Me
ete
JOURNAL
OF
MORPHOLOGY
FounDEpD By C. O. WHITMAN
EDITED BY
dhe oSig ) JEGILINI( GSH Fe University of Illinois Urbana, Ill.
WITH THE COLLABORATION OF
Gary N. CALKINS Epwin G. CoNKLIN C. E. McCuiunea Columbia University Princeton University University of Pennsylvanis
W. M. WHEELER WILLIAM PATTEN Bussey Institution, Harvard University Dartmouth College
VOLUME 32
MARCH, JUNE, SEPTEMBER 1919
THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA
CONTENTS
No. 1. MARCH
Cart G. Hartman. 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. Bight text fig- ures and twenty-two plates. . So Spats rcaneio aio OEE NOI oi
Epwarp PHeEeLps ALLIS, JR. The nee and ne nasal Pee tes in ie a OSHA SiS NAHM syb-qR OANA DIKAS ARES ae ro ccia a ae Geer e Ao Gin aebioe atin dane c
No. 2. JUNE
Epwarpb Puetps Auuis, JR. The myodome and trigemino-facialis chamber of fishes and the corresponding cavities in higher vertebrates. Four plates Ghwemuyan ime lfielITes) iio feed 2 apres Sy em ereatee aye! Cia) days mal cee ae ne. ek ae
ArtHur WILLIAM Meyer. On the nature, occurrence, and identity of the plasmeance lls7Ot HORACE i nee ecco a yrcadat ist clsa comets tt gla oes ree a ater eens
ApoupH R. RincoeN. The development of the gastric glands in Squalus acan- thias. Three plates (seven figures).. fs eee etic Geel ce caeerentarars 6
GitMAN A. Drew. Sexual activities of Ne squid, ative peal (Les. ) “The spermatophore; its structure, ejaculation, and formation. Six plates (forty- (OMAR CAE DSTSD | eee NEP ane Cay SR ste ta us age oye eae SHU) ll a eI Le tS
No. 3. SEPTEMBER
Witiiam M. GotpsmitH. A comparative study of the chromosomes of the tiger beetles (Cicindelidae). One hundred twenty-seven figures (ten plates). ... BENNET M. ALLEN. The development of the thyreoid glands of Bufo and their normal relation to metamorphosis. One plate (six figures) and one text JORA DHS) 5 Bs teu aes ce Ata opener Nass RRS Let ba een 1 OS noe ce eee cele Waro Nakanara. A study of the chromosomes in the spermatogenesis of the stonefly, Perla immarginata Say, with special reference to the question of synapsis. Three plates (fifty-one figures) .. ot ones STA : Stipnney I. Kornwavser. The sexual cheteererenes of the twee Thelia bimaculata (Fabr.). I. External changes induced s eats theliae (Gahan). Fifty-four text figures .. 2 SOE COONS Sr REE RN eaais css ope Toxuyasu Kupo. The facial oe Ge et abe Tiaaeae: ae text fics and three plates. . niece Tne Nem Sica Beeege : CLARENCE L. TURNER. “The Scone ne in ate spermary oe he ee epee foumpapure se. 30. sew tas a tyne cre
ili
145
207
327
. dol
379
437
. 489
. 509
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. 637
. 681
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 24
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
CARL G. HARTMAN The Wistar Institute of Anatomy and Biology and the University of Texas
CONTENTS
PART III. DESCRIPTION OF NEW MATERIAL ON MATURATION, CLEAVAGE, AND
ENTODERM FORMATION
Er OC WE ENO Mixers: S50. |: Sta Ee Os Ca Re POS RR ye ES Ce aes Gee TeravOny LEMALKS terest reer oe, so PIER oicmerceners Goo aire eked nicl ce oe (Sy lB GST rrope Oren Moe tian PeWA tmietonr lorie Aare met icics Gibt Uaemne Eon aera Riche, cictovc sak Scola ame eee Cu Viatenralvanditechmiquessseesnc..: poor secrete rite a ree a d. External changes in the female opossum at ovulation
Maturation and cleavage to the formation of the blastocyst................ See AEN PERO MALI AMY COD emvatiane tac jac elect Me cP ceo oinpetn eases oi ROME: eheb uly slain’. ifn abl sate sac we tecve Sasa 2 Sepa ten aia weer sictansese «ptt CaphheryouneMberin ese resis ew eater oa se Mn ee, ceases, ES eee edtheshirsty Clea aoe e ciens hey ected See eae teks renee arene exch ccna aks: sepa oe en hersecondsclea Varese seme varus Metneh san Vif nepal deh auc gti ne Senet f. On the origin of the crossed arrangement of the first four blastomeres. . g. Comparison of the 4-celled egg of the opossum and of Dasyurus...... h;. Deutoplasmolysis‘or the elimination of yolk. .2 3.5.22. 6 ee 2 olan i. Later cleavage to the formation of the blastocyst..................... j2.On the: fate omthe timstrtwo blastomenress ess. cis os. ce nc nel ene ore
The formation of the entoderm
SAG ENE Lr alliak bases yay aera Me Pe ted revere i hat LR ES ee lia sc Sg ARS etn oh aN a bhe youngest unilamnar! pla sbocysiseyact cas cee x: -/ «evoke cle olotele en epee Pebhe rst ENCOGSLIN MAG UME EGOS 8st oho cusehe cca sjeus' x cas) sameeren urchaebdle . The detachment of the entoderm mother cells.......................:: . The proliferation of entoderm confined to one pole of the egg......... AeA CRO LM CAL Alc Creer am ey Meee eth: eer d tes 5 ctenc) ah = ,ere.chsuoieted epee os accchs keys . Included: cells which may not be entodermal........ . cece vo csao >= Hurthenpolanrrdiienrentiatlones ya. a. ei 4ci- <ccias clteteite teksts =) orto , Thetemtbnyonte ares, supenicial in postion... ..-.....” sameprmeh acca ee
1
= Doo po Qo»
2 CARL G. HARTMAN
jARRHeyprimmbiviexento dena... savrdcie cise ac c/s ore SIO OIC en eee 65 ky Furtherierowth-of the blastocyst... i. 5i\ssetoa: «+ cece setae erie pees 65 1. The end of entoderm formation and the spreading of the entoderm.... 68 m. The maximum. attenuation of the trophoblast......................... 70 n. The cause of the spreading of the entoderm.............:....-2:ss-«¢+ 71 o. The changing shape and position of the blastocyst.................... 71 De Some, abnormal: egg: 72.251 ee a eee wee ee er nee ele) oof cog. eee ney Ree 72 PART IV. THE BILAMINAR BLASTOCYST General description............... SELLER EEE ese Soh ie seals onlee eee 73 geekhemmatertali ss ac scene MEGAMI ined 5 ls 2 gee 73 b. The development as seen in the living eggs.........................- 73 he just completed bilamimar blastaeyste yess sere 6 85 &. os uns ee 76 Tia Dhe embryonic ectoderm 72-5 Seen eh eee: ee 76 ba Lhetrophoblasticiareay.c.04o eee COA Oe oe ee 78 ¢: Phe entoderm: ..52. 25 cate ets Oa 1d oe) oo ch ee 78 ‘The: t=1mm “blastocyst. o%, tistce ac. tee teres ee a eR ics, 3 tice 79 as General desoription: 2.3 ations. ko ne mers ok Oar Es mee 79 b. The bilaminar blastocyst according to Belen yi oy Sar a Ae eR 82 c. /DheAl=mm. blastocyst according ton Miainotw-7,...21ae pe eee See ee 82 ‘Eheilatesbilaminar blastocyst parce 2044 eee eee Cer ee 84 a. General-deseription(-s: -cc1oe een ae Bore ee ohare so. ee 84° by’ The: central light field in the embryonic area. .........2...-.62544.-) ee 85 e. Modifiedentodermalicells;ee. 4. eee ee eee ark Cotiasateeecdc 86 d. The ectoderm of late bilaminar blastocysts............ ae Buia EES 86 é€. Yolk spherules in eetodermpandsentoderm:....-<. 5. 55..04:6.e ete eeee 87 f,. Mesoderm tormationsiniguapedm. cen cesre ss a. eee ae ae ee 88 UMN AT YF 3.8 Po ieee a ey As Se eins EE ea 2 Ck ee ee 89 Tatera ture cited 2... on<4 oS Wee RTs OA One eon) oe She. ae een 97 Explanation of fistires c's. (eee cs & ee ie pees ee eee ee 98 Bates... Cecte btkcucae eee ee a Se ae Si Rae ee ne va See ee ee 99
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 preliminary 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
DEVELOPMENT OF THE OPOSSUM 3
stages, were also collected during 1916, and at this time many more eggs and embryos were sacrificed for a series of physio- logical 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 ex- perience 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 requi- site 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 Aimée 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 photo- graphs 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
4 CARL G. HARTMAN
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 Hill (10) on Das- yurus. Mention was also made of Caldwell’s discovery (’87) of the shell membrane enveloping the marsupial egg (Phasco- larctus), and of a short paper by Professor Minot (’11) on the bilaminar blastocyst of the opossum. Simultaneous with the publication 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,
DEVELOPMENT OF THE OPOSSUM Da)
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 char- acteristic 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 num- bers 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 in- terpretation 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 suc- ceeding transitional stages, I rejected the view that these are true entoderm mother cells and considered them of ‘nomor- phological 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.
26: CARL G. HARTMAN
But the ages 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 ap- parently determines on the basis of one experiment in which he secured what I regard as fragmenting 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 based 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 there- fore too low. The average number per litter is 11.5, the ex- tremes are 1 (No. 94) and 22 (No. 346’), not taking into con- sideration 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 eggs used in the present study. The data for Nos. 21 to 144
“IJ
DEVELOPMENT OF THE OPOSSUM
TABLE 1
Summary of eggs
Nn 4 f i=] ia] ° B ae 3 | 3 Zoo | Bi {Vee mor) ||| 8 = NUMBER OF NORMAL 5 A = RAS 4 =) 2 EGGS = A < z & BNE n es] me & Ho 2 sino co) Dp 5A 5 =] a a I A. Previously reported: litters Nos. 21 to 144, 21 different animals 1, From pregnant animals............... 35 248 130 | 378 2. From pseudopregnant animals........ 2 0 37 37
B. New material: litters Nos. 173 to 415, 45 different animals
3. From pregnant animals at first opera- Grone (eftuberus)) ss sce ces eerie: ole 22 166 97 263 (63% normal) 4. From second operation (right uterus) eggs used for this article............ 14 107 79 | 186 ; (57.5% normal) 5. Later stages mentioned in summary, Secondeoperatlomeemsaeeeiers ss caer 16 137 39 | 176 (78.4% normal) 6. From pregnant animals, proportion es-
Cin abe Cheeta no er OAR Aer on Ge 2 os ie 16 120 62 | 182 (Estimated)
7. From pseudopregnant animals........ 15 0 156 | 156
Motaleitemshleeoe 4snanduOwenne sea sees | Lod 641 368 |1009
(63.6% normal)
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. With- out 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
8 CARL G. HARTMAN
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 53 days later, and these are designated as No. 320’. The same system applies to 299 and 299’, 292 and 292’, ete. 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 ‘he figures illustrating the respective litters of eggs. An asterisk (*) is placed after the figure or plate containing heliotype illus- trations of eggs photographed in Ringer’s solution in the living state.
Animals used inthe study. No. 21. Killed three days after attempted copulation; mature ovarian eggs (fig. 1, pl. 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 almost killed by female Jan. 26, indicating that oestrus had passed. Killed Jan. 27; large undischarged follicles with ripe eggs (fig. 1, pl. 13).
No. 40. Blastocysts near end of entoderm formation with greatly attenuated non-formative area (figs. 3 and 4, pl. 18).
No. 43. Eggs 0.8 to 1 mm., blastocysts bilaminar throughout (figs. 4 and 4A, pl. 20), except several like those of No. 40 (fig. 1,
No. 46. 2- to 5-celled eggs (text fig. 4, P).
No. 50. Gianna vesicles of about 50 to 70 cells with none or with one to several entodermal mother cells (figs. 1 and 3, pl. 7; fig. 11, pl. 13; figs. 2 and, spl: 16).
No. 52. From pronuclear to.4-celled stages, but mostly unseg- mented eggs (fig. 21, pl. 14).
No. 54. About same as preceding.
No. 55. Numerous bilaminar blastocysts about 1 mm. in diam. (figs: 2, 2A, and 6,spleZ)):
No. 56. Unfertilized tubal ova still devoid of albumen layer (fig. 3, pl. 13; figs. 7 and 14, pl.-14).
No. 58. Undivided, unfertilized uterine eggs.
No. 76. Tubal ova with small trace of albumen (fig. 2, pl. 13; figs. 8, 1 13; and 15°to 177 ple):
No. 81. Fertile eggs, all 4-celled (text fig. 4, N).
DEVELOPMENT OF THE OPOSSUM 9
No. 82. Bilaminar blastocysts like those of No. 50 (figs. 1 and 1A, pl. 20).
No. 88. Four 4-celled eggs (figs. 8, 11, and 12, pl. 15) and three young blastocysts! (figs 12 and 19, pl. 16).
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, pl. 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, pl. 7; figs. 3, 7 to 11, 18, 21, and 22, pl. 16; fig. 13, pl. 22; compare also page 36, Hartman, 716).
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 days before; ovary hypertrophied; eggs subnormal in size (figs. 9 and 10, pl. 15).
No. 144. Blastocysts more advanced than those of No. 88; at- tenuation of non-formative area well under way (figs. 1 to 3, pl. 17).
QOOOo®O
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, pl. 3) and one is 3-celled (text fig. 4, L; fig. 8, pl. 15).
No. 173’. At 8 p.m., Jan. 19 (interval 24 hours) about 12 just com- pleted blastocysts were secured from right uterus; no entodermal mother cells present (fig. 5, pl. 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; pseudo- pregnant; 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, pl. 18; figs. 7 and 7A, pl. 19 and accompanying text fig. 1). The measurements of the eggs of litter 175’ are here given as made in salt solution:
1 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 twenty- four hours. There is, of course, a possibility of error on my part due to mixing of labels in this case.
10 CARL G. HARTMAN
Through shell........ 0.
7 BU 0.70 0.70 0.70 0.68 0.64 Through blastocyst.. 0.54 55
0 0.55x0.4 0.50 0.44 x0.4 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 @Gien18; pli is ehes4 pli:
No. 189’. Killed at 10:30 p.m. same day (interval 123 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 unfer- tilized eggs (fig. 20, pl. 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 210); plaalserhio ssl stale):
No. 191’. Jan. 26, 9 p.m., removed right uterus, leaving ovary (interval 3 days, 53 hours); 11 eggs: 4 bilaminar blastocysts, 1.4 mm. in diam. in alcohol, almost no albumen; 7 unfertilized eggs. Animal died Feb. 5; no wound infection.
No. 192. Operated Jan. 23, 4:30 p.m.; 12 eggs: 4 unfertilized; 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 14-celled stage with two cells in telophase (fig. 8, pl. 3; fig. 9, pl. 13).
No. 193’. Removed remaining uterus Jan. 26, 8:45 p.m. (interval 3 days, 2¢ hours); 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 l-mm. blastocyst was dead and one of 1.40 mm. has imperfect embryonic area; several unfertilized eggs (figs. 1 and 2, pl. 10; figs. 1,.9, 9A, 9B, 9C; pl. 22).> Ammaldied’ Jan.) 29 hot 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 opera- tion; 18 eggs: 9 unfertilized; 9 vesicles with entoderm only at em- byronic area, stage intermediate between Nos. 356 and 352 (fig. 5,
DEVELOPMENT OF THE OPOSSUM ct
pl. 12, figs. 13, 14, 15, pl. 17 and accompanying text fig. 2). Measure- ments in salt solution and in fixing fluid are as follows: In Ringer’s solution (average 0.66 mm. and 0.34 mm.).
‘Rhroueheshelle seen On7s) (0268 0265, 0065) 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 Aceto-osm.—biochr ithroueheshelle ener On COR SonOsoon Room: OFG0R OL50ORGOM O55 0s54 Through blastocyst...... O34 10:32) 0533 0.3) 0733 10.34 10.32 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, pl. 14; some 2-celled (text Wed hy tOrd wud. spl. bss hou plik) s one 3- celled (text fig. 4, 7); others 4-celled (fig. 8, pl. 13: ; fig. 7, ple 15); one recorded measurement of whole egg is 0.44 mm. ‘through shell membrane, 0.15 through ovum.
OGOO oO ©
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 record 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 measure- ment 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..; 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 O70rmmra hes, 29° Ti and-12,-pl. 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, Tespectiv ely; one of the latter 2 mm. with area 1.32 x 1 mm.
10; CARL G. HARTMAN
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, 10A, and 10B, pl. 19).
No: 208’. Killed Jan. 31, 1:45 p.m. (interval 26% hours); right uterus yielded 8 eggs: one unfertilized, 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, 2.85 mm.
No. 214 (D. marsupialis). Received from south Texas, Feb. 1; operated Feb. 2; a dozen or more undivided, unfertilized eggs, a slight 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, numer- ous 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 lined with ento-
2QODOQVOP
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: <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; ento- dermal lining complete (least advanced, figs. 3, 3A, and 3B, pl. 20; most advanced in figs. 7 and 7A, pl. 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 disin- tegration (fig. 6, pl. 13; fig. 19, pl. 14).
No. 287’. Jan. 18, 6 p.m. (interval nearly 33 days); 13 clear, hyaline eggs, disintegration evident in ovum.
No. 290. Copulation during night of Jan. 11 to 12; motile two- headed 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, abnormal (compare No. 290 (8), fig. 2, pl. 6); 2 eggs with normal bilaminar vesicles occupying about one-half of the egg (compare 290 (4), fig. 2, pl. 6; fig. 6, pl. 12).
No. 290’. Killed Jan. 18, 4:30 p.m. (interval 192 hours); 8 eggs: 2 unfertilized; one retarded blastocyst; 5 apparently normal bilaminar
DEVELOPMENT OF THE OPOSSUM 13
blastocysts a little over 1 mm. in diameter; no preparations made of this litter (figs. 2 and 3, pl. 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, pl. 1*; figs. 1 Deore Tand.G, ple i6):
No. 292’. Killed Jan..21, 10:40 p.m. (interval 4 days); 7 vesicles about 3 mm. in diameter, late primitive-streak stage; one unfertilized ege@; one degenerating young bilaminar blastocyst (fig. 6, pl. 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, pl. 2*; figs. 5 and 6, pl. 15).
No. 293’. Killed Jan. 22, 7:30 a.m. (interval 33 days); 17 eggs: 8 unfertilized, one defective; 8 bilaminar blastocysts like those sketched insiext meure. 3, , KH, (igs 2. pl: 2° )2 9 =
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 blasto- cysts at one pole, all rather abnormal; 2 apparently normal bilaminar blastocysts like F, text fig. 3 (fig. 1, pl. 11*).
No. 294’. Killed Jan. 20, 7 a.m. (interval 343 hours); 14 eggs: 7 unfertilized; one 1.4 mm. in diameter and 4 small degenerating blasto- cysts; 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, pl. 11*). No preparations were made of this litter.
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 ento- dermal proliferation (fig. 7, pl. 2*; figs. 7 and 8, pl. 6; figs. 6 and 20, ple 16):
No. 298’. Killed Jan. 23, 12 m. (interval about 33 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 unfertilized eggs (eggs in utero, fig. 8, pl. 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 (ie 3) pli hess Grandetepl 3, ties. 13 and 14, pl. 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 blas- tocysts; 3 abnormal blastocysts; 5 unfertilized eggs (fig. 4, pl. 1*; figs. 1 and 2, pl. 6; fig. 5, pl. 10; figs. 7 and 8, pl. 12; fig. 16, pl. 13).
No. 303. Caught Jan. 19; Jan. 20, pseudopregnant, 7 degenerating eggs a week old (fig. 7, pl. 11*); killed Feb. 1, the mammary glands still very thick, almost as in pregnancy.
14 CARL G. HARTMAN
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, pl. 3; fig. 2, pl. 15); one egg is 3-celled (text fig. 4, K; fig. 4, pl. 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*, pl. 10; fig. 17, pl. 13; figs. 2 and 2A, pl. 20; figs. 1 and 1A, pl. 21).
No. 307. Jan. 21, 3:30 p.m.; 11 eggs removed from left Fallopian tube (fig. 7, pl. 1*; figs. 2 to 6, 9, and 10, pl. 14).
No. 307’. Killed Jan. 27, 9:15 a.m. (interval 52 days); 10 eggs, unfertilized and fragmenting (fig. 8, pl. 1*).
No. 3138. Caught Jan. 19; Jan. 22, 10: 30 p.m., 9 tubal ova, with considerable albumen (figs. 1* and 3, pl. 3; figs. 12 and 18, pl. 14).
No. 313’. Animal died during the night; the 11 eggs taken from remaining oviduct had more albumen than the ‘313’ litter; eggs poorly fixed (fig. 5, pl. 13).
No. 314. Copulation a.m., Jan. 20; spermatozoa recovered; Jan. 23, 7:30 p.m., 9 eggs (fig. 1*, pl. 5; fig. 1, pl. 6); of the 5 eggs sectioned one is a blastocyst of 26 cells (fig. 2, pl. 5), 2 contain about 30 cells each (fig. 4, pl. 6), and 2 are abnormal (fig. 10, pl. 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. 6, pl. 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, pl. 1*).
No. 320’. Jan. 30, 9:25 a.m. (interval 53 days); 17 eggs: 4 unfer- tilized; 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, pl. 1*).
No. 321’. Jan. 25; litter of foetuses near term accompanied by the «cee shown in fig. 9, pl. 11*; these eggs are, therefore, nearly 10 days
No. 332. Jan. 26, 21 eggs (9 plus 12), degenerating, unfertilized, in middle stage of pseudopregnancy (fig. 10, pl. 11*).
_ No. 336. Jan. 27, 9 p.m., 14 eggs (one was lost before photograph-
ing); the 6 preparations made are young blastocysts of 17, 26, 29, 30,
mi a 32 cells, respectively (all figures on plate 4*; figs. 18 and 19, 15
No. 336’. Killed Feb. 1, 5:45 p.m. (interval nearly 5 days); s 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 unfertilized, one 8-celled and six 16-celled; two eggs sectioned are 15- and 16-celled, respectively (fig. 9; pl. 1*; figs. 3* and/4*% ple peal Gp. 5),
No. 337’. Feb. 1, 10:30 p.m. (interval 44 days); 14 eggs: 12 blas- tocysts, about 2.5 to 4.5 mm., first appearance of medullary groove; 2 defective blastocysts (eggs in utero, fig. 10, pl. 1*).
DEVELOPMENT OF THE OPOSSUM lex
No. 339. Jan. 28, 3:30 p.m., 8 eggs (fig. 6*, pl. 9): 2 unfertilized; 5 eggs with more or less abnormal, round, thick-walled blastocysts at one pole (fig. 2, pl. 6; fig. 15, pl. 18; figs. 5, 5A, and 6, pl. 19); one quite normal thin-walled blastocyst with entoderm spread to equator (fig. 2, pl. 6; figs. 6 and 6A, pl. 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 unfertilized eggs (fig. 3, pl. 21).
No. 342. Received Jan. 27; Jan. 28, 9:30 p.mM., 19 eggs; of the 4 sectioned specimens two are defective and the other two are blastocysts of 26 and 28 cells, respectively (figs. 6* and 7*, pl. 5; fig. 20, pl. 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 unfertilized; one small defective blasto- cyst; one blastocyst with defective embryonic area; 9 normal bilaminar blastocysts about 1 mm. in diameter, embryonic areas 0.64 to 0.76 mm. Gi ple Zoaties o. pl. 21):
No. 343’. Killed 7 hours, 20 minutes later (73? days after copula- tion); 8 eggs, of which 3 are unfertilized, 5 normal 1.8-mm.blastocysts ‘just preceding proliferation of mesoderm; embryonic areas 1 to 1.1mm. in diameter (fig. 6, pl. 2*; fig. 2, pl. 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, pl. 16); 7 are normal blastocysts showing early differentiation of embryonic and non-embryonic areas; the most ad- vanced contains 124 ‘ectodermal’ and 45 entodermal cells (figs. 5*, 6%, and27, pl. Si fies. 14 tor l7, pl. 16).
No. 344’. Killed Feb. 1, 8:30 p.m. (interval 3 days, 32 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 unfertilized; one dead blastocyst; 8 normal 1.5 mm. blastocysts, embryonic areas about 1 mm.; the other eggs retarded and defective (igs; ply 25):
No. 346’. Killed next morning at 6:35 o’clock (interval 9$ hours); 22 eggs: 11 unfertilized; 11 blastocysts ranging up to 2.2 mm. in te all in early primitive-streak stages (fig. 4, pl. 2*; fig. 22, pl. 13).
No. 347. Jan. 29, 9:45 p.m.; 15 eggs: 4 unfertilized; 11 normal blastocysts partly or entirely bilaminar (fig. 5*, pl. 9; figs. 5 and 5A, 7 and 7A, pl. 18; figs. 3, 8, and 8A, pl. 19).
No. 347’. Jan. 30, 10:15 p.m. (interval 123 hours); 17 eggs: 4 un- fertilized; 13 bilaminar blastocysts measuring 1.1 to 1.24 mm. in alcohol (fig. 1, pl. 22).
No. 349. Front foot wounded in trap; Jan. 30, 3:45 p.m., 5 eggs: 2 unfertilized; one unilaminar blastocyst (fig. 4, pl. 8); 2 blastocysts with spreading entoderm (fig. 3*, pl. 8; fig. 12, pl. 17).
JOURNAL OF MORPHOLOGY, VOL. 32, No. 1
16 : CARL G. HARTMAN
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., 23 hours later; 14 eggs with a little albumen found in right oviduct (fig. 2*, pl. 3; fig. 4, pl. 18).
No. 352. Jan. 30, 5 p.m.; 16 eggs: 9 unfertilized; of the 7 remaining, 3 are like eggs No. 40, the others less advanced and perhaps not quite normal (figs. 1* and 2, pl. 9; fig. 14, pl. 13; fig. 2, pl. 18; fig. 8, pl. 21).
No. 352’. Killed Jan. 31, 8 a.m. (interval 15 hours); 20 eggs: 8 un- fertilized, one egg with dead vesicle; one egg with two vesicles; the remainder bilaminar vesicles fill one-half to three-quarters of the egg, which measured fresh about 0.75 mm. (fig. 4*, pl. 9; fig. 4, pl. 10; fig. 4, pl. 19).
No. 358. Jan. 30, 7:45 p.m., 16 eggs: 5 unfertilized;