water-on-the knife method, which gives incomparably better results than ribboning the series wdth the rotary microtome.
6. Unfertilized eggs. Since, unfortunately, the vast majority of the eggs removed from animals in captivity are unfertilized, it will not be amiss to mention them here. Such eggs remain in the uterus for many days, undergoing progressive degeneration before being discharged or absorbed. For the first two or three days they are not easily distinguished from normal uterine eggs. The first sign of degeneration is the breaking up of the ovum into masses of various shapes and sizes (fig. 5, pi. 11) and the ovum may flatten out into the shape of a crescent (fig. 6, pi. 11). Gradually, too, the eggs increase in opacity (fig. 7, pi. 11) and become covered with white concretions (fig. 8, pi. 1; figs. 8 to 10, pi. 11), so that they are only too prominent when one opens the uterus hoping to find embryos. The eggs shown in figure 9, plate 11, accompanied foetuses near term; hence these eggs are at least nine days old.
7. The illustrations. The drawings (plates 12 to 22) were made on Ross stipple board No. 2 and reduced to one-sixth the original size. Korn's lithocrayon No. 1 (a paraffin-carbon pencil) gave the best results, since to be reproduced by the line process the dots must be absolutely black, a result not easily attained with a graphite pencil.
The drawings on plate 12 were made free hand by Mr. T. H. Bleakney from stained specimens in oil of wintergreen, the size being calculated from photographs of the living eggs. The eggs shown in text figure 4 were mostly drawn from wax models made after the Born method, X 600. This figure was also drawn on Ross board No. 2, but was reproduced one-third of the original size. All other drawings, except a few where especially mentioned, were made from sections and were drawn as nearly like the specimens as possible, imperfections and all. The attempt was made to reproduce not only the form, but also the texture of the specimens, and for this purpose the Ross board has proved to be a delicate and responsive medium.
The smaller drawings were outlined with the camera lucida at a magnification of X 300, X 1200, X 3000 (reduced to X 50, X 200, X 500) ; the larger sections of blastocysts, since they had to be drawn at a magnification of 1200 which resulted in drawings m.ore than a meter long, were first sketched X 400 with a Leitz-Edinger drawing apparatus, then photographed and the negative finally projected to the desired magnification by the Edinger apparatus. To facilitate measurements, the scale of a stage micrometer was drawn beside the first sketch made and appears upon the negative made from it.
The photomicrographs were made with Spencer lenses, which afford a flatter field for photographing than the Zeiss microscope, lenses. Attention is especially directed to the photographs of living eggs. More than 500 different eggs are represented in the hehotypes. All of the photographs on plates 1, 2, and 11 and many other figures are magnified eight times and some are at a higher magnification.
The photographs are unretouched and are reproduced as exactly like the original as was possible with the process employed.
To secure an absolutely black background for the photographs taken by reflected light, we found it best to use a black watchglass. The eggs are removed from the uterus and placed in a deep watch crystal in clear Ringer's solution free from dirt and blood-cells. The watch crystal is now set into the watch-glass which must also be filled with the solution; for it is absolutelyessential that there should be no air space for the reflection of light between the transparent glass holding the eggs and the black glass serving as a background.
In the photographs of preparations, as well as in the drawings, for ease of comparison, magnifications of 50, 200, and 500 have been adhered to with few exceptions. In this connection plates 12 and 13 are especially adapted to serve as a resume of the stages covered in this paper. Comparison of these two plates shows that the young eggs shrink greatly on account of their delicate albumen layer.
Altogether the twenty-two plates accompanying this paper contain over 750 representations of more than 600 different opossum eggs, mostly, of course, in groups on the photographs. The drawings and the photomicrographs of preparations number some 240 of over 180 different eggs.
d. External changes at ovulation in the female opossum
In common with many other wild animals, the opossum does not breed well in captivity. I have worked with hundreds of animals kept in cages or in large rooms, isolated or in groups of dozens or of a hundred or more; yet the number of observed copulations that I have to record is disappointingly small. Many births have taken place in the cages, but the cases of .pseudopregnancy outnumber the cases of true pregnancy many times over.
In spite of careful personal attention to the habits of the captive animals, I was unable during the first two years' collecting to determine from outward signs the sexual state of the female. In this regard I was at first forced to agree with Selenka who says: *'Ohne operative Eingriffe ist tiber die Trachtigkeit eines Weibchens keine Gewissheit zu erlangen, da man weder durch Tasten mit dem Finger die weichen Uterushorner auffiden kann, noch auch an den Milchdriisen eine Veranderung wahrnimmt, bevor nicht die Embryonen nahezu ausgewachsen sind." I have since learned, however, that Selenka was wrong in his statement concerning the mammary glands. For, during the 1916 season, I found that by simple palpation of the mammary glands within the pouch I was able to diagnose with a high degree of accuracy the state of the internal reproductive organs, so responsive are the glands to the physiological changes going on in the animal just before and after oestrus. By this method one is enabled to select from the animals on hand those that are likely to furnish eggs or embryos. Thus out of the hundred animals Nos. 300 to 400, used at the height of the breeding in 1917, only a half-dozen failures are recorded. A typical case of misjudgment is that of No. 326, in which '5-mm. vesicles' were predicted and ripe follicles found in the ovary; or No. 354 in which 'bilaminar blastocysts' were expected and the animal was found in pro-oestrus. Sometimes a later stage than the one predicted will be found, a's is, of course, to be expected from individual variations that are general in all . physiological processes. The method has resulted in the saving of a great deal of time, effort, and material, especially during the last breeding season.
Unfortunately, however, the physiological changes which the mammary and other reproductive organs undergo are identical immediately after oestrus whether pregnancy ensues or not. This holds true for the mammary glands more than for the other organs, and it is impossible during the first five or six days to distinguish externally between pseudopregnancy and pregnancy. As ovulation is always spontaneous, the internal organs behave the same in both conditions. The vaginal loops begin to retrogress even before ovulation. The uteri are almost maximum in size when the minute eggs first reach them; in pregnancy they remain bright red and turgid and possess a peculiar luster like polished red agate; but, if the eggs are unfertilized, the uteri, after four or five days, become dull and dark red and then flaccid and collapsed. The cor'pora lutea are somewhat more persistent in true pregnancy. But the mammary glands continue development even after the degeneration of the corpora and the involution of the uteri are well under way.
e. Are the eggs of operated animals normal
The question may well be asked whether we are dealing with normal material in the case of eggs removed some time after an abdominal operation under anesthesia or whether such treatment of the mother affects the development of the eggs unfavorably. It should be emphasized at the outset, however, that whatever answer we give to the question does not affect the conclusions reached in this study, which is supported by an abundance of material from freshly killed animals and by a large assortment of specimens removed from animals at the first operation. It should also be noted that the time interval between the two operations was in many cases only a few hours or a half-day, so that in this material, too, the chances of modifying the normal course of development of the eggs were reduced to a minimum. From a careful examination of my notes and a scrutiny of both classes of material I have concluded that there is no evidence pointing to deleterious effects of the operation, and I here present some of the facts that have led me to this conclusion.
In the first place, the condition of the operated animals was as good or better than that of non-operated cage animals; for the former were the choice specimens, vigorous in health and sexually active. As stated above, I am now able to determine, with a high degree of accuracy, the near approach of oestrus in the female opossum. A surprisingly large number of females are captured (and by the terms of our contract with the hunters must be purchased) which are either too old or too sick to breed. Specimens with deep, infected wounds, intestinal diseases, xerophthalmia (McCollum), or other nutritional disturbances do not come into heat. Only once or twice have I seen females with badly infected wounds continue in the oestrus cycle like a normal animal; but I have records of dozens of cases in which the normal sexual processes were interrupted by wounds or disease during pro-oestrus or dioestrus. Pregnant females, however, pass successfully through the period of gestation even in the same condition that prevents the onset of oestrus. On the other hand, operated animals recover quickly, often eating heartily several hours after the operation. Their wounds heal readily and the animal comes into heat again even after two operations and after double hysterectomy, in the same manner as if the abdomen had not been opened. Certainly, if Bischoff a century ago was able to secure as many as six different stages of normal embryos from one rabbit, without anesthesia or asepsis, successively opening the abdomen and ligating off segments of the uterine horns "until inflammation set in," then a very simple operation on the opossum under modern surgical precautions should have no deleterious influence on the embryos.
If, now, to test the matter further, we compare the proportion of normal ' eggs secured at the first operation (table 1 above) with the proportion from the second operation, we find 63.1 per cent normal (item 3, table 1) for the former and for the latter 67.4 per cent (items 4 and 5, table 1), an unexpectedly but quite accidentally large percentage of normality for the operated animals.
These figures, however, include under 'abnormal' all unfertilized eggs, which should be left out of consideration, since we are here testing the effect on the development of the eggs and embryos. We must, therefore, count only the dead and defective fertilized eggs in given litters, selecting comparable stages. Table 2 gives this data for bilaminar vesicles of litters in which every egg was studied; and cases from the second operation are selected in which at least one day had intervened after the first operation. Table 3 gives similar data for primitive-streak stages up to 5 mm. in diameter. The litters are arranged more or less in order of relative stage of advancement.
From a study of tables 2 and 3 it is apparent that there is a high rate of mortality in the eggs of the opossum, both of operated and unoperated animals, but that wholly normal litters occur in both classes. Some cases are of special interest. No. 334, for example, yielded a perfect litter of eggs from the left uterus, but only a single abnormal vesicle 20 hours later ffom the right uterus. On the other hand, No. 344 yielded 7 normal and 2 abnormal eggs of an early stage and after an interval of three days a perfect litter of 7 normal vesicles. Litter No. 339 from the left uterus is largely abnormal, whereas No. 339' from the second operation was largely normal. Both batches of eggs from animal No. 294 were mostly abnormal, possibly on account of a temporary interference with the circulation of the uteri when the animal was twisted out of its lair
TABLE 2 Number of abnormal eggs at the bilaminar stage
LITTER NUMBER
INTERVAL
NUMBER OF NORMAL EGGS
NUMBER OP ABNORMAL EGGS
TOTAL NUMBER
OP FERTILIZED
EGGS
a. At first operation
days
339
1
5
6
205
8
1
9
343
9
2
11
346
8
5
13
353
11
11
360
10
1
11
Total
47
14
23%
61
b. At second operation
299'
4
6
3
9
293'
•J 2
8
1
9
339'
1
6
1
7
306'
4f
7
1
8
191'
3i
4
4
Total
33
6
10.8%
37
after a method employed by hunters and applied in this case. Litters No. 175' and No. 194', twenty-five and twenty-eight days, respectively, after the removal of the left uterus, contained no abnormal eggs.
Later embryos and foetuses present facts similar to those just indicated for the younger stages. To refer to special cases shown in table 4, No. 349, which had furnished only 3 normal eggs out of 5 from the left uterus, had 9 large, normal embryos and one dead embryo in the right uterus three and one-half days later, and this in spite of the fact that the animal was unusually small and had one of its legs wounded in a trap. But of the six large embryos yielded by the left uterus of No. 379, an apparently normal female, only one was normal; but 18
TABLE 3
Number of abnormal eggs at primitive-streak stages
LITTER NUMBER
INTERVAL
NUMBER OP NORMAL EGGS
NUMBER OF ABNORMAL EGGS
TOTAL NUMBER
OP PERTILIZED
EGGS
a.
At first operation
180
5
5
338
14
4
18
380
8
2
10
334
11
1
11
211
10
1
11
211'
12t2 hours
8
8
Total
56
8 11%
63
b. At second operation
days
208'
1
7
1
8
292'
4
6
1
7
320'
51
11
2
13
337'
4i
10
2
12
344'
3
7
7
356'
3
6
6
298'
^
3
3
6
ISO'
1
11
1
12
334'
1
1
1
Total
61
11 15.3%
71
hours later all of the 6 embryos in the other uterus proved to be normal.
As such cases could be multiphed, the facts are that there is a mortality in the opossum ovum at all stages and that the death rate is not affected by the abdominal operations such as employed in our experiments.
Ỉ 4 Niimher of abnormal embryos of later stages
NUMBER OF
TOTAL NUMBER
LITTER NUMBER
INTERVAL
NUMBER OF NORMAL EMBRYOS
ABNORMAL EMBRYOS
OF EMBRYOS
207
11
1
12
207'
2h days
3
7
10
226 (left)
3
4
7
226 (right)
all
291
10
10
291'
1 day
7
7
379
2
4
6
379'
18 hours
6
6
314'
5§ days
6
6
336'
5 days
6
2
8
342'
7 days
9
2
11
349'
3^ days
9
1
10
360'
3 days
IS
2
20
Maturation and Cleavage to the Formation of the Blastocyst
a. The ripe ovarian egg
Since the publication of my former paper I have not seen the first maturation spindle, for the ova of all large follicles thus far studied in numerous series of ovaries have either germinal vesicles in the resting stage just preceding maturation or have already given off the first polar body. Recently collected ovaries containing large follicles are now being prepared and will be discussed in connection with a paper on the corpus luteum.
The ripe ovarian egg is broadly elliptical in form, but may be nearly spherical, as some dissected specimens indicate. Measurements were previously stated to average 0.165 x 0.135 mm. or larger than any tubal or uterine ova. This is large in comparison with Eutherian ova, but small in comparison with the egg of Dasyums, which measures 0.21 x 0.126 to 0.27 x 0.26 mm. The ova shown in figure 1, plate 13, and in figure 1, plate 14, are unusually large, even for ovarian eggs, measuring .183 X 0.156 mm. (average 0.175 mm.) and 0.185 x 0.15 (average 0.167) mm., respectively. That the ovarian ova are on the average larger than the tubal or the uterine ova would seem to be the case from the few measurements of ovarian eggs that have been made. There is, of course, considerable variation in the sizes of different eggs, both of the same litter and of different litters (fig. 2, pi. 3, figs. 2 to 6, pi. 13).
The ova are surrounded by a well-defined zona pellucida, within which the polar body is found. This is given off usually at one of the ends of the somewhat elongated egg (fig. 1, pi. 13), but it may be found near the equator. The polar body is small and flattened, and contains chromatin matter and a minimum of cytoplasm. The chromosomes of the egg nucleus lie in the cytoplasm near the polar body, mostly more or less discrete and arranged in an equatorial plate. In this condition the egg reaches and traverses the Fallopian tube.
The ovarian egg is, therefore, essentially like the tubal ovum presently to be described in greater detail. There is no polar differentiation recognizable except for the location of the polar body. It is important to note also that the yolk has no tendency to accumulate at one pole of the egg, as is so strikingly the case in the mature ovum of Dasyurus and to a slight degree in certain Eutherian eggs (bat, armadillo). Herein lies the first striking difference between the eggs of Didelphys and the Australian Dasyurus.
b. The tubal ovum
1. Material, ovulation., secretion of albumen and shell membrane. Eggs were removed from the Fallopian tubes of five animals, but in no case was insemination observed. Unfortunately, none of the thirty or more eggs sectioned contains any trace of a spermatozoon. This stage has, therefore, not yet been observed in the case of any marsupial.
Eggs were found in both tubes of female No. 56; and, since both batches of eggs had practically no albumen deposited on their surface, they must have been discharged simultaneously from both ovaries a short time before their removal. None of these eggs possesses any granulosa cells nor was any semblance of a 'corona radiata' ever observed on any tubal ova. Their naked condition when discharged is positive evidence that Selenka ('87) was in error when he considered the shell membrane of uterine eggs as the modified granulosa cells — an error made despite Caldwell's correct interpretation of this structure.
Litter No. 76 was taken from one Fallopian tube only, the opposite one not yet having received the ova, which had, however, been discharged from the ovary on that side, as indicated by the presence upon it of fresh corpora lutea. I infer that the eggs must have been lost in the body cavity. Since the eggs secured from the right uterus had already been provided with a small quantity of albumen, one may assume that they had anticipated the eggs from the other ovary by a short space of time.
No. 351 had ovulated at 5 p.m., when the animal was first opened, but no eggs were found in the left Fallopian tube, they having also been lost in the handling of the organs. Two and a half hours later the right tube contained eggs which had a distinct albumen layer on all sides (fig. 2, pi. 3). If this represent the amount of albumen deposited in two and a half hours, it would require twenty-four hours for the eggs to traverse the Fallopian tube and receive their entire quantum of albumen. Professor Hill thinks that the eggs of Dasyurus pass through the tube very quickly, since he has never found a whole litter of eggs in the tube. But the uterine eggs of Dasyurus are very scantily provided with albumen; in fact, never relatively more than the incomplete deposit around the eggs of my litter No. 351 (fig. 2, pi. 3).
Eggs No. 307 (fig. 7, pi. 1) show a greater deposit of albumen on one side of the egg. Since this is true to some extent in all the eggs found in the upper part of the tube, and since, later, the albumen is of about the same thickness on all sides (fig. 1, pi. 3), the eggs are probably rolled about slowly as they pass through the Fallopian tube.
The shell membrane is doubtless secreted and added to the surface of the albumen in the lower part of the oviduct. Insemination must of necessity take place soon after the eggs enter the tube before albumen is deposited; for spermatozoa are found in some eggs throughout the albumen and most often nearest the ovum. The eggs of litters Nos. 336 and 356 have enormous numbers of spermatozoa entangled among the lamellae of the albumen; in figure 12, plate 13, for example, the spermatozoa are seen to occur in thick clusters as well as scattered singly throughout the albumen.
Usually an ovum is necessary to afford the stimulus for the secretion of the albumen; but in one case a rounded mass of epithelial cells proved adequate, and there was produced a structure without an ovum, the cell mass replacing the latter in the center of the egg. Epithehal cells from the wall of the Fallopian tube, enmeshed within the albumen, are of common occurrence. In another case an ovum and a cell mass and in a third case two ova were included within the same egg envelopes. Both of these latter ova would be likely to develop, if one may judge from the cases of double ova in the blastocyst stage shown in figure 2, plate 1, and in figure 4, plate 9. An egg of a parasitic roundworm once found among tubal ova did not seem to afford the adequate stimulus for the secretion of albumen.
2. Size and shape. The ova vary greatly in size and shape, not only among the different Utters, but also among the eggs of a single litter. They are elliptical, rarely spherical in shape, as may be seen from the figures in plates 3 and 14. The average size of thirty-one preparations on the slide is 0.122 x 0.104 (av. 0-.113) mm. Twelve eggs of litter No. 56 average 0.128 x 0.109 (av. 0.118) mm.; this list includes two whole mounts which are nearly round and measure 0.131 mm. (fig. 7, pi. 14) and 0.135 mm., respectively, the latter being the largest tubal ovum in the collection. The maximum length of elliptical eggs of this batch is 0.142 mm., the maximum width 0.120 mm. The average of eight eggs of batch No. 76 is 0.134 x 0.113 (av. 0.123) mm.; the maximum length is 0.148 and maximum width 0.125 mm. But this batch includes also two very small eggs measuring 0.100 x 0.085 (av. 0.093) mm. and 0.090 x 0.083 (av. 0.087) mm., the latter being the smallest egg in the collection. Three eggs of batch No. 307 average 0.126 x 0.116 (av. 0.121) mm. in the preparations; the size of the fresh eggs of this batch, shown in figure 7, plate 1, cannot be given because the magnification of the photograph is not known. The two living eggs of batch No. 313 shown in figure 1, plate 3 measure 0.119 x 0.105 (av. 0.112) mm. and 0.106, respectively; the average of eleven eggs of batch No. 313', as photographed in the living state, is 0.108 x 0.099 (av. 0.103). In the preparations, three eggs of batch No. 313 measure 0.105 x 0.091 (av. 0.098) mm., indicating some shrinkage of the eggs in the histological processes. Batch No. 351' (fig. 2, pi. 3) average 0.110 x 0.096 (av. 0.104) mm. in the living state; three preparations from this batch measure 15 per cent less, nainely, 0.102 x 0.076 (av. 0.089).
S. The distribution of yolk. The opossum egg, in common with the eggs of other marsupials, is rich in yolk or other lipoid deposit, which partly accounts for their larger size (figures on pi. 14). The fat occurs in the form of granules or spherules, many or perhaps all of which stain black with osmic acid. Eggs fixed in Bouin's fluid show numerous vacuoles from which the fat is dissolved in clearing. The fat content of the eggs renders them much less transparent; but in the living state the globules may be seen in the egg and they also appear distinct near their outer limits of distribution in the photographic negatives . taken by transmitted light in salt solution. Thus the negative from which figure 1, plate 3, was made shows in detail oil globules quite similar in distribution to those shown in figures 12 and 18, plate 14. A study of the fresh eggs and photographs of them convinces me that the fixed and sectioned specimens accurately show the true details of these eggs, little altered by the histological processes.
Three more or less distinct regions are typically recognizable in many of the ova (fig. 12, pi. 14). There is a marginal zone, sometimes very narrow, consisting of granular cytoplasm, nearly devoid ol fat granules. Beneath this is a more or less diffuse zone of oil globules, which may be very small or very large or medium in size, as seen from the figures on plate 14. Some litters show considerable uniformity in this respect (No. 76) and in others there is variation within the litter (No. 313, figs. 12 and 18, pi. 14). The outer surface of this zone of fat globules is often marked by a delicate region which may break down in fixation (light zone in figs. 12 and 19), reminding one of the delicate deutoplasmic pole of the egg of Dasyurus. In the living opossum egg this region is a light band interrupted here and there with oil globules coming near the surface. The third region is the large central portion of the egg which is rather uniformly granular and contains few oil globules or vacuoles.
The tubal ovum, like the ovarian ovum, exhibits no polar concentration of yolk, which is in striking contrast to the unsegmented ovum of Dasyurus, in which the deutoplasm is gathered in a mass at the vegetative pole of the egg and is bodily extruded just prior to the first cleavage; whereas in the opossum the yolk is thrown out from both ends or from all sides in greater or less amounts, as the sequel will show.
4. The polar body. The first polar body, which is present in all ripe ovarian eggs and in all tubal ova, lies in a spindle-shaped depression under the vitelline membrane. It is never large, containing a modicum of cytoplasm, in contrast to the prodigality with which yolk and cytoplasm are eliminated from the egg in cleavage. The polar body is usually of such a peculiar color and homogeneous texture that it is easily recognizable in eggs fixed in Bouin's fluid; but if the fixing fluid contain osmic acid the polar body is seldom recognizable. The chromatin is usually a deeply staining irregular mass.
Both polar bodies are soon absorbed, disappearing as distinctive structures in early cleavage. Except for a slight difference in size, the two polar bodies are practically identical. I have seen them in numerous eggs in cleavage, especially in 4-celled eggs. The oldest stage which contains two objects that I take to be polar bodies is a blastocyst of 34 cells. The polar bodies are caught between two blastomeres of the vesicle (fig. 1, pi. 16). The larger of the two is shaped like a bent spindle and resembles in outline the space which it occupied while still crowded in the usual periovarial space before cleavage. I have never seen polar bodies so large that they appear in cross-section like those figured by Spurgeon and Brooks ('16).
5. The chromosomes. The spindle for the second maturation division is formed soon after the giving off of the first polar body, and in this condition the egg reaches the Fallopian tube. The vesicular or resting stage does not seem to intervene between the two maturation processes. Insemination was not observed. In the absence of spermatozoa, the ovum reaches the uterus unchanged, except for the accession of the egg envelopes. Thus the young uterine eggs Nos. 58, 287, and others have chromosomes practically indistinguishable from those about to be described for tubal ova.
Three preparations from batch No. 307 (fig. 7, pi. 1; figures on pi. 14) are especially favorable for a study of the chromosomes and for determining their number. These are still scattered along the clearly defined spindles, the equatorial plate being delayed in its formation. Some of the spindle fibers are thick and beaded as though they were derived from the fibers of the preceding division. One spindle is contained in a single section (fig. 6, pi. 14). There are clearly twelve chromosomes in each of these eggs. In all other tubal ova the chromosomes are closely arranged in a more or less definite equatorial plate and are difficult to count; but I am sure that the number is twelve in five or six cases, and I can count at least ten or eleven chromosomes with distinctness in all cases. Hence I am prepared to state that twelve is the reduced number of chromosomes in the egg of the opossum.
Figures 11 and 14, plate 14, represent the usual appearance of the chromosomes in these specimens, and in these two cases twelve chromosomes can be clearly made out. Figure 13 shows a side view of a spindle in which eight chromosomes are seen and short fibers are clearly outlined. The three sections shown in figures 15 to 17, plate 14, were cut tangentially through the egg, hence the polar body is cut longitudinally and the chromosomes are seen in polar view.
The chromosomes in every case are short and thick, never characteristically rod-shaped. Some are hollow squares with rounded corners, others more "perfectly ring-shaped. In side view several appear as short, thick rods, slightly constricted in the middle; others bent or cupped so as to appear narrowly kidney-shaped. The spindle is usually situated in a granular area free of vacuoles or fat globules; or, in other words, in the region of the spindle, the central and the marginal granular regions are bridged across. The polar body is usually placed near the chromosomes, as seen in the figures. In one case one chromosome was extruded with the polar body (fig. 8, pi. 14).
c. The young uterine egg
1. Size and shape. The appearance of young uterine eggs is well illustrated by the photographs in plates 1, 2, 5, 11, and others, which represent them with fidelity just as they were removed from the uterus. In size the eggs, as measured through the shell membrane, are subject to considerable variation among the different litters as well as to some extent within a given fitter. Thus litter No. 342, consisting of about the 26-celled stage, average 0.7 mm., whereas the 4-celled eggs of litter No. 293 average 0.57 mm. and the eggs of litter No, 292, which are young vesicles of some 100 cells, measure 0.55 mm. Again, litters No. 336 and 337 average 0.73 and 0.50 mm., respectively, although they are in nearly the same stage of advancement and the ovum proper is of about the same size in the two litters. The differences in diameter among the eggs is therefore a difference in the quantity of albumen deposited about the ovum. Figure 1, plate 12, represents an average unsegmented uterine ovum.
2. The albumen. The albumen is laid down in delicate, concentric lamellae around the ovum (pi. 13 and others). In the living state it is usually of nearly the same density throughout the layer (fig. 2, pi. 4) or it may be more concentrated about the ovum in young eggs (figs. 3 and 4, pi. 5).
At first the albumen is extremely poor in protein content, for on fixation it usually gathers immediately about the ovum and the thin and delicate shell membrane collapses and follows close upon the albumen (fig. 5, pi. 5). This phenomenon is apparent on comparing plate 12, which represents the living condition, with plate 13, on which corresponding stages are shown from sections on the slide. The shrinkage of the ovum proper or the whole egg in later stages is comparatively slight; only the albumen of the younger egg suffers great collapse. It follows, therefore, that the albumen layer gradually increases its protein content (figs. 14 and 17, pi. 13), and the shell membrane likewise growls in thickness and resistance. The uterine 'milk' doubtless supplies the material thus absorbed. This holds true for unfertilized eggs also, which continue to grow in diameter and in density of albumen and shell membrane for a week or more. Figures on the thickness of the shell membrane have previously been given (Hartman, '16) and are not repeated here. It is subject to great variation, as may be seen from the various drawings in the plates, where the shell menibrane is represented in correct proportions.
3. The unsegynented ovum. Unless insemination has taken place, the uterine differs from the tubal ovum only in the possession of completed albumen and she'l envelopes (fig, 19, pi. 14). My collection contains a number of litters- of such eggs. The first polar body and the second maturation spindle are as in the tubal ova, although in some case the chromosomes begin to show a clumping and are surrounded by a light area. The chromosomes fragment sooner or later, however, and the chromatin breaks up and rearranges itself into round lumps simulating nuclei in the resting stage. The cytoplasm breaks up also, some fragments taking one or several 'nuclei,' others none. Sometimes the fragments are equal or nearly equal in size, so that such eggs may easily be taken for cleavage stages (fig. 5, pi. 11). I have never seen a mitotic figure in such degenerating eggs. The eggs shown in the photograph in figure 8, plate 1, remained in the uterus until near the end of the sixth day after ovulation.
4. The promiclear stage. Several eggs were studied at this stage, although I did not secure an entire litter of eggs containing pronuclei. The pronuclei at first lie at the periphery of the ovum in a homogeneous granular area devoid of fat globules. Eventually they come to lie near the center of the egg where the first cleavage spindle will form. The chromatin of the pronucleus is in most cases very diffuse and stains weakly. Two figures of the stage are give<n in the writer's former paper and fi-gure 20, plate 14, is an o\aim with two nuclei which differ from the nuclei of the other members of this litter (2-, 3-, and 4-celled eggs); hence I regard this egg as in the pronuclear ftage.
d. The first cleavage
1 . The first cleavage spindle. No new material containing the frst cleavage spindle has been obtained recently. In figure 21, plate 14, I have redrawn specimen 52 (3) as a composite of four sections, which were taken obliquely through the spindle, which lies in the central yblk-free zone of the egg. The fat vacuoles are evenly distributed at the poles and some of the yolk has already been extruded, chiefly on one side of the egg.
2. The 2-celled stage. The early cleavage material in the writer's collection was, however, considerably increased by recent accessions, namely, from the following litters: No. 173, which furnished 3- and 4-celled eggs; No. 203, which furnished four 2-celled, one 3-celled, and the rest 4-celled eggs; and No. 306, which furnished, besides a number of eggs that were unfortunately lost or misplaced, two 2-celled and one 3-celled egg. All of these litters are the product of the left uterus and in each case a later stage was removed from the right uterus, which is good evidence that we are here dealing with normal fertilized material.
Of the 2- and 3-celled eggs models were prepared and drawings made from the models, which are shown in text figure 4.1, J and P of this figure were drawn from eggs mounted in toto in balsam.
The blastomeres of the 2-celled eggs are usually flattened as if by mutual pressure upon their contact surfaces. They may be of equal size and shape and practically identical, or they may be unequal, as the drawings in the figure amply show. If they differ in size I rather believe this difference to be secondary and not to unequal cleavage, that is, to the greater amount of yolk extruded from the smaller blastomere. Thus, in egg No. 203 (13), shown in I, text figure 4, one blastomere has given off a large mass of yolk at each end, but the aggregate of the masses in the two halves of the egg is as nearly equal as in the adjoining f gure of a sister egg.
A study of the eggs in serial section fails to reveal either a qualitative difference between the two blastomeres or the slightest indication of polarity within the blastomeres themselves (fig. 4, pi. 3; figs. 1 and 2, pi. 15). The yolk granules occur in equal numbers and sizes at the two poles and the nuclei are centrally placed. The distribution of the yolk granules is indeed quite similar to that of the undivided egg, namely, in a zone toward the margin of the cell (figs. 2 to 4, pi. 15), and this holds true also for the blastomeres of the 16-celled stage and even later (fig. 17, pi. 15). In no case is it possible- to distinguish a more deutoplastic 'vegetative' pole and a relatively yolk-free 'animal' pole in any stage of segmentation.
There would seem, however, to be a qualitative difference in the blastomeres, as evidenced by the more precocious division of one of them in the formation of the 3-celled stage, which in later divisions leads to the 6- and the 1 2-celled conditions.
e. The second cleavage
1. The 3-celled stage. The 3-celled egg differs from the 2-celled stage only in the more rapid division of one of the blastomeres (figs. 3 and 4, pi. 15). In all the 3-celled eggs the large blastomere has two nuclei (text fig. 4, K, L, M) and in one egg the cytoplasmic division is initiated, as indicated by a constriction around the cell. The interesting point in these eggs lies in the position of the blastomeres to one another, especially in eggs Nos. 306 (3) and 173 (8); for the lines joining sister nuclei are almost absolutely at right angles to each other. The usual position of the blastomeres of the 4-celled stage (text fig. 4 N to P; figures on pi. 15) is, therefore, already anticipated in the 3-celled egg. The shifting of the blastomeres in egg No. 203 (3), shown in text figure 4, M, is rather along the original plane' of the 2-celled stage, and such an egg might develop a 4-celled egg like that shown at O, whereas an egg like No. 173 (8), shown at L, would be sure to develop into the typical ovum with cross-shaped blastomeres, as in figures 5 and 6, plate 15.
2. The 4-celled stage. If the number of specimens which the collector happens to secure of a given stage be any criterion of the relative length of time which the egg remains in that stage, then according to my collection the 4-celled condition of the opossum egg is not passed very quickly. For I have more than five dozen, mostly excellent preparations of this stage, and have other eggs still unsectioned. Three whole litters (Nos. 293, 299, 320) furnished only 4-celled eggs so far as these have been studied. However, this preponderance of 4-celled eggs is probably quite accidental. Inasmuch as cleavage proceeds irregularly after the 4-celled stage, it would not be fair to compare the number of 4-celled with the number of 8-celled eggs, for example, for a litter preponderatingly 8-celled would be sure to contain 6-, 7-, and perhaps 10- and 12-celled eggs also (compare Nos. 85, 117, 342).
The 4-celled egg of the opossum is typically Eutherian in the cross-shaped arrangement of the blastomeres. This is quite evident from the figures presented, some of which are drawn from models, others from in toto preparations and from sections (text fig. 4 and pi. 15). The arrangement of the blastomeres is such that no section can possibly pass through the centers of all the four blastomeres. If the imbedded egg be so oriented that a section cutvS two blastomeres the other two have a chance to be similarly cut (figs. 6 and 7, pi. 3; f^gs. 11 and 12, pi. 15). Sometimes three blastomeres are found in one section and a single one in another section (figs. 9 and 10, pi. 15). In figure 5, plate 3, and figures 7 and 8, plate 15, the knife passed through the centers of two blastomeres, the top of a third, and the bottom of the fourth. I have also studied 4-celled eggs in the living state under strong illumination and have clearly seen that the crossed arrangement of the blastomeres, sometimes with slight deviations from 180°, is normal for the opossum egg.
The four blastomeres of any one egg are usually of the same size; hence one can seldom differentiate a pair of large and a pair of small cells, and I have searched in vain for any other trace of polarity in these eggs aside from that afforded by the occasional presence of the polar bodies which, with the shifting of the cells, has little meaning (figs. 11 and 12, pi. 15). Moreover, the blastomeres are always spherical, except when very large, in which case they are flattened on contact surfaces by mutual pressure (figs. 7 and 9, pi. 15). The entire ovum measures through the zona the same as the undivided tubal or uterine egg. Among the various litters of eggs there is, however, a remarkable variation in the relative size of the blastomeres, which depends upon the amount of yolk extruded. The egg represented in figure 7, plate 15, has a minimum of eliminated yolk and the largest blastomeres; figure 14 represents the other extreme; figure 11 the intermediate condition. The extent of yolk elimination would seem to be hereditary, for in each batch of eggs the blastomeres of the individual eggs are approximately of the same size; thus, in No. 203 they are all very large, in No. 299 all extremely small. Both types are, however, normal, for sister ova in the right uterus in each case were allowed to develop and produced normal blastocysts.
The eliminated yolk in the 4-celled eggs seems to be characteristic of this stage. It occurs in small rounded lumps of about equal size, uniformly distributed (figs. 5, 6, and 7, pi. 3; figs. 7, 8, 11, 12, and 14, pi. 15).
f. The origin of the crossed arrangement of the first four hlastomeres
Since in the Eutherian ova there is very little yolk to be eliminated, even in cases, such as the bat, where the phenomenon has been described by Van der Stricht, the blastomeres of the 4-celled stage fill the space within the vitelline membrane rather snugly. It has therefore been suggested that mutual pressure is responsible for the shifting of the blastomeres and that in the crossed arrangement they occupy the minimum space in the egg. A glance at the specimen photographed in figures 6 and 7, plate 3, will convince one, however, that this mechanical explanation is inadequte, for certainly here one cannot speak of mutual pressure of the blastomeres, for they are not even in contact, and yet in such eggs the shifting also takes place. Hence we must look for other causes of the shifting movement.
It is, of course, quite possible that there is no shifting at all, but that the cleavage planes cut the two blastomeres of the 2-celled egg at right angles, as has been suggested by Professor Hill ('10, p. 31). According to this assumption, one of the blastomeres would be divided meridionally, the other equatorially, and the crossed arrangement would obtain from the beginning. Indeed, a study of the 3-celled eggs described above would seem corroborative of this view, for here the definitive arrangement has already manifested itself. But two facts make this theory untenable. First, a number of 4-celled eggs and one 3-celled egg I find to deviate less than 180° from the parallel arrangement; hence for these one would under the theory have to postulate a backward shifting toward the parallel postion.
But conclusive evidence on the point is furnished by eggs Nos. 306 (1) and 306 (2), in each of which both blastomeres are in mitosis. In the latter the spindles are exactly parallel, as shown by lines connecting their ends in D, text figure 4. In the former egg (A and B) the spindles in the blastomeres are 36° removed from the parallel. These observations seem to indicate that division begins in both blastomeres of the 2-celled egg in a single cleavage plane and that secondarily a shifting sets in early in the process of division.
Fig. 4. A and B, two views of egg No. 306 (1); C and D, two views of egg No. 306 (2); in both cases both cells are in mitosis and the lines run through the ends of the spindles (compare fig. 2, pi. 15, and fig. 4, pi. 3). E and F, two views of egg No. 203 (8) (compare fig. 1, pi. 15). G and H, two views of egg No. 203 (4) (compare fig. 7, pi. 13). I, No. 203 (13); one blastomere only has given off a large amount of yolk. J, egg No. 203 (11). K, egg No. 306 (3) (compare fig. 4, pi. 15). L, egg No. 173 (8) (compare fig. 3, pi. 15). M, egg No. 203 (3). N, No. 81(6). O, No. 299 (7) (compare fig. 13, pi. 15). P, No. 46 (7). I, J, and P, drawn from total preparations; all others from wax models.
There is a third possibility, as described by Sobotta ('95) for the mouse. According to this author, if I follow him correctly, one of the two blastomeres of the 2-celled egg divides meridionally, but the other blastomere has the division spindle at right angles to the first cleavage plane. In other words, the cleavage plane of the first blastomere to divide stands at right angles to the first cleavage plane, whereas in the second blastomere it is parallel to it. In such a case some shifting is also necessary to bring about the typical crossed arrangement of the 4-celled egg. This method does not obtain in the opossum, as is seen from my description above.
This point would appear to be further complicated by Spurgeon and Brooks ('16), who describe and figure cleavage stages, derived apparently from two female opossums. According to these authors, the second cleavage plane passes through both blastomeres equatorially and not meridionally, and thus a fourth method is suggested. I would cheerfully accept the authors' conclusions, but for the fact that the eggs described by them do not appear to me t© represent normal fertilized eggs. I believe their specimens to be fragmenting and unfertilized eggs that "have been in the uterus three or four days. My reasons are as follows: 1) Cases of fragmenting eggs are extremely common in cage animals and such eggs may fragment into regular pieces resembling blastomeres of eggs in cleavage, as I have seen repeatedly in hundreds of such eggs (compare my photograph in fig. 5, pi. 11). 2) In their illustrations some of the blastomeres have an additional peculiar nucleus and many of the nuclei are very eccentric in position. Multinucleated 'cells' and those with nuclei placed at a distance from their centers are quite characteristic of fragmenting eggs. 3) The 'polar bodies' represented are peculiar for their large area in cross-section and for their position at a distance from the periphery of the egg. 4) The authors do not figure any of their 4-celled eggs, of which they secured four along with other stages, although they present drawings of six 2-celled and other eggs. 5) The photographs given by the authors in their figures 12, 13, and 14 I recognize from my experience with hundreds hke them as typical pictures of degenerating eggs; for example, in the thickness of the shell, which suffers little collapse in fixation; in the peculiar stringy, not uniformly concentric character of the albumen, and in the character of the ovum itself, where fragmentation is quite apparent. 6) Finally, the size of the eggs as stated by the authors, 0.75 to 1.5 mm., is far above that of normal eggs in cleavage and entirely in agreement with my own specimens of fragmenting eggs. I must, therefore, conclude that the eggs described by Spurgeon and Brooks do not represent normal cleavage in the opossum.
I would conclude, therefore, that both blastomeres of the 2-celled opossum egg divide meridionally, but that they shift their position during division so that the resulting 4-celled ovum possesses the typical crossed arrangement.
g. Comparison of the 4-celled egg of the opossum and of Dasyurus
It is seen from the foregoing that the 4-celled stage of the opossum is typically Eutherian, at least in the arrangement of the blastomeres, and quite different in every recognizable way from the egg of Dasyurus, in which, as described in Hill's beautiful monograph, the second cleavage is shown to be meridional, dividing the egg into four equal cells which exhibit the same polar differentiation as the 2-celled egg, for each blastomere possesses a larger, vegetative pole and a smaller, relatively yolk-free animal pole.
Precisely such an egg is described by Selenka ('87) for the 4-celled stage of the opossum. I can reaffirm my former statement that this is a case of an unfertilized egg undergoing pseudosegmentation or amitotic fragmentation, in which the four pieces or 'blastomeres' (pseudoblastomeres) happen to be of equal size. I have seen such eggs dozens of times. Figure 5, plate 11, is a photograph of a litter of eggs, palpably fragmenting, but showing one '2-celled' and one '4-celled' stage which might easily be mistaken for normal cleavage. This matter is again mentioned and the photograph presented as further evidence that there is a decided difference between the normal 4-celled egg in the opossum and that of Dasyurus. What has been described by various authors as 'parthenogenetic cleavage' in ovarian eggs of mammals may often be merely a fragmentation process similar to that here described for the opossum. I have also found just such fragmenting eggs in atretic follicles of opossum ovaries.
h. Deutoplasmolysis or the elimination of yolk
In the eggs of both Dasyurus and the opossum the extrusion of yolk proceeds in the manner that one might predict from the distribution of the yolk in either case. In the ripe egg of Dasyurus the deutoplasm is collected in a mass at one pole where it is bodily extruded when the first two blastomeres round up during the first cleavage. In the opossum the yolk, being peripherally distributed, is given off from any or all sides. This happens, in small amounts, as early as the pronuclear stage and in larger amounts at the first cleavage. At each cleavage stage some yolk is left within the blastomeres, and it is probable that with each succeeding division of the blastomeres some additional yolk masses are eliminated. There seems to be no regularity of time in the elimination of the yolk, just as there is no regularity in the relative amounts eliminated; but the greatest quantity seems to be given off between the 2- and the 4-celled stage. The blastomeres may be very large and full of yolk or very small and proportionally yolk-free; and, since considerable cytoplasm is thrown off with the yolk, this would seem to indicate that a relatively unimportant role is played by the peripheral cytoplasm in the normal processes of the cells. But the fate of the yolk is in all cases the same: it is eventually digested and resorbed, so that in the bilaminar stage only a few granules occur among the cells of the embryonic area, as will be pointed out later.
As the eliminated material contains both cytoplasm and yolk granules, it would seem that whole portions of the cells are dropped bodily. The appearance of these cast-off masses in the various stages may be seen from the drawings. With trichloracetic fixation, blastomeres and yolk blend into an almost uniform mass, so that the limits of the cells are recognizable with difficulty (compare fig. 13, pi. 15, with fig. 14, eggs from the same litter).
The yolk eUmination in marsupials is, of course, striking in that the mass involved is very large, and this is as one would expect from the phylogenetic position of the group, as has been so ably discussed by Professor Hill. This phenomenon has, however, not entirely disappeared among the Eutheria, as Van der Stricht's fine study of the bat ovum amply proves. This author has shown that there is a polar distribution of the yolk in the bat egg and this undergoes elimination, a process called deutoplasmolysis by the author. A similar condition is found in the ovum of the armadillo by Newman ('12), but this author's statement that the similarity in the distribution of deutoplasm in the eggs of the armadillo and of Dasyurus argues for the low phylogenetic position of the Edentata loses some of its force from the fact that the egg of Didelphys, a marsupial, does not exhibit a polar concentration of fat.
i. Later cleavage to the formaiion of the blastocyst
An extended description of the later cleavage of the opossum egg was presented in my former article (Hartman, '16), to which the reader is referred for details here omitted. The new material collected in 1916 and 1917 contains eggs from 8 to 26, 28, and more cells; all corroborative of the former account. These eggs were also carefully studied in the living state and were photographed in salt solution at high and low magnifications, and the assurance may be given that the fixed and sectioned material accurately represents the true morphological relations. This is well borne out by the photographic reproductions of living eggs and of sections made from them as shown on plates 4 and 5.
The later cleavage is represented in the collection by eggs with every number- of • blastomeres from the 4-celled stage in which two blastomeres only are in mitosis (figs. 5 and 6, pi. 15) to the fully formed blastocysts of about 32 to 36 cells. Cleavage proceeds very irregularly after the 4-celled stage, which explains the fact that the 8-celled and the 16-celled eggs are only slightly in the plurality (compare litter No. 85). There is a retardation in division of cells at one pole of the egg, presumably among the lineal descendants of one of the first two blastomeres. In models of 10- and 12-celled eggs the larger cells are grouped at one pole, but, aside from this fact, there is nothing that would point to a polar differentiation, and in the 16-celled stage even this criterion is lost.
After the 4-celled stage is passed, the ovum of the opossum behaves no longer as a typical Eutherian, but as a marsupial ovum. In the former the blastomeres of the successive divisions cling together to form a solid mass or 'morula,' which is soon overgrown by a layer of cells, Rauber's layer or the trophoblast. The mass within is the 'inner cell mass' which gives rise to the embryo and its envelopes. The blastocyst is formed by the appearance of a cavity between the trophoblast and the inner cell mass at the lower pole of the egg.
In the marsupials the morula stage is absent. Already in the 2- and 4-celled opossum eggs the space between the blastomeres represents, potentially, the blastocyst cavity, for at the 16-celled stage, or even earlier, the blastocyst cavity is clearly indicated. As early as the 6-celled stage the blastomeres manifest a tendency to migrate to the zona pellucida and to apply themselves to the wall of the omm (fig. l5, pi. 15). In 12and 15-celled eggs the blastomeres are usually well flattened out at the periphery, as seen in figure 8, plate 3; figure 9, plate 13, and figure 16, plate 15. At the 16-celled stage it is exceptional to find rounded cells, and models of such eggs show the outer surface of the blastomeres molded against the curvature of the surrounding albumen (compare figs. 17 and 18, pi. 15).
It thus happens that the eliminated yolk comes to lie within the cavity of the blastocyst, for the blastomeres migrate to their places against the wall of the ovum and here undergo further division and further flattening until they come into mutual contact and thus complete the blastocyst wall, leaving the yolk within the cavity. Figure 19, plate 15, is a section through an ovum of 26 cells; figure 20 through one of 28 cells. In both cases there are gaps in the wall of the blastocyst, indicating that this is not yet complete. The same is true of two eggs of 30 and 32 cells, respectively, in which the gaps are fewer in number (fig. 3, pi. 4). In figure 10, plate 13, and figure 1, plate 14, are shown sister ova of 32 and 34 cells, respectively; their walls are practically continuous and the blastocyst may be considered complete. Occasionally more advanced blastocysts still have gaps in their walls, as, for example, the one shown in figure 6, plate 6, which has 46 cells. We may say, however, that, on the average, the blastocyst wall is completed when the 32-celled stage is reached or soon thereafter. No polarity is evident in the egg, the cells being of uniform size and structure throughout. Not long after this the entoderm formation is initiated.
Hence, in the opossum the blastocyst is completed at a much earlier stage than in Dasyurus, where the blastomeres of the 16-celled egg are arranged in two superimposed rings at the equator of the egg. To form the blastocyst wall they must proliferate and migrate toward either pole, and the blastocyst is not completed until the gaps at the two poles are closed. In the opossum, on the contrary, to complete the blastocyst all that is necessary is the closing of the gaps between the cells which are early distributed more or less evenly at the periphery. The just completed blastocyst of Dasyurus contains more than three times the number of cells (90 to 130) than does the corresponding stage of the opossum, and it is three times as large.
At this stage in the opossum, neither the ovum nor its envelopes have increased perceptibly in size (pi. 12). The albumen layer lies over the ovum as thickly as before, again in striking contrast with the condition in Dasyurus, in which the albumen layer is completely resorbed when the egg has reached the 16-celled stage. The opossum blastocyst is completed about thirty hours after the beginning of cleavage; in one case (No. 314) such eggs were found three and one-half days after copulation.
j. On the fate of the first two blastomeres
In the Eutherian ovum it seems probable that one of the first two blastomeres is destined to form the inner cell mass, the other the trophoblast, as was first pointed out by van Beneden (75). If, then, Hill be correct in his interpretation of the embryonic area of marsupials as being homologous with the inner cell mass of Eutheria (a view in which I join), one might suppose that the 2-celled stages in the two groups of mammals are also homologous. But that this does not hold in the case
Fig. 5. To illustrate the probable fate of the two blastomeres of the 2-celled egg. Polarity is indicated in B, D, and E by the more rapid cell division at the upper pole. In F, a 16-celled egg, and G, one of 40 to 50 cells polar differ of Dasyurus seems clear from the scholarly work of Professor Hill. In Dasyurus the most reasonable interpretation of the facts is that the upper poles of the two blastomeres form the embryonic area and the lower poles the non-embryonic area. If this view be correct, then both blastomeres contribute equally to the embryo and to the trophoblast, or, in other words, the upper halves of the two first blastomeres of Dasyurus are together homodynamous with an entire blastomere of the Eutherian ovum, the lower halves homodynamous with the other blastomere. There would seem, then, to be a fundamental difference between the 2-celled Metatherian and the 2-celled Eutherian ovum.
The question arises: Does the opossum ovum follow, in its behavior, the egg of Dasyurus, with which the opossum is phylogenetically more closely related, or does it follow that of the Eutherian ovum, to whose indeterminate type of cleavage it is strikingly and unexpectedly similar?
I have previously taken the latter position, namely, that the formative area very likely arises from one of the blastomeres, as in the Eutheria. If one follow a series of models of opossum eggs in successive stages, such as shown in text figure 5, A to H, one may visualize the formation of the blastocyst. We may safely assume that there are an upper and a lower pole in the eggs A to E, as evidenced by the difference in rate of division, aside from various other differences which may occur between the first two blastomeres (in size, amount of yolk extruded, rate of division). In the 12-celled egg there are eight smaller cells
entiation is lost, soon to be reestablished by the appearance of entoderm. It seems not unreasonable that the upper pole of H is the product of one of the two cells in A.
(2 X 4) and four larger cells (1 x 4), and it is evident that such an egg arose by one division from the 6-celled stage. Polarity is, therefore, indicated at least to this extent. The four undivided cells may next divide, establishing the 1 6-celled stage, in which polar differentiation is lost (F), not to be resumed again until the entoderm begins to proliferate at about the 50- to 60-celled stage (between G and H in the figure).
It is, therefore, impossible to bridge over the brief gap between the 16-celled stage, where polarity is lost, and the 50-celled stage, where it is resumed, and all that may be said is that it seems more reasonable to assume that all of the slowly dividing cells are of one kind and have one destiny and that all of the rapidly dividing cells are of another kind and have a different destiny. One has the choice between this view and the alternative, that a part of each type of cell goes into the formative and a part into the non-formative region.
Because of the short period in the opossum egg in which polar differences are lost, it is, therefore, impossible to demonstrate cell lineage in the cleavage of the opossum egg. But the same statement may be made with reference to the egg of Dasyurus, as pointed out by Hill himself, for, in the Dasyurus blastocyst, polar differentiation is lost during the long period of growth from 0.6 to 3.5 mm. Hill says ('11, p. 46): "It might therefore be supposed that the polarity, which is recognized in early blastocysts, and which is dependent on the pronounced differences existent between the cells of the upper and lower rings of the 16-celled stage, is of no fundamental importance, since it apparently becomes lost at an early period during the growth of the blastocyst. Such an assumption, however, would be very wide of the mark .... and, indeed, in view of the facts set forth, is an altogether improbable one." There is not the slightest doubt that Professor Hill's view is the reasonable and the probable one. Upon the same grounds, the 2-celled opossum egg is not homologous with the 2-celled egg of Dasyurus, but rather with the 2-celled Eutherian egg.
Several abnormal opossum eggs are instructive in this connection, for they are indicative of polar differentiation in young blastocysts normally devoid of evidences of polarity. They are abortive attempts on the part of one-half of the egg in each case to form a blastocyst wall and they were found in litters of eggs made up for the most part of normal young blastocysts. Figure 3, plate 16, represents a section through an egg in which one-half of the blastocyst, consists of twenty cells, which have flattened normally, whereas the other half consists of eleven cells which are still rounded as in an earlier cleavage. Similarly, one-half of another egg (fig. 4, pi. 16) seemed to develop normally, the other half containing cells with fragmenting nuclei. In still another the normal half is beehive-shaped and surrounds two very large and three small cells (fig. 10, pi. 21). Egg No. 356 (2) has two large blastomeres at one pole of the blastocyst (fig. 14, pi. 22) and egg No. 88 (6) has a retarded blastomere enclosed within the blastocoele (fig. 13, pi. 22).
These cases perhaps indicate that the cells at one pole of the blastocyst are all of a distinct type, and it is not a far cry from eggs of 32 cells to the 2-celled stage, nor is it an unreasonable assumption, in view of the facts presented, to derive cells of each type from one of the two blastomeres.
The Formation of the Entoderm
a. General
In his classical work on Dasynrus, Hill has described an apparently new method of entoderm formation in mammals. His account is specific and definite, for the entoderm may be traced from certain unique cells which appear in the blastocyst wall when the egg has attained a diameter of about 4 mm. Within the embryonic area of such eggs a number of small ectodermal cells become modified, leave the blastocyst wall, and migrate to the inner surface to become the definitive entoderm. A similar process was independently discovered in the armadillo blastocyst and described in detail by Patterson ('13), who showed conclusively that also in this Eutherian mammal the entoderm forms not by delamination of cells on the surface of the inner cell mass, but by migration of the cells from the embryonic ectoderm of the monodermic vesicle.
Selenka, in his work on the opossum, naturally also speculated upon the method of entoderm formation in this species. His ideas were based upon one defective 8-celled egg and on two blastocysts of 42 and 68 cells, respectively. He believed that the lower half of the 8-celled egg consists of entodermal, the upper half of ectodermal cells. Each of his two youngest blastocysts has a large cell included within the blastocyst cavity, and one of his figures is almost identical with my specimen No. 314 (2), shown in figure 2, plate 5. This included cell, which he calls 'Urentodermzelle,' Selenka believed to be a migrant from the lips of the 'blastopore' at the 'entodermal pole' of the egg.
In my previous publication I reported upon 44 normal young unilaminar blastocysts, in 39 of which there occurred one or more cells within the blastocyst cavity, as well as other enlarged and modified cells still within the wall (pis. 7 and 16; compare Hartman, '16, p. 36). I conjectured that the free cells might have arisen by accidental inclusion of a blastomere in about the 16-celled stage (compare fig. 17, pi. 15) or by proliferation from the large cells within the blastocyst wall, since frequently a number of cells would be united into a column projecting into the cavity (figs. 3 and 8, pi. 6). These cells appeared to come from various points in the blastocyst wall.
My next stage consisted of considerably advanced unilaminar blastocysts (compare figs. 1 to 4, pi. 18), in which I found entoderm in various stages of differentiation, including certain few cells that appeared to come out of the formative area of the blastocyst in precisely the same manner described for Dasyurus by Hill; and I figured cases in point.
With these two considerably separated stages before me, I concluded that the entoderm arose, as in Dasyurus, after the formative area had become well differentiated, and hence I considered the included cells of the young stages as of 'no morphological importance.'
Since publishing my report on these young blastocysts, I have been fortunate enough to collect an unbroken series of transitional stages between the just completed unilaminar blastocyst and the just completed bilaminar stage, and of espe€ial interest are litters Nos. 344, 356, 194', and 349, of which I possess numerous preparations (pis. 8, 9, 16, 17). I also have more than five dozen additional young unilaminar eggs of the stage previously described, so that I now have before me 100 such preparations, besides a considerable number which I did not consider necessary to section. In these blastocysts I again find the persistent occurrence of the peculiar included cells such as previously described, which my new material now teaches are the true entodermal mother cells of the opossum. What I had previously described as entoderm formation marks the end and not the beginning of this process. The true entoderm formation begins in blastocysts containing 50 to 60 cells within the blastocyst wall; that is, these large modified cells in the blastocyst wall, which proliferate after becoming free, or even in situ, constitute the first entoderm mother cells. This I am now able to show from the study of a closely graded series of stages, as abundantly illustrated by my drawings (pis. 16 to 18) as well as by photographs of preparations and of living eggs (pis. 6 to 9).
b. The youngest unilaminar blastocysts
It has been shown above that the blastocyst arises by the early migration of the blastomeres to the periphery of the ovum, where they flatten out against the zona pellucida or the albumen layer. By further division and spreading, the cells come into mutual contact, obliterating the spaces between them. The blastocyst is completed at the 32-celled stage or immediately thereafter. At first there is no evidence of polarity in the blastocyst, all of the cells being of the same structure and thickness throughout.
c. The first entoderm mother cells
At about the 50- or 60-cell stage, on the average, certain cells within the blastocyst wall undergo modification in situ. They become larger jutting out more or less into the blastocyst cavity. On their inner surface they may be rounded (ENT^, figs. 6 and 7, pi. 16), or they may display an extended tip as if undergoing amoeboid movement (fig. 5, pi. 16). Some eggs show this tendency only to a slight degree in one or several cells; in others one or two cells will show more decided enlargement, projecting as much as two-thirds of the radius of the blastocyst into the .cavity (fig. 4, pi. 7). These cells are the first entoderm mother cells in the opossum and can be traced in every graduation from earliest differentiation until they become detached from their place in the wall. Most of these cells are to be recognized only by their size and shape, since they have the same staining reactions as other unmodified cells and they contain apparently the same number of yolk granules. But if they remain some time in the wall, they elongate greatly and take a much darker stain, as in figure 4, plate 7. This elongated type of cell is common in the collection. If the attachment of such a cell in the wall continues, it may give rise by cell division to columns of three, four, or more cells, as numerous examples serve to indicate (figs. 3 and 8, pi. 6, and figs. 20 and 21, pi. 16).
d. The detachment of entoderm mother cells
It more commonly happens, however, that the entoderm mother cells leave their place in the wall soon after attaining their maximum size, and their behavior at this time constitutes perhaps the most remarkable phenomenon in the entire development of the opossum egg. Their performance at this stage is little short of spectacular. Such partly or wholly detached cells are present in nearly every egg of litter No. 88, which covers this critical period in the formation of the entoderm by a series of more than two dozen preparations, and there are identical cells in numerous other excellent preparations from various litters. The cells, moreover, have such a characteristic appearance that I should term them the more typical entoderm mother cell of the opossum.
After a period of growth the entodermal cell rounds up on all sides. In this way its contour no longer conforms to the curvature of the ovum, and as a result, the contact with the adjoining cells is broken — the cell seems to roll out of its place, as it were, into the blastocyst cavity. But the gap thus formed does not long remain, for the vacant spaces are filled at once by a flowing in of the surrounding cells. This is clearly seen at A, figures 7 to 11, plate 16, which specimens were not selected originally with this point primarily in view, but they. illustrate the phenomenon without exception. The entoderm mother cells, when they leave their place in the wall, do not, therefore, leave gaps that may be called 'blastopores' (Selenka), and such gaps as occur in earlier stages, with or without included free cells, are due to a different cause, as was shown above. Somewhat more advanced stages, moreover, still proliferate cells of the 'same type, as will appear below (pi. 17).
The newly formed entoderm mother cells are sometimes found in mitosis (fig. 10, pi. 16, and fig. 8, pi. 17) ; indeed, in egg No. 88 (11) six of the nine entoderm mother cells are in process of ceU division, although most of them have not yet left the blastocyst wall (fig. 22, pi. 16).
It is thus apparent that the entoderm mother cells, found in variable numbers within the blastocyst cavity, arise from cells leaving the blastocyst wall and also as a result of their multiplication before, during, and after their migration into the cavity. The specimens figured here as well as numerous others afford ample evidence of these developmental processes.
The process in the opossum is essentially the same as obtains in Dasyurus, for, at a given stage in both forms, certain cells in the superficial unilaminar wall become modified and migrate into the interior of the vesicle. In the opossum we have an approach to the Eutheria in the early differentiation of the entoderm; hence we may consider the Dasyurus as exemplifying the more primitive, the opossum the more specialized condition.
e. Proliferation of entoderm confined to one pole
The small blastocysts of about 0.15 mm. in diameter referred to above cannot be oriented for sectioning, and hence the plane of the sections is entirely a matter of chance. It thus happens that the sections taken tangentially or obliquely through the ovum present, in some cases, very confusing pictures; for in such specimens the entodermal proliferation appears to take place promiscuously from various parts of the egg, and the polarity, which is very apparent in favorably cut series, is thus obscured. In the former the entodermal proliferation is palpably confined to one pole; to ascertain the arrangement in the latter it is necessary to make idealized reconstructions in the proper plane. This I did from series of camera-lucida drawings. Five such reconstructions are shown in figures 18 to 22, plate 16. In every case, without exception, the entoderm proliferation is confined to one pole, in some cases to exactly one-half of the blastocyst. We may, therefore, now speak of embryonic and non-embryonic areas, for there is no longer any doubt as to their identity: the embryonic area is marked by the position of the entoderm mother cells and the polarity of the ovum is definitely reestablished.
A study of the young blastocysts just considered, as well as immediately succeeding stages, seems to show, moreover, that the first proliferation of entoderm takes place more actively on the margin of the future embryonic area, for one often finds them most numerous on opposite sides, as shown, for example, in figures 11, 18, and 22, plate 16, and figures 8, 10, and 11, plate 17.
The blastocyst now contains two types of cells: 1) those clearly entodermal in destiny, as just described, and 2) the peripheral or enveloping layer, which, of course, gives rise to all of the ectoderm, embryonic and trophoblastic. All of the cells at the lower pole are ectodermal, being trophoblastic. But the epithelial cells at the embryonic pole, since they will for some time still continue to- proliferate entoderm mother cells, are potentially both ectodermal and entodermal and should better be called entectoderm until the entoderm is fully formed. They are, however, also potentially mesoderm, as my next paper will clearly show.
The ovum of the opossum has not grown much in volume since its discharge from the ovary, being still less than 0.15 mm. in diameter (pis. 12 and 13), which is in striking contrast with the blastocyst of Dasyurus, where, at the appearance of the first entoderm mother cells, the vesicle is nearly 4 mm. in diameter. In the opossum the period of growth follows the formation of the entoderm, but in Dasyurus this is preceded by a long period of growth. The process of entoderm formation in Dasyurus may, therefore, be studied from surface mounts of pieces easily cut from the blastocyst wall, as well as from serial sections; but the opossum egg at this stage may be studied in section only, for it is small, covered with a thick layer of albumen, and is densely packed with more or less opaque yolk. The vesicular structure can well be made out from in toto preparations, but for detailed study such preparations are worthless.
f. Time of appearance
From the foregoing it is apparent that size is as yet no criterion to the differentiation among the blastomeres. The nu-mber of cells seems, therefore, to be the best means of establishing the stage in question. With this in view, a careful count was made of the number of cells in thirty-three flawless series. By making camera-lucida drawings of each series sketching in the nuclei, superimposing the successive drawings, and eliminating duplicates, it is believed that the counts are quite accurate. This data is presented in table 5.
This table shows that there is a rough correlation between the number of cells and the extent of entoderm proliferation. Extreme variations occur, however, as, for example, in the sister eggs Nos. 298 (1) and 298 (3), which have four entodermal mother cells each, but the latter totals twice as many cells as the former (64 and 124, respectively). But it may be stated in general terms that entoderm proliferation usually begins when the blastocyst is made up of 50 or 60 cells.
It is thus apparent that in the early differentiation of entoderm the opossum again approaches more closely than does Dasyurus to the condition in the Eutheria. Thus in the absence of polar differentiation in the undivided egg (with due consideration to certain exceptions as the bat and armadillo), in the crossed arrangement of the blastomeres in the 4-celled egg; in the more or less indeterminate type of cleavage; in the early proliferation of entoderm — in all of these characters the opossum egg resembles that of the Eutheria. But in the absence of the morula stage and in the method of entoderm formation from definite entoderm mother cells arising from the unilaminar entectoderm the opossum closely resembles its relative Dasyurus as described by Hill. It is, of course, possible that Dasyurus represents the more typical development among the marsupials, as would appear also from Hill's description of some vesicles of Macropus and Parameles, in which the entoderm is laid down in vesicles less than 1 mm. in diameter, just as in the opossum.
TABLE 5 Cell counts in young opossum blastocysts
IDENTIFICATION NUMBER
NUMBER OP ENDODERMAL MOTHER CELLS
NUMBER OF
CELLS IN WALL
OF
BLASTOCYST
TOTAL
NUMBER OF
CELLS
FIGURES AND PLATES WHERE ILLUSTRATED
314 (2)
2 (?)
28
30
2, V
314 (5)
32
32
4, VI
191 (2)
32
32
10, XIII
191 (5)
34
34
1, XVI
292 (4)
46
46
6, VI
88 (5)
50
50
50 (3)
52
52
88 (13)
62
62
50 (7)
70
70
2, XVI
50 (5)
1
64
65
1, VII
83 (5)
1
52
53
12, XVI
50 (8)
2
61
63
5, XVI
88 (10)
2
50
52
88 (23)
2
55
57
10, XVI
298 (1)
4
61
64
7 and 8, VI
298 (3)
4
20
124
20, XVI
356 (3)
—
—
100
13, XVI
88 (20)
5
60
65
88 (7)
5
82
87
3, VI, 21, XVI
83 (1)
5
106
111
19, XVI
88(1)
6
72
78
«
88 (17)
6
97
103
2, VII; 18, XVI
298 (5)
8
118
126
6, XVI
50 (4)
9
59
68
3, VII
88 (11)
9
94
103
22, XVI
88 (3)
10
59
69
6, VII
88 (21) .
10
60
70
8 and 9, XVI
88 (16)
10
72
82
4, VII; 11, XVI
88 (9)
11
95
106
7, XVI
344 (14)
19
174
193
16, XVI and 17
344 (11)
23
141
164
15, XVI
344 (7)
45
124
169
7, VIII
356 (4)
42
241
283
6 and 7, XVII
356 (5)
48
201
249
10 and 11, XVII
g. Included cells which may not he entodermal
It sometimes happens that a blastomere in early cleavage becomes displaced, fails to attain its proper pos tion at the periphery and thus comes to be surrounded by its fellows. I have several 16-celled eggs with one such misplaced blastomere (fig. 17, pi. 15). Another egg. No. 314 (2), shown in figure 2, plate 5, has two included cells. One of these in the section figured is near a gap in the blastocyst wall, and it might be supposed that it had migrated from the unoccupied space. But this egg is made up of only 28 cells, a stage at which the blastocyst would hardly be expected to be completed. The cells were probably accidentally included at a somewhat earlier stage and are probably not typical entoderm mother cells. The included cell in ovum No. 83 (5) shown in figure 12, plate 16, has every characteristic of an entoderm mother cell except that it is unusually large. Some other included cells, as, for example, those in figures 10, plate 21, and 13, plate 22, are clearly undivided blastomeres of an early cleavage stage, as previously pointed out. The true entoderm mother cells are quite distinctive and are not readily mistaken for abnormal cells.
h. Further polar differentiation
After the proliferation of entoderm mother cells is well under way, the differences between the embryonic and the nonembryonic areas of the blastocyst become more and more pronounced. The former becomes marked by the large size of its cells as well as by the presence at that pole of entoderm mother cells ; the non-embryonic portion becomes progressively more and more attenuated. These changes are readily understood from plates 16 and 17. The increase in size of the blastocyst is largely due to the spreading of the non-embryonic or trophoblastic ectoderm, and as a result the embryonic area comes to occupy a more and more restricted proportion of the surface of the vesicle, and this process continues until the formation of the bilaminar stage has been completed. The change in proportionate number of embryonic and trophoblastic cells is apparent from the few examples given in table 6.
If a comparison be made between the facts shown in table 6 and the illustrations referred to therein, it is apparent that the increase in the number of cells and the differentiation proceed pari passu. In figure 15, plate 16 — a longitudinal section through ovum No. 344 (11) — the formative area is roughly marked out by the position of the entodermal cells and the yolk and coagulum surrounding them; there is some thinning out of the trophoblastic cells. Ovum No. 344 (14) is shghtly more advanced. It was cut tangentially to the formative area, to which nine of the twenty-two sections belong, the limits of this area being determined by the presence in the ninth section of
TABLE 6
Number of embryonic and trophoblastic cells number of cells
IDENTIFICATION NUMBER
TOTAL NUMBER
ENTODERMAL CELLS
EMBRYONIC
ENTECTO DERM
TROPHOBLASTIC ECTODERM
ILLUSTRATIONS, (FIGURES AND PLATES)
344 (11) 344 (14) 356 (4) 356 (5)
164 193 283 249
23 19 42
48
71
76 101 126
70
98
140
75
15, XVI
16, and 17, XVI 6 and 7, XVII 10 and 11, XVII
the last entodermal cells. The further differentiation between the two areas, as seen in litter No. 356, is quite apparent from a glance at plate 17.
In the eggs of litter No. 344, of which I have seven excellent preparations, the vesicular structure was quite apparent in the living state as well as after fixation, but it was not possible to show this in the photographs taken after staining them, since the albumen also absorbed considerable stain (fig. 2, pi. 6). Two eggs were, however, photographed alive in Ringer's solution and are shown in figures 5 and 6, plate 8. The former was taken in side view and shows the yolk and coagulum hanging in the vesicle like a bunch of grapes; in the sections of the egg taken longitudinally the relations were found to be as in life (fig. 7, pi, 8), The other eggs shown in figure 6 was photographed with the embryonic area uppermost ; the dark spot in the center is the rather opaque yolk mass.
In the eggs of Utter No. 356 the polarity was always apparent in whatever medium they were placed, whether in Ringer's solution immediately on removal from the uterus or in alcohol after fixation; hence these eggs were readily oriented for sectioning. One of these eggs was photographed by transmitted light in salt solution and is shown in figure 1, plate 8. It is a perfect sphere, situated in the center of the egg as in younger stages (fig. 4, pi. 12; fig. 1, pi. 6). The embryonic area is an opaque mass at one pole and the trophoblastic area is a thin layer making up the rest of the vesicle. This egg is typical of all of this litter (fig. 1, pi. 6), all of which measure 0.17 to 0.20 mm. in diameter through the vesicle. Fixation and staining have not changed the relation of structures essentially and even the distortion due to imbedding is very slight (compare figs. 1 and 2, pL 8; fig. 3, pi. 9, and fig. 1, pi. 6).
A somewhat transitional stage between Nos. 344 and 356 is furnished by litter No. 144 (figs. 1 to 3, pi. 17). These eggs were overfixed in Carnoy's fluid, but are instructive and corroborative of the trend of development described above.
In all of these litters (Nos. 144, 344, and 356) the entoderm mother cells are still being formed, as the figures in plates 16 and 17 amply show {ENT\ ENT'). The cells are of the same type as those previously encountered, namely, rounded and in process of leaving the periphery. Because of the greater density of the cytoplasm, these cells often take a deeper cytoplasmic stain than do the neighboring cells, from which they also become separated by a more definite cell membrane. Occasionally the entodermal cells are united into a column, as at ENT", figure 5, plate 17, reminding one of such rows of cells in the younger stages (fig. 3, pi. 6).
In these eggs, too, the margin of the embryonic area seems to be the region of greatest proliferation. Thus figure 10, plate 17, represents a section near the margin of the area and shows a line of primitive entodermal cells; while figure 11, a section nearer the middle of the series, shows entodermal cells only at the margin.
At the stage just described there is still a considerable quantity of yolk and coagulum in the egg. This is usually collected near the inner surface of the embryonic area as well as among or within the cells of the area, more rarely also in the trophoblastic cells. Occasionally the yolk is collected in a large spherule, as in egg No. 356 (11); this spherule measures 0.04 mm. in diameter and a portion of it, cut tangentially, is shown at Y, figure 4, plate 17.
i. The embryonic area superficial in position
The question may arise whether there appears at any time over the embryonic area a transitory layer that may at all be compared with Rauber's layer in the Eutherian egg. Professor Hill has homologized the embryonic area of the Eutherian egg with the inner cell mass and the non-embryonic area with Rauber's layer, and hence he uses the term 'trophoblastic' to designate the latter. According to this view, the embryonic cells lie upon the surface of the ovum from the beginning and are potentially ectoderm, entoderm, and mesoderm; in other words, the embryonic cells are never covered with trophoblastic cells. I believe this to be the true interpretation of the facts. Since my collection includes an unbroken series of critical stages on this point, if there were such a layer, it could not escape detection. Nowhere is there the slightest suggestion of a transitory layer of cells. Mitoses are always present in the superficial layer (pi. 17), disintegrating cells never. The very method of blastocyst formation, as described in these pages, precludes the probability of a trophoblastic cover over the embryonic area, which is differentiated very soon after the establishment of the blastocyst. For, if the upper half of the unilaminar opossum egg is not embryonic, it is trophoblastic and there can be no embryonic area; in which case we should be forced to derive the embryo from the trophoblast, a manifest absurdity. The preceding and the succeeding stages all show that in the blastocysts last described, the superficial cells at the upper pole are ectodermal except a few which are destined to form entoderm mother cells.
j. The primitive entoderm
In the stages thus far described the entodermal cells are still round to polygonal and only occasionally does a cell flatten out upon the surface of the mass as at ENT^, figure 4, plate 17. We may call these cells primitive entodermal cells {ENT~) as distinguished, on the one hand, from the large entoderm mother cells from which they arose (ENT^) and on the other, from the typical, flattened definitive entoderm into which they are about to develop. The primitive entoderm, through rapid cell division, becomes more or less crowded and shows a tendency to become two or three cells deep, as early as the stage represented by litter No. 356 (pi. 17).
k. Further growth of the blastocyst
When the blastocyst contains less than 200 cells, of which about 20 would be entodermal (litter No. 344, pi. 16), the embryonic and the trophoblastic areas each make up about one-half of the blastocyst wall. When the number approaches 300, including 40 or 50 entodermal cells (litter No. 356, pi. 17), the latter area has greatly extended so that the embryonic portion occupies a third or less of the blastocyst wall. The increase in size of the blastocyst is, therefore, to be attributed largely to the spreading and attenuation as well as a more rapid multiplication of trophoblastic cells (table 6).
The eggs of htter No. 194' are illustrative of the further development in the direction just indicated and follow close upon the eggs of litter No. 356. The vesicle has grown from about 0.23 mm. in diameter as the maximum for litter No. 356 to 0.34 mm. in litter No. 194', or about double the diameter of the ovum at cleavage. The blastocyst is still situated in the center of the egg, which has, however, not yet increased in volume of shell membrane (text fig. 2; fig. 13, pi. 13; fig. 5, pi. 12).
The development of the trophoblastic ectoderm is seen to have continued in the direction indicated above, so that in this litter of eggs it now occupies about three-fourths of the circumference of the blastocyst. Little more need be said of this layer. It becomes progressively more attenuated until it may have the appearance of endothelium, and even at high magnifications appear as a sharp narrow line with here and there a swelling which marks the location of a nucleus (fig. 1, pi. 18). The region may come to occupy from four-fifths to five-sixths of the entire circumference of the blastocyst; and this again constitutes a point of contrast with the egg of Dasyurus, in which the formative area occupies, in section, from one.-third to one-half of the blastocyst wall.
In general, the marsupial trophoblast does not differ markedly in structure from that of Eutherian vesicles, but more interesting and important changes take place in the embryonic area of the opossum blastocyst. For a short period, which includes the stage represented by litter No. 194' (figs. 13 to 15, pi. 17), these changes now appear to be chiefly of two kinds: 1) further proliferation of entodenii mother cells from the peripheral layer, and 2) multiplication of all types of cells.
The former process gives every evidence of having slowed down considerably since the preceding stage, the cells which can be identified as migrating inward from the superficial layer being of comparatively rare occurrence. Such cells are shown at ENT^, figs. 14 and 15; they stand with their long axes at right angles to the surface of the area and project inward among the primitive entodermal cells now every^vhere closely applied to the ectoderm. It is clear that entodermal proliferation from the superficial entectoderm is approaching the end.
As a result of the cell multiplication, the embryonic area has become crowded, so that in places it is three and occasionally four cells deep; and it may be stated parenthetically that this is the only stage before the formation of the mesoderm that the blastocyst wall is anywhere more than two cells deep, as the sequel will show. At this stage there is no regular arrangement of entodermal cells into an epithelium, and, even in the superficial layer, regularity is only approximated (figs. 13 to 15, pi. 17). The cells which are not in contact with the albumen are as irregular in shape and size, at least in my specimens, as they are in arrangement; only the nuclei preserve a uniformity of size and structure.
The superficial cells for the most part are clearly embryonic ectoderm, and all of the nuclei seen below this layer are primitive entoderm. Most of them possess rounded nuclei, and only here and there in the sections is there any indication of cells which tend to flatten out into definitive entodermal cells (EN T^, fig. 15). In this respect there has been little progress since the preceding stage.
Litter No. 194', was found four days after copulation or about two days after the beginning of cleavage.
A somewhat more advanced stage is represented by litter No. 349, one of which is shown photographed in the living state in figure 3, plate 8. It measures 0.32 mm. through the vesicle. Figure 4 is a section through the youngest egg of the litter and belongs to an earlier stage corresponding to litter No. 344. One of the two eggs like the one in figure 3 was sectioned, the other was accidently broken and was used for study in toto. The two are in essential agreement. The formative area, shown as a distinct opacity in the living egg (fig. 3, pi. 8), is larger in area than in the litter just described; the trophoblastic area is thick-walled and less extended than would be expected at this stage. The embryonic area is crowded, the cells being three and four cells deep in some places. A large number of cells of all types — embryonic ectoderm, primitive and definitive entoderm — are in mitosis, chiefly in the spireme stage, as though a wave of cell division had spread over the entire area. Here and there an entoderm mother cell is still in process of formation. The definitive entoderm has begun to differentiate and to spread beyond the area (ENT). In surface view the embryonic area is approximately round and is sharply marked off from the surrounding trophoblastic ectoderm. This description shows that the entoderm is present in a watch-crystal-shaped mass at one pole of the egg in vesicles of 0.30 to 0.35 mm. The mass is thicker in the middle, being even three to four cells deep. Only the outer superficial layer is ectodermal, the massed cells beneath being all entodermal.
The opossum blastocyst differs, then, both from the corresponding stage of Dasjoirus, on the one hand, and of the higher mammals, on the other. In Dasyurus the entodermal cells flatten out and spread singly as they are formed and never pile up in a mass as in the opossum. There would seem to be in the opossum a nearer approach to the Eutherian ovum in its possession of a kind of 'inner cell mass' (fig. 15, pi. 17; fig. 1, pi. 18).
But there are fundamental differences. For in the Eutheria the entoderm seems to arise only from the cells on the inner surface of the inner cell mass, presumably from a single layer. The outer or superficial layer of the blastocyst is Rauber's layer; between the two is the embryonic ectoderm, a layer of cells variable in thickness and at first irregularly dispersed. Thus, if figure 1, plate 18, represented an Eutherian egg, the superficial layer would constitute Rauber's layer; beneath would be the irregularly disposed ectoderm (cells marked 'ENT^'), and only the innermost layer would be the entoderm. In the opossum there are only two layers: 1) embryonic ectoderm, a superficial layer, one cell deep, and 2) all the remainder which is entodermal. If there seem to be more than two layers, as in the figure just referred to, the outer layer is the ectoderm, the inner the differentiated entoderm, and between the two a mass of cells which, are still undifferentiated or primitive entoderm which are yet to spread and form entoderm. The opossum is, therefore, fundamentally like Dasyurus; but the resemblance is obscured by the temporary massing of entodermal cells, the resulting picture superficially resembling an Eutherian vesicle with spreading inner cell mass.
l. The end of entoderm formation and the spreading of the entoderm
In my previous publication I described some vesicles from 0.3 to 0.5 mm. in diameter, of the type shown in figures 1 to 4, plate 18, in which certain cells seemed to migrate from the embryonic ectoderm to take their place among the entodermal cells. I interpreted these cells as entoderm mother cells and presented a number of cases which closely parallel the process of entoderm formation in Dasyurus as described by Hill. Indeed, Hill states that in Macropus the primitive entodermal cells are already recognizable as cells situated internally in the blastocyst of 0.35 mm. and in Parameles in vesicles of about 1 mm., which would seem to correspond very well with the condition in the opossum. Certain cells drawn in figures 3 and 4, plate 18, might conceivably be proliferating entoderm. If this be true, then certainly these sporadic cases are the last stragglers in the stream of entodermal cells which arise from the entectoderm. The climax in the formation of entoderm in the opossum, however, occurs long before this, namely, in blastocysts between 0.15 and 0.30 mm. in diameter.
The differentiation of primitive entodermal cells into definitive entoderm takes place with rapidity soon after a diameter of 0.34 mm. is attained (litter No. 194'), so that in vesicles of about 0.50 mm. the entoderm has largely assumed its squamous structures and lies closely appressed against the simple unilaminar ectoderm. As soon as this differentiation is well under way, the entoderm at once migrates beyond its region of origin toward the opposite pole of the vesicle. These changes are readily observed in some typical examples furnished from litters Nos. 194', 349, 40, 43, 175', 339, 299', and 347.
In litter No. 194' as described above, the spreading of the entoderm has scarcely begun. In litter No. 349 (fig. 12, pi. 17) a few cells have flattened decidedly, while the majority are still in the condition of indifferent primitive entoderm. The tendency to spread is exhibited on the entire margin of the area. As soon as the entodermal cells have differentiated, they at once stain much darker, a characteristic which they maintain in sharp contrast to the ectoderm throughout the bilaminar stage; this is true without exception.
A somewhat later stage is represented by egg No. 43 (7), which is large, and has a greatly attenuated trophoblastic area, and represents the normal condition at this stage. The section in figure 1, plate 18, is the tenth of thirty-five sections through the embryonic area; that is, lies to one side of the midline. The definitive entoderm would seem from this section to clothe the entire inner surface of the area, but a study of the series discloses the fact that the entoderm has differentiated only in spots, chiefly at the periphery of the area. That these changes take place chiefly in the periphery first would appear also from ovum No. 339 (3) shown in figures 6A and 6 and in figure 2, plate 6. At ENT^ is a group of cells which run through ten sections; they are primitive entodermal cells not yet differentiated. So also in figure 8 (egg No. 175' (2) ) the entoderm has already advanced considerably toward the equator, although there are still some undifferentiated cells near the middle of the area. Similar undifferentiated cells are also seen in other vesicles, as at ENT^, figures 3 and 4.
Litter No. 352 consists of small blastocysts which fall into the stage under discussion. The group of eggs was photographed fresh in Ringer's solution by transmitted light (fig. 1, pi. 9). The vesicles with their more or less opaque embryonic areas are very evident. In the largest specimens the entoderm has entirely differentiated, except in one (fig. 14, pi. 13; fig. 8, pi. 21) in which a large blastomere has retarded the spreading of the cells (compare fig. 2, pi. 9, and fig. 2, pi. 18). In these eggs the entoderm has also advanced some distance toward the equator of the blastocyst.
m. Maximum attenuation of the blastocyst wall
The trophoblastic ectoderm, it was seen above, begins its thinning and spreading process soon after the proliferation of entoderm begins (in 0.15-mm. ova) and reaches its maximum when the formation of new entodermal cells from entectoderm ceases, and the entoderm begins to line the lower hemisphere (0.50-mm. blastocysts). The spreading and attenuation, however, affect the embryonic as well as the trophoblastic area and takes place rapidily while the entoderm is migrating to the opposite pole. In all of the eggs of litters 175' and 347 (pi. 18) these facts are clearly shown. Blastocyst No. 175' (9) is an extreme case in point (figs. 7 and 7A, pi. 19). In succeeding stages the embryonic area thickens progressively but slowly, until it reaches its maximum in blastocysts 1 to 1.5 mm. in diameter, after which it remains more or less constant until the embryo begins to differentiate.
n. Cause of spreading of the entoderm
In the spreading of the entoderm the chief factor is the active migration of the entodermal cells. The passive spreading, due to the enlargement of the vesicle, as had been suggested in the case of other mammalian vesicles, is, in the opossum, a negligible factor. An inspection of plate 18 will make this clear, for the vesicles are about as large when the entoderm begins to spread (fig. 1) as when it has reached the opposite pole (fig. 5). In fact, comparison of eggs of the same litter (figs. 5 and 7) show that the size of the vesicle bears no relation to the extent of the entoderm. My observations on numerous eggs at this stage (Nos. 347 and 299') go to show that, when once begun, the spreading of the entoderm proceeds rapidly. I have not been able to demonstrate amoeboid movements in the cells, but processes sometimes occur on entodermal cells at about this stage (fig. 2, pi. 19). .
o. Changing position of the vesicle in the egg
In most of the eggs which mark the early stages in the spreading of the entoderm, the vesicle still occupies practically the center of the eggs as in previous stages (text figs. 1 and 2). In litter No. 352 (fig. 1, pi. 9) the tendency of the vesicle to approach the shell membrane is already manifest to some extent; also in litter No. 175' (fig. 7, pi. 19). In all later stages the vesicle occupies an eccentric position and in most cases it is in immediate contact with the shell membrane. This contact is established, therefore, for the first time about the beginning of the bilaminar stage, or about four days after the beginning of cleavage. This delay is in contrast, again, with the egg of Dasyiirus, in which the blastomeres in the 16-celled stage have already establshed contact with the shell membrane on all sides, the albumen of the egg, always limited in thickness, having entirely disappeared. The formative area almost always reaches the shell membrane first, the exception being very rare. The albumen, therefore, becomes concentrated at one pole, gradually decreasing in amount with the growth of the blastocyst. Henceforth the stage of advancement of the blastocyst may be gauged by the amount of albumen which appears as a crescent in the egg when viewed from the side or when seen in a longitudinal section (eggs No. 299', in figs. 1 and 2, pi. 6; fig. 5, pi. 10, etc.). That this eccentric position is not an artifact due to fixation or other causes, is shown by the fact that in the living egg the blastocysts are situated in exactly the same position as after fixation, as the photographs (fig. 4, pi. 1; figs. 4 to 6, pi. 9) show.
p. Some abnormal eggs
Since future workers on the opossum are likely to encounter abnormal material, it is not amiss to describe several abnormal eggs of about the stage just described.
In the group of eggs in figure 2, plate 6, a number of such abnormal specimens are shown: Nos. 294 (1), 294 (2) and 294 (3) and 339 (4). The last mentioned is the least abnormal of all. In the living state the vesicle was spherical and remained so throughout the process of imbedding (figs. 5 and 5A, pi. 19). A similar egg is shown in figure 15, plate 13, and the embryonic area of a third in figure 6, plate 19. These eggs, all from one litter, are in close agreement and the relation of ectoderm and entoderm is as in normal eggs of this stage (compare pi. 18). But the wall of the vesicle consists of unduly inflated cells with very diffuse cytoplasm and often large nuclei. Egg No. 339 (3) is exceptional in this litter, for its normal appearance; it doubtless represents the normal stage to which the others should have attained (fig. 6, pi. 9; fig. 2, pi. 6; figs. 6 and 6A, pi. 18).
The other more abnormal eggs referred to above show even at low magnification evidences of abnormality (fig. 2, pi. 6). The vesicles are not plump and rounded, but more or less shriveled and are surrounded by a large 'perivitelline space.' That the abnormalities are not due to the method of fixation is shown by the appearance of the living eggs, of litter No. 294 reproduced in figure 1, plate 11. In sections made of these eggs the walls are composed of cells swollen to enormous volume and are extreme cases of the condition shown in fig. 5A, pi. 19. Many cells are in mitosis, with the chromosomes strewn about pell-mell throughout the cell. Similar eggs are also met in normal litters (fig. 4, pi. 9). The 'pear-shaped vesicle' described by Selenka and figured in his Tafel XVIII, Fig. 1 u. 2, was doubtless an egg of the type just described.
Part IV. The Bilaminar Blastocyst
General Description
a. Material
The various stages in the bilaminar blastocyst of the opossum are represented in my collection by an unbroken series separated from one another by minutes rather than hours of development. Two hundred and thirty-five normal eggs were secured from thirty-two litters of twenty-five different animals; hence it may be assumed that, the following description gives in detail the normal opossum egg duriiig these stages. One hundred and fifty eggs were sectioned or were dissected for study of surface views; and the former include many that were carried through the imbedding and sectioning process without collapse and with the minimum of shrinkage.
b. The living eggs
The general trend of development during this period may be followed by reference to the photographs of living eggs presented in plates 1, 2, 9, and 11 of this paper.
All of the eggs still lie free in the lumen of the uterus (fig. 10, pi. 1 ; fig. 8, pi. 2) and are distributed as in the preceding stages, often grouped near the os uteri; hence one should not speak of the 'implantation' of the eggs even at the 2-mm. stage. The shell membrane has attained considerable thickness (Hartman, '16) and throughout the stage in question maintains the shape of a perfect sphere.
Before the entodermal spreading is well under way the blastocyst occupies approximately the center of the egg (fig. 1, pi. 9). Before the entoderm has reached the opposite pole of the blastocyst the embryonic area has almost or quite come into contact with the shell membrane (compare figs, 3 and 4, pi. 19), giving the blastocyst a decidedly eccentric position, It now fills one-half or less of the egg and has the shape of a bi-convex lens (fig. 5, pi. 10). This migration of the blastocyst may be due to the increased metabolism of the more voluminous cells of the embryonic area, as a result of which the albumen is here more rapidly digested and absorbed. This position is maintained in the subsequent stages (compare eggs No. 299', fig. 1, pi. 6).
The size of the entire egg containing the youngest bilaminar blastocysts with just closed entodermal sac is very little greater than the youngest uterine eggs, although the albumen has become denser and the vesicle wall has become considerably differentiated. Thus, for example, the diameter of the eggs in figures 4 to 6, plate 9 (all bilaminar blastocysts), is only a little greater than that of eggs in cleavage stages (figs. 1, 3, and 5, pi. 1). Again, the two litters shown in figures 3 and 4, plate 1, exhibit an evident, but not striking growth in volume, although they have developed from the 4-celled stage in the former to young bilaminar blastocysts in the latter in a period of four days, or 40 per cent of the entire period of gestation!
In all of the young bilaminar blastocysts the embryonic area is plainly outlined and distinctly marked off at the junctional line from the trophoblastic region. This differentiation increases with the growth and development of the egg.
From the beginning, the bilaminar stage is essentially the period of growth; this period thus follows the formation of the entoderm, whereas in the Dasyurus it precedes as well as follows the process. At first the blastocyst grows faster than the shell membrane, for gradually the albumen disappears before the advancing trophoblastic area. This growth would seem, therefore, to affect the trophoblastic more than the embryonic area, which latter lies in contact with the shell membrane from the earliest bilaminar stage. The embryonic area, however, easily keeps pace with or even exceeds the rate of growth of the entire vesicle; for in the 1-mm. eggs it is proportionally larger than in certain younger stages. It would seem, then, that, despite the absence of albumen, the area receives sufficient nutriment for vigorous growth or is indeed better supplied by virtue of its superficial position in the egg, with the secretion of the uterine glands.
Eggs about 0.8 mm. in diameter, as illustrated by litter No. 306', removed four and one-half days after the beginning of cleavage, still contain considerable albumen which is readily visible at all positions of the eggs and may be seen on the photographs of the eggs (fig. 7, pi. 10; fig. 17, pi. 13; fig. 1, pi. 21). When the diameter of 1 mm. is reached, the quantity of albumen has been considerably reduced and is mostly confined to the trophoblastic region below the equator of the egg (fig. 2, pi. 21). In living specimens of such eggs the albumen is visible as a narrow crescent, only when viewed from the side, and is not visible in photographs of living eggs (fig. 5, pi. 2). The reduction of albumen continues, and when the diameter of the egg approaches 2 mm. and the mesodermal proliferation is about to begin there is only a thin film of albumen left (fig. 6, pi. 2; fig. 20, pi. 13). The amount of albumen at any stage is, of course, variable. It may still occur in small amounts in early primitive-streak stages (fig. 4, pi. 2; fig. 22, pi. 13).
The opossum blastocyst, therefore, begins as a perfect spheer at about the 32-celled stage. It maintains this shape until the definitive entoderm begins to spread, when the blastocyst assumes a biconvex form, flattened in the direction of the egg axis, and lies with the formative area against the shell membrane. The spherical form is again attained when the trophoblastic area has reached the shell membrane, which occurs almost completely when the egg is 1 mm. in diameter, more perfectly at a diameter of 1.5 to 2 mm. It then maintains the spherical rom until, through crowding of large litters in the pregnant uterus, the vesicles are somewhat misshapen through mutual pressure.
The embryonic area of the larger blastocysts, due to its protoplasmic differentiation, now stands out clearer, so that it is plainly visible in living eggs. It is recognizable in photographs of living eggs, but much more clearly in photographs o' eggs immersed for a few minutes in the fixing fluid (fig. 2, pi. 11).
The size of the embryonic area varies greatly, even in proportion to the total surface area of vesicles in the same litter. In general its diameter occupies between one-fifth and onefourth of the circumference of the egg, occasionally a little less than one-fifth, sometimes nearly one-third. But it never reaches the equator as in Dasyurus. An average 1 mm. blastocyst is shown in figure 9, plate 21. Various measurements are given under the legends of the eggs illustrated.
Aside from these details, little may be learned from a study of the living egg, and we must turn to preparations for more intimate details of structure. The progress of development will be followed by describing first the youngest stage, then the 1 mm. blastocyst, and lastly the blastocyst just preceding the proliferation of mesoderm.
The Just Completed Bilaminar Blastocyst
The bilaminar stage may be said to begin when the entoderm has migrated to the trophoblastic pole of the egg opposite its point of origin, thus forming a closed sac within the ectoderm. This stage was attained by most of the eggs in litter No. 299' about four days after the beginning of cleavage. The vesicle occupies about one-half of the egg contents.
a. The embryonic ectoderm
In the youngest bilaminar blastocysts the embryonic area is approximately circular in shape and clearly visible, but not as clear-cut nor as definitely and neatly circular as in later stages. In surface view of preparations the junctional line between the two areas can always be made out (figs. 11 and 12, p'. 19); but in sections some difficulty is experienced in trying to determine with exactness the marginal cells of either area, for the cells of the embryonic area have not yet assumed that density of protoplasm characteristic of later stages. The embryonic ectoderm is at first comparatively thin, consisting of a single layer of somewhat flattened cells as in figures 5 to 7, plate 18, and 3 and 8 to 10, plate 19. The area gradually thickens and the cells become cubical in section (fig. 4, pi. 10, fig. 4, pi. 19). In the younger eggs the nuclei are, therefore, further apart, but, as they multiply, they become more and more crowded until they are almost or quite in contact (compare fig. 3B, pi. 20, and fig. 12, pi. 19). In surface views the area is studded with mitotic figures (fig. 3B, pi. 20).
The surface views presented in figures 3B, plate 20, and 12, plate 19, of which the latter is the more advanced, show that the junctional fine between the embryonic and the trophoblastic areas is quite definite, sometimes being marked by a continuous sharp line around the entire area. Figure 3B, plate 20, is especially instructive in this connection, since it represents in surface view (X 500) a portion of the same area shown in figure 3A in section (X 200); it is the portion removed before imbedding from point A, figure 3. There is, neither in the section nor in the surface view, any great dfference of tone between the two areas, but the junctional line {XX) or margin of the embryonic area may easily be located where the ectodermal nuclei become less crowded. The junctional line is always more clearly defined in specimens fixed with a fluid that will bring out the cell membranes, as in figure 12, plate 19. It is seen that the line is formed of the cell membranes of contiguous cells bordering the areas. It is a perfectly definite structure : the marginal cells of the two areas do not intermingle nor do transitional cells occur between the two types.
b. The trophoblastic area
The cells of this area are very attenuated in the late unilaminar stage, but as they multiply they also thicken and increase their volume. The thickening is often most pronounced at the vegetative pole where numerous mitoses may occur (figs. 5 and 7, pi. 18). This is a matter of some interest, for in larger blastocysts this region may continue to be more thickened than the remainder of the trophoblastic area; or certain 'blisters' may occur there, such as will be described under the 1 mm. blastocyst (0, figs. 2 and 3, pi. 21).
In all stages the cytoplasm of the trophoblastic cells is very diffuse and loosely reticular, but in the early bilaminar stage especially it tends to break down into strands and ret'culum, leaving the cell membrane collapsed and wrinkled. Only around the nuclei is there a denser mass of well-fixed cytoplasm and the nuclei themselves maintain their form and structure as perfectly as in the embryonic area (fig. 2, pi. 20). Normally, the trophoblastic layer follows the curvature of the albumen layer to which it remains closely applied, as is apparent from a study of the living eggs and photographs of them and as the best preparations show (fig. 4, pi. 1; fig. 17, pi. 13). Frequently, however, the vesicle collapses more or less in this area, leaving as an artifact a space between the vesicle and the albumen (fig. 4, pi. 10; ART, fig. 1, pi. 19). At this stage the albumen seems to be most dense nearest the shell membrane and often very loosely layered near the vesicle, which would account for the more frequent breaking down of the albumen at this point in the specimens.
c. The entoderm
Soon after completely hning the blastocyst wall the entoderm is everywhere the same, passing over the junctional line without change. This condition remains so throughout the bilaminar stage, except for certain modified cells to be mentioned in connection with older blastocysts. At first the entoderm is extremely delicate so that it appears in section to be discontinuous, because in the fixing fluid portions of the cells break down. That this is the correct explanation is seen from surface views of good preparations, as in figure 11, plate 19, in which only the entodermal cells are shaded. They are seen to be connected by fibrous strands, the coagulated portions of the delicate cells. The entoderm may, therefore, be considered practically continuous. In most surface mounts the entoderm appears like a mottled surface, for the cells are thick in the middle, hence darker, and shade ofT almost into nothingness toward their edges (fig. 2, pi. 19).
The entoderm invariably stains darker than the ectoderm. It is interesting to note that an entodermal cell in mitosis is darker, often very decidedly so, than its fellows in the resting stage (fig. 2, pi. 19) ; while on the other hand an ectodermal cell is usually much lighter when in mitosis (fig. 12, pi. 19), so that in some surface views dividing cells look like holes in the wall. Yolk granules abound among the cells of the embryonic area, both ectodermal and entodermal, and occur occasionally also in the trophoblastic region.
A somewhat older egg (figs. 4 and 4A, pi. 20) is presented because of the degenerating cells included within the cavity. Such cellular remnants have been noted in apparently normal vesicles of Eutheria (Hartman, '16, pp. 46 and 47). In this egg, too, the albumen is definitely arranged in three layers of varying density, a condition noted also in a few other specimens.
The 1 mm Blastocyst
a. General description
The typical 1-mm. blastocysts contained in litter No. 343 (fig. 6, pi. 2) were removed seven and a half days after copulation. It has already been pointed out that only a small amount of albumen still remains in these eggs and the vesicle has become very nearly a perfect sphere. The embryonic area is now more sharply marked off from the surrounding trophoblast and lies like a cap at one pole of the egg (fig. 9, pi. 21). It is. in fact, occasionally in alcoholic specimens raised in relief above the surface of the ovum hke a bhster, a condition probablydue to its greater density and resistance to shrinkage as compared with the trophoblastic area.
The growing contrast between the two regions of the egg, which is now as clear-cut in sections as in whole mounts, is due to the increasing difference in the structure, as well as to the number of the formative cells. These are taller, much more crowded, and contain a denser and more granular cytoplasm, and this contrast in the types of cells is a constant character, no matter what the fixation, and the differences that exist among the specimens are those of degree only. These points are evident from an inspection of figures lA, 2A, and 4 to 7, plate 21, which were drawn as nearly as possible in imitation of the tone of the specimens.
While there is great variability in the thickness of the embryonic areas in 1-mm. blastocysts, it is true that in most cases the area has become considerably thickened as the vesicle has grown in volume and as the area- has increased in diameter (pi. 21). The cells have become mostly tall cubical to columnar and in the embryonic area are now nearly or quite as much crowded together as in older stages (compare fig. 12, pi. 19, and fig.T2A, pi. 22).
The embryonic ectoderm is arranged strictly in a single layer, never stratified or pseudostratified. The nuclei are practically on a level throughout, and this is one of the points of contrast with the blastoderm of other mammals. Mitotic spindles usually stand with their axes parallel to the surface of the egg (fig. 7, pi. 21). Frequently the cell that is in mitosis juts out above the level of the ectodermal layer (fig. 5, pi. 21). as in the blastocysts of the rabbit and other mammals, and such cells almost always stain less deeply, a fact that applies both to sections and to surface views.
The trophoblastic area has also developed more mass and thickness, proportionally quite as much as. the embryonic area (figs. 6 and 7A, pi. 21). Typically it is about 8 to 10 /^ in thickness. It is usually uniform in structure at all points and fits closely to the albumen (fig. 6), except when artificially separated from it in fixation (fig. 7A). In the 1-mm. blastocyst it will endure fixation better than in younger stages. In all cases, in contrast with the uniform granulation of the embryonic area, the trophoblastic cells are reticulated and often possess coarse meshes or appear highly vacuolated. In extreme cases, especially when fixed in aceto-osmic-bichromate, the trophoblastic area may be greatly swollen; but this may also sometimes liappen even in so reliable a fluid as Bouin's, as in figure 4, plate 22. The trophoblastic area is thus much more affected by fixation than the embryonic area.
While, as a rule, the trophoblastic area is rather uniform in thickness throughout its extent, there are frequent exceptions which deserve special mention. The area may gradually thicken toward the lower pole (fig. 4, pi. 22), or there may be a thick mass of cells jutting out into the albumen, and even touching the shell membrane at that point. In such cases the entoderm is continuous over the mass. In still other cases the ectoderm at the extreme lower pole is depressed outward into a pocket which may also come into contact with the shell membrane (0, figs. 2 and 3, pi. 21). The entoderm bridges over this cavity in a continuous layer and does not follow the ectoderm into the pocket. In the whole egg the pocket is quite evident and looks like a blister on the vesicle. These structures can scarcely have any special significance, since they are not of constant occurrence, nor are they situated at a point of special future importance.
As in both younger and older stages, the entoderm is a continuous layer lining the entire cavity of the blastocyst. It consists of a very attenuated layer of large squamous cells quite typical of the corresponding stage of all mammals. The cytoplasm is mostly gathered near the center of the cell, where the nucleus lies. In surface views the entodermal nuclei usually appear larger than the ectodermal and the chromatin granules in them are more evenly distributed. They can be recognized by this difference as well as by the depth of focus required to see them. The entoderm always has a stronger staining reaction than the ectoderm; I find no exception to this rule.
b. The bilaminar blastocyst according to Selenka
In his 'Studien' ('87) Selenka briefly describes two opossum blastocysts of about 1.1 mm. in diameter. The lithographs presented by him are idealized drawings, reconstructed from his sections, which I judge to have been considerably shrunken by the tre'atment to which they were subjected. The illustrations give the correct relation of the structures except for the diffuse junctional line, although Selenka is in error as to the homology of the 'Granulosa membran,' as he terms the shell membrane.
c. The 1 mm. blastocyst according to Minot
In 1911 the late Professor Minot published a description of six 1-mm. blastocysts of the opossum, of which two were fixed in Flemming's fluid and four in Zenker's and of the latter, two were fixed in situ with the uterus.
He gives an adequate description of the embryonic area of this stage, as did Selenka in 1887. Of especial interest, however, is Minot's description and interpretation of certain cells in the trophoblastic area. In a vesicle which he dissected and mounted flat on a slide he found numerous large light areas apparent as 'minute round holes' when viewed with a hand lens. He interpreted these areas as gaps in the ectoderm filled with entodermal cells which thus reach the surface at these points. Corroboration was found in the study of the serial sections. The author furthermore draws a comparison between these large lightly staining entodermal cells, which rise to the surface in the trophoblastic region of the opossum egg, and the small darkly staining entoderm mother cells which appear in the embryonic ectoderm of the Dasyurus blastocyst.
While the preparation of the present paper was in progress I had the privilege of studying Professor Minot's specimens at the Harvard Medical School. As I expected, the serial sections of eggs fixed and sectioned in toto with the uterus are badly shrunken and filled with coagulum in a manner which never occurs in eggs removed from the uterus and treated separately.
The specimens are unique, too, in that the entoderm is somewhat hghter in stain than the ectoderm, as described by Minot.
The surface mount is nicely fixed and is, histologically, an excellent preparation. The light areas are as described by Minot, and they are even more striking in the specimen than in his figure 2B. It is my judgment, however, that the vesicle in question is not entirely normal, for the reason that among all of my numerous specimens, I have never encountered any possessing such large light spaces. It is true that normally small lightly staining areas occur in almost all opossum vesicles of about this stage; and they usually mark the presence of cells in mitosis (figs. 12 and 12A, pi. 22), or cells that have just divided or are preparing to divide, and they are especially prominent in specimens fixed in bichromate mixtures. But they never attain such size as in Doctor Minot's unusual specimen. I have no explanation to offer of the phenomenon; I saw no evidence of degeneration of cells at those points.
Again, among all of my specimens I have looked in vain for entodermal cells coming to the surface in bilaminar blastocysts, either in surface views or sections; and I am certain that normally this does not occur. I have convinced myself, however, that also in the Harvard specimen the entoderm is nowhere at the surface and that Doctor Minot was in error in his interpretation. In the first place, by careful focusing with the oilimmersion lens the (ectodermal) nucleus within the light area is in several instance seen to be superimposed over an entodermal nucleus. Furthermore, if one plot the entodermal nuclei of the embryonic area, it is seen that they are uniformly and continuously distributed, entirely without reference to the abovementioned light areas. These areas are without doubt ectodermal and not entodermal. Hence Minot's comparison between the supposedly superficial entodermal cells in the trophoblastic area of- the opossum with the entoderm mother cells of the unilaminar blastocyst of Dasyurus is a futile one.
The entoderm, therefore, never comes to the surface in the bilaminar stage of the opossum egg. Entodermal cells in Dasyurus and in the opossum, and doubtless in all marsupials, occupy the superficial position only as undifferentiated entoderm mother cells from which all of the entoderm is destined to be formed. The formative area is, from the beginning, potentially ectoderm, entoderm, and mesoderm, giving rise first to the entoderm and later in quite a similar manner to the mesoderm, the residue becoming definitive ectoderm. The trophoblastic area consists of a single layer, the ectoderm, until lined with the entoderm arising from the embryonic area.
The Late Bilaminar Blastocyst
a. General description
Passing now to the later stages, we note that superficially the blastocyst appears to have changed but little, except in size (compare figs. 3, 5, and 6, pi. 2). The embryonic area remains prominent at the upper pole and less and less albumen remains at the lower pole. Important changes in the blastocyst wall are, however, to be discovered from a study of the sections.
In blastocysts of 1.2 to 1.5 mm. the ectoderm, both embryonic and trophoblastic, has attained its maximum thickness for the bilaminar stages. Two eggs shown in plate 22 illustrate this point. Egg No. 353 (4), shown in figures 3, 3A, 3B, 3C measured 1.22 mm. in alcohol. The embryonic area consists of tall cells, for the most part of the columnar type; the trophoblastic area is the same as in smaller blastocysts above described. Practically the same holds true for egg No. 360 (4) (fig. 6), which measured about 1.3 mm. in diameter (compare stereogram fig. 8, pi. 10). The wall of a somewhat smaller egg, No. 347' (1), 1.1 mm. in diameter in alcohol, has a somewhat thinner embryonic area (fig. 5, pi. 22). There is, however, considerable variation in this respect, even within the same litter of eggs of equal size.
In all of the larger blastocysts the entoderm can be followed as a continuous layer completely lining the vesicle. Except where pulled away in the preparations, the entoderm fits closely aigainst the ectoderm and is always distinctly recognizable at all points (fig. 6, pi. 10).
b. The central light field in the embryonic area
When the diameter of the egg approaches 1.8 mm., certain changes of importance have taken place, for in such eggs the first prohferation of mesoderm is usually observed. The eggs of litters Nos. 189' and 353' are of this size; in the last litter mesodermal cells occur and the primitive streak is faintly indicated {M, fig. 21, pi. 13); but the other litter lacks a few minutes of development to have reached this stage.
The premesodermal changes in the blastocyst are best illustrated by a typical and favorably sectioned example, namely, egg No. 193' (2), measuring about 1.4 mm. in alcohol. This is one of the two eggs shown in figures 1 and 2, plate 10. In surface view there is within the embryonic area, a large light field, more plainly visible by transmitted light. Such areas have been described for other mammalian vesicles of a corresponding stage. They are usually somewhat eccentric, sometimes very considerably nearer one side than the other. I am convinced that the point where the light field comes nearest the margin of the area markes the posterior portion of the embryonic area, and I have therefore, in figure 9, plate 22, oriented the surface view of such an egg with the posterior end down. At a slightly later stage, perhaps an hour later, the primitive streak would have appeared as a faint tongue-shaped clouding projecting upward from the lower margin of the formative area into the light field in question, as in the eggs of litter No. 353', to be described in the next number of these studies.
The light field is due entirely to a thinning of the embryonic ectoderm {T, fig. 9A, pi. 22), a condition already seen in small eggs in figure 6, where at T the area is palpably thinner than at either end of the section. At T, figures 4A and 7, the sections also pass favorably to show this central thinner field. In this region, too, the nuclei are usually farther separated, whereas at the margins they are so crowded as to form a continuous chain like a string of beads, although not quite so uniformly arranged.
c. Modified entodermal cells
In many of these larger eggs the entoderm undergoes sKght differentiation at one point. Over the junctional line which marks the border of the embryonic area there is often a group of entodermal cells which attract attention by virtue of their number, the roundness of their nuclei and the volume of the cytoplasm (ENT, fig. 9A). They are sometimes found in eggs of 1 mm. (ENT, fig. 5, pi. 21), more often in larger blastocysts (ENT, figs. 4A, 6 and 7, pi. 22). Similar entodermal cells have been described by Van Beneden for the bilaminar blastocyst of the rabbit. He states that they mark the anterior end of the area and that the future primitive streak appears at the opposite side. In the preparations made from litter No. 353', in which there occurs the first anlage of the primitive streak, these cells appear in the region where the mesodermal cells are found, hence not in the anterior, but in the posterior portion of the embryonic area. I shall treat this subject further at a later date.
d. The ectoderm of late bilaminar blastocyst
As was stated in the preceding section, the entoderm attains its maximum thickness in vesicles of about 1.3 mm. diameter (figs. 3 A and 3B, pi. 22). Larger vesicles may have thinner formative areas or they may remain about the same, although they are apparently more slender because of their length in sections. In some of the eggs, especially in litters Nos. 193' and 343', the trophoblastic areas are as greatly attenuated as in the 0.8-mm. stage (figs. 1 and 9A, pi. 22). Sometimes the trophoblastic area becomes gradually thicker towards the lower pole (fig. 4, pi. 22), or there may be ectodermal pockets or 'blisters' at this point, as described above in connection with the 1-mm. stage.
As seen in surface view, the distribution of cells is practically the same as in the 1-mm. blastocysts (figs. 12A and 12B, pi. 22). All of the cells of the embryonic ectoderm are crowded closely together and are darker than the trophoblastic cells because they are thicker and uniformly granular, but the cell boundaries are not as apparent as those of the large flat trophoblastic cells. The nuclei of the latter are flatter, but of uniform roundness, unchanged by mutual pressure, and possess fewer chromatin granules and larger light spaces than the embryonic nuclei, otherwise the nuclei of the two areas are very much ahke. The entodermal nuclei, as a rule, appear larger in surface view than those of the ectoderm and they possess a more uniform granulation.
It should be noted that the embryonic ectoderm is still a single layer of cells with the nuclei mostly at nearly the same level. In the corresponding stage of other mammals the embryonic area is considerably thickened as in a pseudostratified epithelium (rat, bat). This simple arrangement has the decided advantage for the observer in that the very first mesodermal nuclei which drop down out of the ectoderm may be located instantly and with certainty.
e. Yolk spherules in ectoderm and entoderm
In many surface views of bilaminar blastocysts round dark objects, usually as large as a nucleus or smaller, frequently meet the eye. Sometimes these bodies stain like the cytoplasm, or they may be much darker in preparations fixed in osmic acid. They are found in both ectoderm and entoderm (figs. lA and 2A, pi. 21; fig. 10, pi. 22), sometimes free, sometimes within the cytoplasm and partly enveloped by the nucleus. The inclusions are often surrounded by a light zone as though they were partly digested and absorbed (fig. 3B, pi. 20) ; indeed, vacuoles, instead of solid masses, in similar situations are not uncommon (F, figs. 11 and 12B, pi. 22).
Blastocyst No. 189' (12) is worthy of special notice. It appeared to be normal in every respect, and the embryonic area, which measures 0.75 mm., was dissected off and stained and mounted intact in balsam. The interesting feature of this vesicle is the large number of these dark bodies that are mostly observed in connection with the entodermal cells. The majority of these cells underlying the embryonic area are each provided with large or small masses, about which the nucleus lies as if about to engulf it. In figure 11, plate 22, are several typical cases drawn with the aid of the camera lucida. At A two bodies are found in connection with a single nucleus; and a similar case is seen in section of a somewhat younger blastocyst at A, figure 10. At B, figure 11, the entodermal cell behaves toward a vacuole as toward a solid mass, a phenomenon by no means rare. The large cell at C seems to have completely ingested a mass, the body in the center being not a nucleolus, but a typical inclusion like those marked Y in the other figures.
I have looked through the whole series of stages from the first appearance of entoderm to the largest bilaminar blastocyst and find that the foreign bodies just described are present in nearly all cases, whether in total preparations or in serial sections. I am convinced that they are only remnants of undigested yolk. If one recall the young blastocyst containing 40 or 50 entodermal cells (litter No. 356) at a stage when the trophoblast has become considerably attenuated (pi. 17) one notes that the included yolk is almost entirely confined to the embryonic area. So in succeeding stages, numerous granules of yolk are found among the embryonic, seldom within the trophoblastic, cells. More such granules are, with some exceptions, found in the younger than in the older blastocysts. Thus, while the albumen melts away before the embryonic area of the young bilaminar blastocysts, the yolk granules maintain their identity in small rounded masses for -a longer time.
f. Mesoderm formation initiated
With the appearance of the first mesodermal cells about six days have elapsed since ovulation, about five days since the beginning of cleavage, or about one-half of the period of gestation, which I am tentatively stating to be ten days in the opossum. The formation of the mesoderm will be treated in the next number of these studies.
Summary
1. Several thousand eggs were removed from several hundred pregnant and pseudopregnant animals during the collecting seasons 1914 to 1917, an average of 11.5 per litter, or 23 from each animal.
2. At least one-third of the average litter of eggs are unfertilized or abnormal (table 1).
3. Six hundred and forty-one normal eggs, from tubal ova to the bilaminar blastocyst, form the basis of the present study.
4. Collection of embryological material from the opossum has become greatly facilitated because of the discovery that the mammary glands of this animal hypertrophy at the approach of ovulation, so that the sexual condition of the female may be predicted with a high degree of certainty, without sacrificing the animal or without loss of time and effort, by simple though trained and practiced palpation of the glands. But the behavior of the mammary glands as well as the other reproductive organs is the same in the early stages of pseudopregnancy and in pregnancy. Ovulation is always spontaneous.
5. A series of photomicrographs of eggs in the living state is presented in the plates 1 to 11.
The development of ten litters for given periods of time is shown in plates 1 and '2 and in figures 1 and 4, plate 9.
Plates 12 and 13 are intended to serve as a resume of the stages covered by the present study.
The development of the opossum egg is illustrated in one series by the photographic plates 3 to 10 and in another series by the drawings plates 14 to 22.
Six hundred different opossum eggs are shown, including 240 illustrations of some 180 different preparations.
6. The rate of development was determined in a number of cases in which the eggs of the right uterus were allowed to develop a given period of time after the removal of the left uterus and its contents. This method contributed in no small part to the success in securing an unbroken series of stages.
7. The stages covered in this paper comprise about the first half of the ten-day period of gestation. More exact figures have not as yet been worked out.
8. The first polar body is given off in the ovary, the second in the Fallopian tube as in other mammals (pi. 14).
9. In the Fallopian tube much albumen and the shell membrane are added to the ovum. It probably requires about twenty-four hours for the passage of the egg to the uterus.
10. The haploid number of chromosomes in the opossum is twelve (pi. 14).
11. At no stage in the unsegmented egg is there any evidence of polarity in the distribution of the yolk, as in Dasyurus, the bat and some other mammalian eggs, although in the opossum the yolk is abundantly present (pis. 13 and 14).
12. The egg varies considerably in size, but on the average it is about 0.12 mm. in diameter through the o\aim and 0.6 mm. through the shell membrane. Some normal eggs attain the diameter of 0.73 mm., owing to the larger amount of albumen deposited (compare fig. 2, pi. 4, and fig. 3, pi. 5).
13. As in probably all marsupials, the egg reaches the uterus unsegmented, hence at an earlier stage than in any of the Eutheria.
14. The pronuclei at first occupy a yolk-free area at the periphery of the egg; then migrate to the yolk-free central portion, where the first cleavage spindle is later to be seen (figs. 20 and 21, pi. 14.).
15. Deutoplasmolysis or elimination of yolk begins at the pronuclear stage, continues at the 2-celled stage, and reaches its maximum during the second cleavage (pis. 3 and 15).
16. The quantity of yolk and surrounding cytoplasm extruded varies greatly, hence the size of the blastomeres varies in inverse ratio to the extent of deutoplasmolysis (pi. 15).
17. Deutoplasmolysis occurs by elimination of masses of various sizes on all sides of the egg, not at any particular spot or pole, as in Dasyurus and the bat (fig. 4, pi. 3).
18. The two blastomeres of the 2-celled stage are usually of the same size, shape and 'structure, or they may differ in size. This difference is probably due chiefly to the difference in the amount of yolk extruded (text fig. 4).
19. One blastomere sometimes anticipates the other in division, and as a result 3-celled eggs are found, but not nearly in as large numbers as eggs in the 4-celled stage.
20. The second cleavage plane is at right angles to the first and the spindles in the two cells lie parallel ; but the shifting of the blastomeres soon begins, so that in the 3-celled stage the crossed arrangement may already be attained (K and L, text fig. 4).
21. The crossed arrangement of the blastomeres in the 4-celled egg is, therefore, in the opossum not due to the direction of the cleavage planes in the second cleavage, but is secondarily caused by the shifting of the blastomeres (compare B and D, text fig. 4).
22. The shifting of the blastomeres is not due to mutual pressure, for in many 4-celled eggs the blastomeres are very small and not even in contact (figs. 6 and 7, pi. 3).
23. There is no morula stage in the marsupials. The blastocyst cavity is virtually present in the 4-celled egg as the space between the blastomeres. In the 16-celled stage, or earlier, in the opossum, the blastomeres have migrated to the periphery and have applied themselves to the zona pellucida. The structure of the blastocysts is clearly indicated. The extruded yolk now lies within the cavity (pis. 4 and 15).
24. The blastocyst wall is usually fully formed at about the 32-celled stage, when all the gaps between the cells are closed by the flattening and multiplication of the cells of the late cleavage stage. This marks the end of cleavage as such, which requires nearly thirty hours of development (fig. 1, pi. 16).
25. During cleavage the only evidence of polarity lies in the difference in the rate of division among the cells at the two poles. The more rapidly dividing cells are probably embryonic and arise from one of the first two blastomeres (text fig, 5).
26. -Definite polarity is established at about the 60- to 70celled stage with the first appearance of the entoderm. One litter at this stage was found six days after copulation, doubtless a case of retarded ovulation, as a later stage was to have been expected (pi. 16).
27. The entoderm arises from entoderm mother cells of very characteristic appearance. They are cells in the blastocyst wall which round up and usually roll out of their place, as it were, into the blastocyst cavity, as in certain invertebrates, or they may remain attached to the wall for some time, in either case multiplying by mitotic division (pis. 7 and 16).
28. The entoderm mother cells all arise from one-half of the egg, the future embryonic area (figs. 15 to 22, pi. 16).
29. The area that remains free of entoderm mother cells is the trophoblastic area; it soon begins to thin and spread out so that the growth of the ovum now begins. Growth is, therefore, at first due to the spreading of the trophoblastic area (pis. 16 and 17).
30. Since the entodermal cells spring from the superficial epithelial layer in the embryonic area, this would better be termed embryonic entectoderm.
31. When the blastocyst has attained a diameter of 0.3 to 0.35 mm., the entoderm is several cells deep, being crowded into a mass which superficially somewhat simulates an Eutherian inner cell mass in the process of spreading. In the opossum only the superficial cells are embryonic ectoderm, all the rest are entodermal (figs. 13 to 15, pi. 17).
32. Such a stage was removed from an animal four days after copulation, or about a day and a half after the beginning of cleavage.
33. The superficial layer of cells is never transitory; it is embryonic ectoderm and not Rauber's layer; it is in active state of mitosis throughout. Rauber's layer is homologous with the non-embryonic or, as Hill has expressed it, the 'trophoblastic' area.
34. The proliferation of entoderm is at an end when the blastocyst has attained a diameter of 0.45 to 0.5 mm., when the trophoblastic area has attained its greatest degree of attenuation (pi. 18).
35. The entoderm now spreads by an active migration of the flattened, definitive entodermal cells toward the opposite pole of the egg (pi. 18).
36. When the spreading is well under way, the blastocyst, previously spherical and centrally placed, is usually flattened like a thick biconvex lens at one pole of the egg, with the embryonic area in contact with the shell membrane (figs. 1 and 2, pi. 6; fig. 4, pi. 10; pis. 12 and 19).
37. Eggs in which the entoderm has just become closed at the lower pole, and which are thus in the beginning of the bilaminar stage, are still about the same size as in the cleavage stages, but the albumen has become more dense and the shell membrane thicker and more resistant (figs. 14 and 17, pi. 13). The albumen disappears with the growth of the blastocyst and egg (pi. 13).
38. The bilaminar blastocyst is simply a double-walled sac consisting of ectoderm without and entoderm within. The two layers are closely applied to each other and to the shell membrane and albumen, and any variation from this condition is due to shrinkage or to abnormality of the egg. There is no 'peri vitelline' space in the normal opossum egg, but frequently occurs in abnormal material (pis. 19 and 20).
39. The bilaminar stage is the period of growth, little differentiation occurring until near the first appearance of mesoderm in vesicles 1,5 to 1.8 mm. in diameter (pis. 19 to 22).
40. The 1-mm. stage was once found seven and one-half days after copulation (litter- No. 343, fig. 5, pi. 2) ; the mesoderm first appears about eight hours later (compare litters 343', 346, 346', 353, 353') ; the 0.8-mm. stage was once removed about five days after the beginning of cleavage (litter No. 306', fig. 17, pi. 13; figs. 1 and lA, pi. 21), and 1.4-mm. blastocysts were found at about four and a half days after the beginning of cleavage (compare litters Nos. 191 and 193, figs. 1 and 9, pi. 22).
41. The embryonic area grows in extent with the growth of the egg, so that in the later bilaminar stage its diameter is about one-fifth to one-fourth of the circumference of the egg (compare pi. 18 and figs. 1 to 3, pi. 21, with figs. 1 to 4, pi. 22).
42. As the egg develops, the embryonic area becomes increasingly more sharply set off from the trophoblastic area (fig. 9, pi. 21). The embryonic ectoderm becomes thicker, the cells cubical to columnar and more densely granular, whereas the trophoblastic ectoderm remains flat and its cytoplasm reticular (pl. 21).
43. The entoderm in the early stages is everywhere the same, consisting of the typical squamous cells with swellings at the nuclei. In surface view the flatter entodermal nuclei, as a rule, appear larger than the ectodermal. The entoderm nowhere comes to the surface of the blastocyst (pis. 10 and 19 to 22).
44. In blastocysts over 1 nam. in diameter the entoderm is often modified at one side of the embryonic area. These cells increase in number, thickness of nuclei, and density of the cytoplasm, and I believe them to mark the future posterior, not the future anterior margin of the embryonic area. The primitive streak will be laid down here {ENT, fig. 9A, pl. 22).
45. These eggs also exhibit a clear field a little to one side of the center in the embryonic area (figs. 1 and 2, pl. 10; fig. 9, pl. 22). This is due to a thinning out of the embryonic ectoderm. Where the light field comes nearest to the margin is the posterior margin of the area, for here the modified entoderm is also found (pl. 22). • _
46. Yolk spherules occur in the bilaminar stage even in the largest specimens. They are remnants of the extrusions of cleavage stages. They are found often within the cells, usually of the embryonic area only, both ectodermal and entodermal, and the nuclei frequently surround the masses as if to engulf them (figs. 9B, 10 to 12, pl. 22).
47. The egg of the opossum is like that of Dasyurus in its possession of a large amount of yolk, in the absence of the morula stage and in the formation of entoderm from entoderm mother cells coming from the wall of the unilaminar blastocyst. But it differs in many regards: in the absence of polarity, in the uniform distribution of the yolk, and in the consequent manner of deutoplasmolysis ; in the indeterminate type of cleavage; in the crossed arrangement of the 4-celled egg; in the early period in which the blastocyst is formed; in the very early formation of the entoderm; in the simulation of an inner cell mass due to the crowding of the primitive entoderm cells, — in these respects
the opossum egg is much more Eutherian than Dasyurine. Of these perhaps the most striking feature is very early differentiation of the entoderm. Since in other marsupials, according to Hill, the entoderm is formed early (Macropus, Perameles) it seems probable that when the other marsupials have been more thoroughly studied it will be found that the opossum is more typical of the marsupials in general and that Dasyurus represents a more primitive, albeit, therefore, an even more interesting form.
Addendum
Since the completion of my manuscript there has come to hand Prof. J. P. Hill's paper on "The Early Development of Didelphys aurita," published in the April, 1918, number of the Quarterly Journal of Microscopical Science. This work is based on eggs secured from six females: one animal furnished unsegmented, unfertilized eggs; another numerous 2-, 3-, and 4-celled eggs; from two others, cleavage stages of 4 to 16 cells were taken, and from two animals bilaminar blastocysts about 1 mm. in diameter.
It appears from this contribution that the developmental stages of the South American opossum, so far as Professor Hill's material goes to show, are closely duplicated by my own specimens of the local species. The same is true of a number of somewhat later stages (primitive streak) which I have secured from the small black D. marsupialis occurring in south Texas and Mexico.
In most respects my own work finds full corroboration as well as interesting extensions in the careful study made by Professor Hill. I wish briefly to refer to several points discussed by our able British colleague.
In his analysis of the 2-, 4-, 8-, and 16-celled stages, he presents additional evidence of polarity in the opossum egg; for he finds that in a large proportion of such eggs the cells are plainly made up of two groups, differing somewhat in size, the smaller cells being considered by Hill as constituting the upper or formative pole of the egg. He also finds that the majority of eggs in later cleavage show an accelerated rate of division at one pole. The evidence of polar differentiation in the opossum egg throughout cleavage seems, therefore, to be complete. Professor Hill recognizes fully the difference between the cleavage of the opossum and of Dasyurus and joins me in deriving the formative and the non-formative areas each from one of the two blastomeres of the 2-celled egg.
As to the method of deutoplasmolysis, Hill considers that" the yolk spheres are budded off from a narrow, clear zone which has made its appearance at the exposed surfaces of the blastomeres" and his "figures shown undoubted yolk spheres in direct continuity with the lighter peripheral zone." I have noted the same phenomenon, although never as pronounced as in the cases illustrated by Hill in his figures 11 to 13, plate 8. In most of my hundred specimens, the smooth, unwrinkled cell membrane can be followed clearly around the blastomeres. Professor Hill is correct in assuming that the egg No. 50 (6) upon which I based my former conclusion on the method of yolk elimination (Hartman, '16, page 23, and fig. 9, pi. 5) is probably not quite normal; in fact, the specimen was considerably retarded in development as compared with its fellows in the 50 to 70-celled stage. Such retarded eggs are always to be regarded with suspicion. I therefore no longer regard yolk elimination as due to the "formation of a new cell membrane, .... at a distance from the original surface of the blastomeres," but believe with Professor Hill that masses of variable size are extruded from different places on the exposed* surfaces of the blastomeres.
On the origin of the crossed arrangement of the blastomeres in the 4-celled egg. Hill presents evidence, which taken by itself, would be conclusive of the fact that the blastomeres do not attain this position by shifting, but assume it from the beginning by virtue of a meridional division of one blastomere and an equatorial division of the other. For out of his ten 2-celled eggs, five have both blastomeres in the process of division and in these the axes of the blastomeres are already nearly or quite at right angles to each other. Three of these eggs have completed their nuclear division, the nuclei being in the resting stage; the other two are in late anaphase.
However, two of my own specimens, both of which contain short spindles in each blastomere, cast doubt upon Hill's view as stated above, with which I had agreed before I came into possession of the eggs (from No. 306). For in one of these eggs the spindles are exactly parallel; in the other they deviate 36° from the parallel.
It is, therefore, apparent that, unless we assume a rapid shifting of the blastomeres during the early phases of the second cleavage, the matter must for the present remain in doubt.
It is interesting to note that D. aurita has two breeding seasons a year, whereas D. virginiana has but one.
Professor Hill states it as his behef that twelve is the reduced number of chromosomes in the opossum, and with this I fully agree.
Literature Cited
Caldwell, W. H. 1887 The embryology of Monotremata and Marsupalia,
Part I. Phil. Trans. Soy. Soc, vol. 178 B. Hartman, Carl G. 1916 Studies in the development of the opossum, Didel phys virginiana L., Parts I and II. Journ. of Morph., vol. 27. Hill, J. P. 1910 The early development of the Marsupalia, with special reference to the native cat (Dasyurus viverrinus). Quart. Jour. Micr.
Sci., vol. 56. MiNOT, Charles R, 1911 Note on the blastodermic vesicle of the opossum.
Anat. Rec, vol. 5. Patterson, J. P. 1913 . Polyembryonic development in Tatusia novemcincta.
Journ. of Morph., vol. 24. Selenka, E. 1887 Studien in der Entwicklungs geschichte der Thiers, Band
4, Das Opossum (Didelphys virginiana). Wiesbaden. Spurgeon and Brooks 1916 The implantation andearly segmentation of the ovum of Didelphys virginiana. Anat. Rec, vol. 10. Van DER Stricht 1909 La structure de I'oeuf des Mammiferes (Chauve-souris, Vesperugo noctula): Troisieme Partie., Mem. de I'Acad. roy. de Bel gique, lie ser., t. 2.
Plates 1 to 11 contain 82 figures nearly two-thirds of which are from photographs of living eggs. Plates 1 and 2 show the development of nine litters for given periods of time. Plates 3 to 10 are arranged by stage of development, and the same stages are shown in drawings on plates 14 to 22. Plate 12 was drawn from 8 specimens as they appeared in the living state. Plate 14 is a resume of the stages covered in this paper as drawn from sections.
Plates
PLATE 1
EXPLANATION OF FIGURES
Photomicrographs of living eggs in Ringer's solution ; the ten figures show two litters from each of five different animals; figs. 1 to 9 X 8; fig. 10, natural size.
1 Litter No. 320; 4-celled eggs.
2 Litter No. 320'; interval 5i days; primitive-streak stage.
3 Litter No. 299; 4-celled eggs.
4 Litter No. 299'; interval 4 days, 3| hours; blastocysts partly bilaminar.
5 Litter No. 292; young unilaminar blastocysts containing 40 to 50 cells.
6 Litter No. 292'; interval 4 days; primitive-streak stages.
7 Litter No. 307; tubal ova with a little albumen on one side.
8 Litter No. 307'; interval 5| days; unfertilized, fragmenting eggs; albumen rather opaque.
9 Litter No. 337; 8- to 16-celled eggs.
10 Litter No. 337'; vesicles with primitive streak and very short medullary groove; photographed in open uterus, natural size.
GENERAL ABBREVIATIONS
A, designates particular portions of ENT"^, undifferentiated primitive en
various drawings to which reference todermal cells (pis. 17 and 18)
is made in the text ENT^, flattened definitive entodermal
.4LS, albumen cells (pi. 16)
ARl , artifact qj^ granulosa cells of discus proligerus
^' , 0, 'blister' in trophoblastic ectoderm
CH, chromosomes
EMB.A, embryonic area ^^' Polar body
EMB, ECT, embryonic ectoderm or SM, shell membrane
entectoderm TR.A, trophoblastic or non-embryonic ENT, definitive entoderm; in plate 18 ^j-ea
it designates the limit of spread of rj.^ ^rp^j^ trophoblastic ectoderm
entoderm; in plates 21 and 22 it re- y ya^^uole
fers to certain specialized entodermal
cells
X, placed at limits of embryonic area
ENT^, entoderm mother cells in wall ^^
of blastocyst (pis. 16 and 17) ^^^ embryonic area of sections, junc ENT\ entoderm mother cells that have tional line of surface views
migrated into cavity of blastocyst Y, yolk masses (pis. 16 and 17) ZP, zona pellucida
PLATE 2
EXPLANATION OF FIGURES
Photomicrographs of living eggs; in Ringer's solution by reflected light; in the eight figures two litters are shown from each of four animals; all figures X 8, except fig. 8, which is X 2.
1 Litter No. 293; 4-celled eggs.
2 Litter No. 293'; interval So days; young bilaminar blastocysts.
3 Litter No. 346; bilaminar blastocysts.
4 Litter No. 346'; interval Of hours; early primitive-streak stage.
5 Litter No. 343; 1 mm., bilaminar blastocysts.
6 Litter No. 343'; interval 7 hours and 20 minutes; bilaminar blastocysts just preceding first proliferation of mesoderm.
7 Litter No. 298; unilaminar blastocysts of 60 to 120 cells with entoderm mother cells.
8 Litter No. 298'; interval 3| days; vesicles in opened uterus; primitive streak and short m.edullary groove. X 2.
PLATE 3
EXPLANATION OF FIGURES
Photomicrographs of tubal ova and early cleavage stages in uterine eggs.
1 Two eggs of litter No 313, photographed in the living state in Ringer's solution by reflected light; considerable albumen has been deposited. X 130.
2 Litter No. 351', photographed in Ringer's solution by transmitted light; a small ring of albumen is seen. X 56.5.
3 Section throught ovum No. 313 (4); 10th section (total in .series 19); polar body shown above; the albumen is darkly stained with Delafield's haematoxylin; yolk stained with osmic acid; Hill's fluid; S/x. X 200.
4 2-celled ovum No. 306 (2); 10th section (total 21); compare C and D, text fig. 4; Hill's fluid; 5m. X 200.
5 4-celled ovum No. 173 (5); 11th section (total 19); aceto-osmic-bichromate; 5m. X 200.
6 and 7 Sections 9 and 14 through ovum No. 299 (5) showing four small blastomeres and much extruded yolk; Hill's fluid; 5m. X 200.
8 14-celled ovum No. 193 (6) ; 8th section (total 17) ; two large cells (of which one is cut longitudinally in section) have undergone nuclear division; acetoosmic-bichromate. X 200.
PLATE 4
EXPLAXATIOX OF FIGURES
Late cleavage stage as illustrated by litter No. 336.
1 The living eggs as photographed in Ringer's solution by transmitted light. X 36.
2 Two eggs of the same litter; the peripheral arrangement of the blastonieres is apparent. X 82.
3 32-celled ovum No. 33G (5); blastocyst still incomplete; 9th section (total 20); Fleraming; 5m. X 200.
4 30-celled ovum No. 336 (4); incomplete blastocyst; section taken through middle of ovum (about 19 sections); Flemming; o/x. X 200.
5 The eggs of litter No. 336 photographed by reflected light in Ringer's solution. X 8.
PLATE 5
EXPLANATION OF FIGURES
Photomicrographs of late cleavage stages; all figures except 2 and 5 were photographed in the living state in Ringer's solution; figs. 1 and 7 by reflected light; figs. 3 and 4, by transmitted light.
1 Litter No. 314. X 8.
2 Ovum No. 314 (2); 13th section (total 19); 28 cells in the incomplete blastocyst wall and 2 cells in cavity, of which only one cell is shown; the cells are highly vacuolated; Bouin; 5,u. X 200.
3 Several eggs from litter No. 337; about 16-celled stage; the peripheral arrangement of the blastomeres is well seen; compare fig. 4, below, and fig. 9. pi. 1. X 82.
4 Litter No. 337; one egg with the albumen has been removed from its shell membrane. X 36.
5 Several eggs in cleavage 'stages and young blastocysts, stained in Delafield's hae;iiatoxylin and photographed in oil of wintergreen; the albumen is darkly stained in some cases; see accompanying text fig. 6 for key. X 16.
PLATE 6
KXPLAXATION OF FIGURES
Mostly uuilanunar blastocysts.
1 Group of young unilaniinar to early bilaminar blastocysts photographed in oil of wintergreen; fixation mostly by solutions containing osmic acid; for identification of individual eggs see accompanying illustration, text fig. 7. X 16.
2 Group of blastocysts stained in Delafield's haematoxylin and cleared in oil of wintergreen; Bouin; see accompanying illustration, text fig. 8 for key. X 16.
3 Section 13 through ovum No. 88 (7), reconstructed in fig. 21, pi. 16; 18 sections in series; 87 cells, including 5 entoderm mother cells; Hill's- fluid. X 200
4 Just completed 32-celled blastocyst No. 314 (5), 9th section (total 19 sections); Hill's fluid;oM; compare fig. 1, pi. 5, and fig. 2 above. X 200.
5 Litter No. 292, photographed alive in Ringer's solution. X 8.
6 Section 10 through the 46-celled. incomplete blastocyst No. 292 (4), also seen in fig. 2 aVjove; 19 sections in series; no entodermal cells; Hill's fluid; o^u. X 200.
7 and S Ovum No. 29S (1); fig. 7, whole egg, X 30, in alcohol (compare fig. 7, pi. 2); fig. 8, 6th section showing column of entoder.n mother cells; 21 sections in series; 64 cells, including 4 entoderm mother cells; Hill's fluid; 5^- X 200.
PLATE 7
EXPLANATION OF FIGURES
Mostly blastocysts with entoderm mother cells.
1 Portion of 13th section through ovum No. 50 (5), showing its only entoderm mother cell; total 21 sections; 65 cells; Bouin; 5^. X 500.
2 Portion of 13th section through ovum No. 88 (17), taken as indicated by parallel lines on fig. 18, pi. 16; characteristic entoderm mother cell is shown; total 18 sections; 103 cells, of which 6 are entoderm mother cells; Hill's fluid; 5m. X 500.
3 Section through 6 of the 9 entoderm mother cells of ovum No. 50 (4); 16th section (total 20 sections); 67 cells; Hill's fluid; 5m- X 200.
4 The 15th section through ovum No. 88 (16), of which the 11th section is shown in fig. 11, pi. 16; total 20 sections; large entoderm mother cell is shown in blastocysts wall; 82 cells, including 10 entoderm mother cells; Bouin; Sm- X 500.
5 23-celled incomplete blastocyst No. 173' (7); 13th of a total of 23 sections; aceto-osmic-bichromate; Sm- X 200.
6 The 12th section through ovum No. 88 (3), showing several entoderm mother cells and much yolk and coagulum; 23 sections in series; 69 cells, of which 10 ar« entoderm mother cells; Bouin; 5m- X 500.
PLATE 8
EXPLANATION OF FIGURES
Photomicrographs showing progress in entoderm formation and polar differentiation; figs. 1, 3, 5, and 6, photographed in the living state in Ringer's solution by transmitted light.
1 An egg of litter No. 356, showing opaque embryonic area and thin trophoblastic area. X 82.
2 Blastocyst No. 356 (7); 16th section (total 32); Flemming 5m; X 200; compare with fig. 1.
3 One of two identical eggs from litter No. 349; opaque embryonic area to the left; compare fig. 12, pi. 17. X 36.
4 Egg No. 349 (5), the least developed egg from litter No. 349; polar differentiation is well under way; retarded in development as compared with fig. 3; 14th section (total 25); Bouin, 5^. X 200.
5 Egg No. 344 (7), lateral aspect, as seen alive; the longitudinal section of this egg is shown in fig. 7. X 82.
6 Egg No. 344 (8), as viewed Avith opaque embryonic area uppermost. X 82.
7 Egg No. 344 (7), in section, seen alive in fig. 5; 10th section (total 23); Flemming; 5 pi. X 500.
PLATE 9
EXPLANATION OF FIGURES
Photomicrographs of late unilaminar and young bilaniinar blastocysts; figs. 1, 4, 5, and 6, photographed alive in Ringer's solution; the first by transmitted light, X 36, the last three by reflected light, X 8.
1 Litter No. 352, blastocysts with bilaminar embryonic area (indicated by dark region at one pole of the vesicle); trophoblastic area very attenuated; compare fig. 5, pi. 12.
2 Section through embryonic area of egg No. 352 (11); 44th section of egg (total 96); 26th section of blastocyst (total 66); 16th section of embryonic area (total 34); entoderm has begun to migrate beyond area; .half-strength Bouin; 5^. X 100.
3 Section 12 of ovum No. 356 (5), of which sections 13 and 18 are shown in figs. 10 and 11, pi. 17; total 21 sections; Bouin; 6m. X 500.
4 Litter No. 352'; interval 15 hours; compare fig. 1; young bilaminar blastocysts; one egg has two blastocysts.
5 Litter No. 347; partially and entirely completed bilaminar blastocysts. '
6 Litter No. 339, a little younger than litter No. 347, shown in fig. 5.
PLATE 10
EXPLANATION OF FIGURES
Photomicrographs of bilaminar blastocysts.
1 and 2 Two eggs of litter No. 193', photographed unstained in alcohol by transmitted light; note light field near center of embryonic area; compare fig. 9, pi. 22. X 30.
3 1.15-mm. blastocyst No. 360 (5), stained in Delafield's haematoxylin, cut in two horizontally and photographed in alcohol by transmitted light. = 16.
4 Egg No. 352' (10), one of the litter shown in fig. 4, pi. 9; 46th section of vesicle (total 92); 29th section of embryonic area (total 54); section is oriented with embryonic area to left; Hill's fluid; 5^. X 100.
5 Egg No. 299' (6); one of litter shown in fig. 4, pi. 1; shown in toto in fig. 1, pi. 6; 61st section of vesicle (total 120); 41st section of embryonic area (total 81); the entoderm has not quite reached the lower pole; Hill's fluid; 5m. X 100.
6 Section of egg No. 360 (5) shown in surface view in fig. 3; section is oriented with embryonic area uppermost; a little albumen is left at lower pole; 55th section of embryonic area (total 121). X 100.
7 Litter No. 306'; interval 5 days, 20§ hours, after beginning of cleavage; photographed alive in Ringer's solution. X 8.
8 Stereogram of ova No. 360 (7), (S), and (9); photographed in alcohol; embryonic area seen in two of the eggs; Zenker. X 6.3.
PLATE 11
EXPLANATION OF FIGURES
Photomicrographs taken alive by reflected light in Ringer's solution; except fig. 2. Figs. 1 to 4, bilaminar blastocysts; figs. 5 to 10, unfertilized eggs. Magnification, X 8, except fig. 5.
1 Littsr No. 294, mostly abnormal blastocysts with still largely unilaminar walls (compare fig. 2, pi. G).
2 Part of litter No. 290', photographed a few minutes after immersion in Hill's fixing fluid; embryonic area is well seen.
3 Litter No. 290'; bilaminar blastocysts (compare fig. 2, pi. 6).
4 Litter No. 294' (interval 34^ hours; compare fig. 1); mostly abnormal bilaminar blastocysts.
5 Litter No. 415; unfertilized eggs in early stage of fragmentation; shows false '1-celled,' '2-celled,' and '4-celled' eggs.
6 Litter No. 318; early eggs in fragmentation; note that the ovum proper is no longer spherical.
7 Litter No. 303, with opaque albumen and fragmenting ova.
8 Litter No. 297; old fragmenting eggs with white concretions on shell membrane.
9 Four degeneration eggs that accompanied foetuses one day before birth; Litter No. 321'.
10 Litter No. 332; degenerating eggs nine or ten days old.
PLATE 12
EXPLANATION OF FIGURES
Resume of stages in the development of the opossum eggs from cleavage to the completed bilaminar blastocysts; drawn from actual specimens cleared in oil of wintergreen, the measurements being made from photographs of the living egg. X 50.
1 The unsegmented uterine egg.
2 The 4-celled ovum.
3 The just completed unilaminar blastocyst of about 32 cells.
4 Blastocyst with polar differentiation well under way; primitive entoderm present; drawn after No. 356 (6) (compare fig. 2, pi. 6).
5 Blastocyst with attenuated unilaminar trophoblastic area, bilaminar only in the embryonic region (after litters Nos. 194', 175', and 352).
6 More advanced blastocyst with spreading entoderm; after No. 290 (4), photographed in fig. 2, pi. 6; the flattened shape of the vesicle is the usual one at this stage.
7 Similar stage with more unusual spherical blastocyst, drawn after No. 299' (2), shown photographically in fig. 2, pi. 6.
8 Completed bilaminar blastocj'st, drawn after No. 299' (1), shown photographically in fig. 2, pi. 6.
PLATE 13
EXPLANATION OF FIGURES
Resume of stages in the development of the opossum egg as drawn from sections of representative specimens from the ovarian egg to the primitive streak stage. X 50.
1 Ovarian egg from specimen No. 28; Hermann's fluid.
2 to 5 Respectively the following tubal ova: No. 76 (S), Hill's fluid; No. 56 (4), Bouin's; No. 351 (1); Hills; No. 313' (1), Bouin's.
6 Unsegmented uterine egg No. 287 (1) ; Hill's fluid; 5 ix.
7 2-celled ovum No. 203 (4); thirteenth section (total 20); 5 ju.
8 4-celled ovum No. 203 (5); tenth section (total 21); Hill's fluid; 5 ti.
9 14-celled ovum No. 193 (6); eighth section (total 17); aceto-osmicbichromate.
10 Just completed blastocyst No. 191 (2); eleventh section (total 18); 32 cells; Boiiin; 6 yu.
11 63-celled blastocyst No. 50 (8), of which one of the two entoderm mother cells is shown in fig. 5, pi. 16; ninth of 17 sections; Hill's fluid.
12 Egg No. 356 (4), the seventeenth section (total 25); compare figs. and 7, pi. 17; 6 IX.
13 Blastocyst No. 194' (3); ninth section of vesicle (total 34); fifth section of embryonic area (total 13); aceto-osmic-bichromate (?); 5 fx.
14 No. 352 (7); detail in fig. 8, pi. 21, q. v.
15 Egg No. 339 (5); thirtieth section of blastocyst (total 65); not perfectly normal; one-half strength Bouin; 5 /x.
16 No. 299' .(5), shown in toto in fig 1, pi. 6; sixty-second section of egg (total 122) and thirty-fifth section of blastocyst (total 83); Hill's fluid; 5 m 17 No. 306' (2), also shown in fig. 2, pi. 20, q. v.
18 Bilaminar blastocyst No. 189 (0), the embryomic area of which is shown in fig. 4, pi. 21. q. V.
19 Bilaminar blastocyst No. 55 (19), showing (at right) a mass of cells at lower pole sixty-sixth section of egg (total 139); twenth-fourth section of embryonic area (total 77); Bouin.
20 Bilaminar blastocyst No. 189' (10) approaching time of mesoderm formation; 130th section of egg (total 282); ninety-third section of embryonic area (total 165); vesicle wall very thin; egg slightly damaged.
21 Egg No. 353' (6), blastocyst with about 140 mesodermal cells; 1.6 mm. in alcohol with embryonic area 1.1 mm.; ninety-second section (total 205); M. mesodermal cells, Bouin; 6 ix.
22 Egg No. 346' (6); section taken through primitive steak; 1.5 mm. in diameter in alcohol; embryonic area 1.1 mm.; Bouin; 5 m; compare fig. 4, pi. 2.
PLATE 14
EXPLANATION OF FIGURES
Maturation and fertilization.
1 Large ovarian egg with discus proligerus, from No. 21; 5 ^t. X 200.
2 Ninth of 23 sections through tubal ovum No. 307 (1); polar body is in nineteenth section; egg is surrounded with thin albumen layer; Bouin; 5 n. X 200.
3 Portion of fig. 2; 7 chromosomes are seen; 5m- X 500.
4 Tenth section of same egg; 5 chromosomes; zona pellucida is a beaded line with darkly staining granules; 5 n.
5 and 6 Eleventh section (total 20 sections) through ovum No. 307 (3); the second maturation spindle has 12 chromosomes; Bouin; 5 ix. X 200 and X 500, respectively.
7 Sketch of ovum No. 56 (11), from total preparation drawn with focus on middle of egg; chromosomes and polar body; Bouin. X 200.
8 Sixth section through ovum No. 76 (1) ; total 23 sections; 6 chromosomes of this section are in ovum and one in polar body; Bouin; 5 m- X 200.
9 and 10 Second maturation spindle of egg No. 307 (2); tenth and eleventh sections (total 23 sections); 12 chromosomes; Bouin; 5 /x. X 500.
11 Portion of ninth section of ovum No. 76 (8); total 15 sections; 12 chromosomes in homogeneous granular area; little albumen at left; Hill's fluid. X 500.
12 Ovum No. 313 (2); eleventh section (total 20); marginal granular zone limited within by reticulated region; oil globules of medium size; a little albumen at left; Hill's fluid; 5 m- X 200.
13 Ovum 76 (6); composite of fifth and sixth sections (total 21); polar body and short spindle with 7 chromosomes 5 n; Bouin. X 2500.
14 Ovum No. 56 (6); portion of fourth section (total 17); there are 12 chromosomes; Bouin. X 500.
15, 16, and 17 Ovum No. 76 (4); 2nd, 3rd and 4th sections tangentially (total 25 sections); polar body and equatorial plate of maturation spindle; marginal granular zone, vacuoles and oil globules; Hill's fluid; 5 ju- X 500.
18 Ovum No. 313 (5) from same litter as fig. 12; 5th section (total 20); large fat globules; albumen layer thick (compare fig. 1, pi. 3).
19 Young unfertilized uterine ovum. No. 287 (5); 9th section (total 16); concentric lamellae of albumen; compare fig. 12; Hill's fluid; 5 /i. X 200.
20 Ovum No. 203 (1) with pronuclei; 10th section (total 20); 5 /x- X 200.
21 Ovum No. 52 (3); composite of sections 12 to 15 (total 20) taken obliquely through first cleavage spindle; a little yolk has been extruded (F); Bouin; 5 n. X 200.
PLATE 15
EXPLANATION OF FIGURES
Cleavage stages; all magnifications X 200.
1 2-celIed ovum No. 203 (8), 11th section (total 20); compare figs. K and F, text fig. 4; Hill's fluid; 5 m 2 2-celled ovum No. 306 (1); 9th section (total 23); compare figs. \ and B, text fig. 4; Hill's fluid; 5 n.
3 3-celled ovum No. 173 (8); 10th section (total 21); compare fig. L, text 4; aceto-osmic-bichromate; 5 m 4 3-celled ovum No. 306 (3); 11th of 22 sections; compare K, text fig. 4; Hill's fluid; 5 M 5 4-celled ovum No. 293 (2), with two blastomeres in mitosis; drawn from clay model; total 18 sections, Bouin; 5 n; compare fig. 1, pi. 2.
6 4-celled ovum No. 293 (4), with two cells in mitosis; drawn from claymodel; Bouin; 18 sections; 5 /x; compare fig. 1, pi. 2.
7 4-celled ovum No. 203 (7); 10th section (total 20); see text; Hill's fluid; 5 yu.
8 4-celled ovum No. 83 (7); 11th section (total 18); blastomeres cut as in preceding; one polar body; Bouin; 5 fx.
9 and 10 5th and 9th sections through ovum No. 17' (7) one of 43 very small eggs from one ovary; total 11 sections.
11 and 12 7th and 14th sections (total 19) through ovum No. 83 (8), with portions of shell membrane and albumen; two polar bodies; Bouin; 5 n.
13 4-celled ovum No. 299 (7), also shown reconstructed in o, text fig. 4; 9th section (total 20); trichloracetic; 5 ft.
14 4-celled ovum No. 299 (5), of which sections 9 and 14 are shown in figs. 6 and 7, pi. 3; 13th section (total 22); Hill's fluid; 5 m 15 6-celled ovum No. 85 (5); 7th section (total 16); blastocyst formation already anticipated; Bouin.
16 15-celled ovum No. 337 (1); 8th section (total 19); one-half strength Bouin; 5 ju.
17 16-celledovumNo.85 (12); 8th section (totaPil); blastomeres still rounded; one misplaced cell; Hill's fluid; 5 m 18 17-celled ovum No. 336 (1); 9th section (total 18) ; Bouin; 5 fx.
19 26-celled ovum No. 336 (2); 11th section (total 17); Bouin; 5 //.
20 28-cellcd ovum No. 342 (1); 8th section (total 18); half-strength Bouin; 5fi.
PLATE 16
EXPLANATION OF FIGURES
The formation of entoderm initiated. All magnifications are X 200, except figs. 5 to 10 which are X 500.
1 Completed blastocyst No. 191 (5); 34 cells; 9th section (total 24); Bouin; 5 m.
2 Large blastocyst No. 50 (7); 70 cells, but no entoderm; 11th section (total 23); Hill's fluid.
3 Half-normal blastocyst No. 88 (18); 8th section (total 19); Hill's fluid; 5 m 4 Half-normal ovum No. 344 (12); 6th section (total 16); half-strength Bouin; 5 n.
5 Portion of 6th section (total 17) through ovum No. 50 (8) showing entoderm mother cell ENTA; 63 cells including 2 entoderm mother cells in wall; Hill's fluid; compare fig. 11, pi. 13.
6 Portion of 8th section (total 22) of ovum No. 298 (5), showing entoderm mother cell leaving its place in blast, wall; 126 cells, of which 8 are free entoderm mother cells and several are in process of formation; Bouin, 5^; cf. fig. 7 pi. 2.
7 Portion of 6th section (total 22) of ovum No. 88 (9); 106 cells of which 11 are free entoderm mother cells; Hill's fluid; 5 /x.
8 and 9 Portions of the 10th and 12th sections (total 18) through ovum No. 88 (21); 70 cells, including 10 more or less detached entoderm mother cells; Hill's fluid.
10 Greater part of 4th section (total 15) through ovum No. 88 (23); 57 cells including the two detached entoderm mother cells here shown; Bouin.
11 Blastocyst No. 88 (16), having 82 cells; 6 of the 10 entoderm mother cells are here shown; 11th section (total 20); section 15, fig. 4, pi. 7; Bouin, 5 n.
12 Ovum No. 83 (5), containing 53 cells, including the one large binucleated entoderm mother cell (?) here shown; 9th section (total 20).
13 Ovum No. 356 (3), most retarded member of litter No. 356; about 100 cells; 12th section (total 20); Bouin; 5 m 14 Ovum No. 344 (4) ; small blastocyst with numerous entoderm mother cells; 8th section (total 18); Hill's fluid; 5 m 15 Longitudinal section of ovum No. 344 (11), showing definite polar differentiation; typical entoderm mother cells; 7th section (total 19); half-strength Bouin; 164 cells:
Embyonic ent-ectoderm 71- cells of which 7 are in mitosis
Trophoblastic ectoderm 70 cells of which 8 are in mitosis
Entoderm 23 cells of which 3 are in mitosis
16 and 17 The 7th and the 16th sections (total 22) taken horizontally through ovum No. 344 (14), slightly more advanced than preceding; fig. 16, through embryonic area; fig. 17, through trophoblastic area; half-strength Bouin; 5 jj.; 193 cells:
Embryonic ent-ectoderm 76 cells, 8 in mitosis
Trophoblastic ectoderm 98 cells, 13 in mitosis
Entoderm 19 cells, 1 in mitosis
18 to 22 Reconstructions from blastocysts to show the polar distribution of entoderm mother cells. Fig. 18, ovum No. 88 (17), 103 cells, of which 6 are entoderm.mother cells; section indicated b}^ parallel lines is shown in fig. 2, pi. 7. Fig. 19, ovum No. 83 (1), 111 cells, of which 4 are free entoderm mother cells. Fig. 20, ovum No. 298 (3), 124 cells, of which 4 are entoderm mother cells. Fig. 21, ovum No. 88 (7), 87 cells, including 5 entoderm mother cells; the section indicated by lines is shown in fig. 3, pi. 6. Fig. 22, ovum No. 88 (11), 103 cells, of which 9 are more or less free entoderm mother cells and 7 of these are in mitosis.
PLATE 17
EXPLANATION OF FIGURES
The formation of entoderm (concluded).
1 The 7th section longitudinally through ovum No. 144' (1); ENT^, dividing entoderm mother cell; 16 sections in series; overfixed in Carnoy. X 200.
2 The 14th section longitudinally through ovum No. 144' (8); 19 sections in series; Carnoy. X 200.
3 Section taken tangentially through embryonic area of ovum No. 144' (10); 14th section (total 19); Carnoy. X 200.
4 and 5 Sections 9 and 11 (total 25) cut longitudinally through blastocyst No. 356 (11); Hill's fluid; 5 m; ENT', primitive entoderm cell tending to flatten out; ENT,^ row of entoderm mother cells similar to those in fig. 3, pi. 6. X 200.
6 and 7 Details of ovum No. 356 (4) shown in fig. 12, pi. 13. Fig. 6, 10th section (total 25), X 200; fig. 7, 16th section, X 500, with spermatozoa in albumen layer; mitosis in embryonic entectoderm; Bouin; 6 y.; 283 cells:
Embryonic ent-ectoderm 101 cells, in mitosis 3
Trophoblastic ectoderm 140 cells, in mitosis 3
Entoderm 42 cells, in mitosis 2
8 and 9 Portions of sections 10 and 17 (total 29) longitudinally through ovum No. 356 (9), shown whole in fig. 1, pi. 6; Flemming; 5 m- X 500.
10 and 11 Sections 18 and 13, respectively (total 21), longitudinally through ovum No. 356 (5), section 12 of which is shown in fig. 3, pi. 9; mitoses in entectoderm; Bouin; 6 m! 249 cells:
Embyronic ent-ectoderm 75 cells, in mitosis 14
Trophoblastic ectoderm 126 cells, in mitosis 11
Entoderm 48 cells, in mitosis 4
12 Longitudinal section of egg No. 349 (2) like the one shown in living stage is fig. 3, pi. 8; 31st section through vesicle (total 43); 16th section through embryonic area (total 28); Bouin; 5 m- X 200.
13 Longitudinal section through ovum No. 194' (4); 7th section through embryonic area, (total 20); Hill's fluid; 7 m- X 200.
14 The 9th of a total of 13 sections through the embryonic area of ovum No. 194' (8). X 500.
15 The 12th of a total of 20 sections through the embryonic area of blastocyst No. 194' (6); 38 sections through vesicle; 7 m- X 500.
130
DEVELOPMENT OF THE OPOSSUM
CARL G. HARTMAN
PLATE 17
••
-' is'" •-,
ENT'
-EMB.ECT
EMB.ECT
131
PLATE 18
EXPLANATION OF FIGURES
Stages from the spreading of the entoderm to the just completed bilaminar blastocyst. Whole sections (figs. 5A, 6A, 7A) X 50; vesicles only X 200; ENT. limits of distribution attained by the entoderm; ENT-, undifferentiated primitive entoderm not yet spread.
1 Blastocyst No. 43 (7); 10th section through embryonic area; Bouin.
2 Blastocyst No. 352 (12); 42nd section through egg (total 98), 33d, section through vesicle (total 80), and 18th section through embryonic area (total 50); half-strength Bouin; 5 n; compare fig. 1, pi. 9.
3 Blastocyst No. 40 (1); 15th section through embryonic area (total 30); Hill's fluid.
4 Blastocyst No. 40 (2); the 19th section through embryonic area (total 36); Carnoy.
5A and 5 Blastocyst No. 347 (2); earliest stage of the completed bilaminar blastocyst; 22nd section through vesicle (total 57); Bouin; 7 fx; compare fig. 5, pi. 9.
6A and 6 Blastocyst No. 339 (3). Fig. 6A, 66th section of vesicle (total 94) and 37th section of embryonic area (total 51 ); fig. 6, 68th section of vesicle ; Bouin ; 5 n; compare fig. 6, pi. 9, and fig. 2, pi. 6.
7A Egg No. 347 (1 ) ; 52nd section through egg (total 127) ; 46th section through vesicle (total 103); entoderm spread to equator.
7 Blastocyst No. 347 (4); nearly the sam.e stage as fig. 7A; 32nd section through vesicle (total 90); Bouin; 7 fi.
8 Blastocyst No. 175' (2); 9th section through embryonic area (total 25) and 30th section through vesicle (total 56); aceto-osmic-bichromate; 6 n.
PLATE 19
EXPLANATION OF FIGURES
Partially completed and just completed bilaminar blastocysts.
1 Longitudinal section through ovum No. 205 (4); 41st section (total 93); Bouin; 7 m- X 50.
2 Surface view of trophoblastic area of an egg from the litter No. 205; entoderm shaded; ectodermal nuclei unshaded. X 500.
3 Detail of embryonic area of blastocyst No. 347 (4), shown in fig. 7, pi. 18. X 500.
4 Detail of section through embryonic area of ovum No. 352' (10), shown in fig. 4, pi. 10; X 500.
5 and 5A Entire section, X 50, and vesicle only, X 200, through the middle of ovum No. 339 (4), photographed in toto in fig. 2, pi. 6; note swollen cells; Bouin; 5 fj..
6 Embryonic area only of similar egg No. 339 (2); Bouin; 5 n. X 200.
7 Blastocyst No. 175' (9), with very attenuated, mostly unilaminar wall; 52nd section through vesicle (total 89); aceto-osmic-bichromate (?); 5 m- X 50.
7a Embyronic area only of same egg. X 200.
8 and 8A Entire section, X 50, and embryonic area (XX), X 200, of ovum No. 347 (5) ; entoderm has not yet reached equator; 30th section of vesicle (total 119) and 18th section through area (total 29); Flemming; 5 m 9 The embryonic area of ovum No. 205 (6); 49th section of blastocyst (total 79); Bouin; 5 n; compare fig. 1.
10 Just completed bilaminar blastocyst No. 208 (1); 56th section through vesicle (total 117); Bouin. X 50.
lOA and lOB Details of embryonic area and trophoblastic area of same egg. X 200.
11 Portion of surface view of ovum No. 205 (7) ; A" A', junctional line; only the entorderm is shaded; ectodermal nuclei unshaded circles. X 500.
12 Surface view at junctional line (XX) of ovum No. 205 (9); entire ectoderm shaded; embryonic nuclei very dark, trophoblastic nuclei very light; entoderm al nuclei intermediate in tone.
PLATE 20
EXPLANATION OF FIGURES
Completed bilaminar blastocyst.
1 Section of embryonic area (A'A') of blastocyst No. 82 (13), nearly like fig. 1, pi. 21; 54th section of egg (total 100) and 38th section of embryonic area (total 58) ; Bouin. X 200.
lA Detail of same. X 500.
2 Blastocyst No. 306' (2) shown in fig. 17, pi. 13; 0.77 mm. in diameter in alcohol; 18th section of embryonic area (total 49) and 63d through vesicle (total 118); compare fig. 7, pi. 10; Hill's fluid; 5 m- X 200.
2A Detail of same near junctional line (X). X 500.
3 Ovum No. 285' (1); 72nd section through blastocyst (total 107); Bouin; 5 n; the portion of vesicle marked by dotted line wds dissected off before inbedding and was stained and mounted in toto (fig. 3B). X 50.
3A Same blastocyst. X 200.
3B Surface view from point A, fig. 3; junctional line XX; ectoderm lightly shaded; entodermal nuclei darkly shaded. X 500.
4. The 92nd section (total 140) through blastocyst No. 43 (10); two degenerating cells at A; Hill's fluid. X 50.
4x\ The embryonic area (XX) of same. X 200.
PLATE 21
EXPLANATION OF FIGURES
The 1-mm. bilaminar blastocyst.
1 Egg No. 306' (3) ; 0.85 mm. in diameter in alcohol ; 44th section of embryonic area (total 70) and the 84th section of vesicle (total 135); Bouin; 5 /x- X 50.
lA Embryonic area (XX) of same section. X 200.
2 Egg No. 55 (20); 0.87 mm. in diameter in alcohol; 57th section of vesicle (total 121) and 33d section of embryonic area (total 77); 0, pocket in trophoblastic ectoderm; Flemming; 6 n. X 50.
2A Embryonic area (A"A^) of same section; at A, unusual crowding of ectoderm. X 200.
3 Egg No. 339' (3); 0.85 mm. in alcohol; 64th section of vesicle (total 118) and 45th section of embryonic area (total 71); 0, pocket in ectoderm; halfstrength Bouin; 6 m- X 50.
4 Embryonic area (AX) of egg No. 189 (6), shown in fig. 18, pi. 13; 74th section of embryonic area (total 94) ; 1.02 mm. in alcohol; Hill's fluid; 5 m- X 200.
5 Embryonic area {XX) of egg No. 343 (4), about 1.0 in alcohol (compare fig. 5, pi. 2, and fig. 9 below); 99th section of vesicle (total 189) and 67th of embryonic area (total 126); Bouin; 5 ju. X 200.
6 A portion of trophoblastic area of a 1.0 blastocyst No. 55 (6), showing remnant of albumen; compare fig. 10, pi. 22; Hill's fluid. X 200.
7 and 7A Sections through embryonic and trophoblastic areas of ovum No. 285' (6); 65th section of vesicle (total 119) and 26th through embryonic area (total 65) ; Hill's fluid; 6 m. X 200.
8 Embryonic area {XX) of ovum No. 352 (7); 35th section of egg (total 80); 24th section of vesicle (total 49) and 13th section of embryonic area (total 21); in alcohol egg measured 0.585 mm. through shell membrane and 0.325 X 0.370 through vesicle; 6 n] Bouin. X 200.
9 Surface view of a typical 1 mm. blastocj'st, showing embryonic area; compare fig. 2, pi. 11, and fig. 3, pi. 10. X 16.
10. Half-normal blastocyst No. 314 (3); Bouin; 5 m- X 200.
PLATE 22
EXPLANATION OF FIGURES
Advanced bilaminar blastocysts, to the beginning of mesoderm proliferation. Figs. 2, 4, and 9 represent eggs only a few minutes removed from the first appearance of mesoderm.
I Egg No. 193' (4), similar to fig. 9 below; a 92nd section of egg (total 184) and 55th section of embryonic area (total 87); Hill's fluid; 7 /x. X 50.
2. Egg No. 343' (2), one of the five shown in fig. 6, pi. 2; 1.5 mm. in alcohol; embryonic area, 0.87 mm.; 130th section of vesicle (total 259) and S9th section through embryonic area (total 161); Bouin; 5 /x. X 50.
3 Egg. No. 353 (4); several hours preceding first appearance of mesoderm; diameter 1.22 mm. in alcohol; 91st section of vesicle (total 135) and 53rd section of embryonic area (total 85); Flemming; 6 m- X 50.
3A and 3B Details of trophoblastic and embryonic areas, respectively, of same section. X 200.
3C A detail of fig. 3B. X 500.
4 and 4A Egg No. 189' (1 ) ; 118th section of vesicle (total 200) and 62nd section of embryonic area (total 116); aceto-osmic-bichromate. X 50 and X 200.
5 Embryonic area {XX) of egg No. 347' (1); 1.1 mm. in alcohol; 74th section of vesicle (total 126) and 55th section of embryonic area (total 82); Flemming; 7 M. X 200.
6 Thick embryonic area {XX) of egg No. 360 (4) ; 71st section of vesicle (total 176) and 41st of embryonic area (total 130) ; T, thinning near middle; Hill's fluid; 5 M- X 200.
7 The 79th section through embryonic area (total 172) of egg No. 189' (9). X 200.
8 Large embryonic area of egg No. 189' (4); 157th section of egg ftotal 311) and 59th section of area (total 169) ; Hill's fluid; 5 m- X 200.
9 Drawing made from photograph of an egg in litter No. 193'; shows central light field in embryonic area; compare figs. 1 and 2, pi. 10. X 16.
9A Section of ovum No. 193' (2); 61st section of embryonic area (total 160). X 200.
9B and 9C Details of portions of fig. 9A. X 500.
10 Portion of embryonic area of 1-mm. blastocyst No. 55 (6), showing yolk granules (F) in ectoderm, and entoderm; compare group A with A, fig. 11.
II Surface view of some entoderm cells from below the embryonic area of egg No. 189' (12), showing reaction of cells to yolk remnants (7).
12A and 12B Surface views, from within, of embryonic and trophoblastic areas of egg No. 189' (11); entodermal nuclei (mostly the larger) are seen above the entoderm; embryonic area measures 0.96 mm.; aceto-osmic-bichromate. X 500.
13 Defective blastocyst No. 88 (6) with large included blastomere; Hill's fluid; 5 m. X 200.
14 Half-normal blastocyst No. 356 (2); Bouin; 5 //. X 200.
Cite this page: Hill, M.A. (2024, April 19) Embryology Paper - Studies in the development of the opossum 3 (1918). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_Studies_in_the_development_of_the_opossum_3_(1918)
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