The Eggs of Mammals (1936) 9

From Embryology

Chapter IX The Growth and Implantation of the Blastodermic Vesicle

The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer
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Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)


In the cinematographs of Lewis and Gregory (1929) the regular cleavage of rabbit ova in vitro is shown to occur at approximately the same rate as in vivo and the formation of the blastocyst is initiated. The rapid expansion of the blastocyst into the typical large blastodermic vesicle (see Plate VII, Fig. 21) does not, however, occur. The attempted expansion is apparently barred by the presence of the relatively rigid zona pellucida and albumen coating so that the blastocyst alternately expands and collapses over a period of many hours until degeneration finally ensues. Br ache t (1912, 1913) had previously shown that ova recovered from the uterus of the rabbit at 5 to 6 days after coitus will develop normally for 24 hours to 48 hours, passing from the tridermic stage to the stage of the primitive streak, with normal development of the ectoplacenta. Rabbit ova enter the uterus between 72 and 75 hours after copulation (Cruikshank, 1797; Assheton, 1894; Gregory, 1930) in the early blastocyst stage and still surrounded by the zona pellucida and the albumen coat. There is a rapid expansion of the ovum at this time due to the infiltration of fluid into the vesicle cavity so that by 96 hours after copulation the blastocyst is easily three times the diameter of the tubal egg. Very soon after the entry of the ovum into the uterus the viscosity of the stretched albumen layer appears to decrease so that its persistence about the large pre-primitive streak vesicle of the 6th day must be due to a marked softening. By the end of the 6th day to the 7th day it disappears completely due probably to its digestion by uterine fluids since it does not disappear in culture-grown ova. The growth in culture of whole vesicles during the period when the albumen and zona coverings still remain is extremely difficult for the ova soon degenerate and often collapse (Waterman, 1932, 1934). As soon as the early primitive streak stage is reached, explantation results in a moderate degree of development. Waddington and Waterman (1933) explanted the embryonic portion of the blastodermic vesicles upon a medium of chicken plasma plus chicken embryo extract and found that the older and more differentiated the embryo at the time of explantation the greater the degree of differentiation in culture. Using the five stages illustrated in Figure 30, the development observed was as follows :

(a) The stage of late pre-primitive streak gives no apparent differentiation as seen in whole mount preparations. Localized thickenings only occur.

(b) The stage of posterior thickening and initial elongation of the embryonic disc develops one or two beating hearts, and localized thickenings after 4-5 days' growth in vitro.

(c) The stage of short primitive streak undergoes marked elongation of the primitive streak and embryonic disc on the 2nd day; two, and in one case three, beating hearts appeared after 2-3 days of culture.

(d) The stage of medium primitive streak gives results comparable to (c) . In several instances brain, hearts, neural tube and somites appear.

(e) The stage of long primitive streak gave rise to embryos with as many as six pairs of somites after 1 day of culture, and the pre-somite and two-somite stages give only slightly, if at all, better development.


Fig. 30. Camera lucida drawings of embryonic areas of the rabbit at the stages of explantation. XG, late pre-primitive streak. XE, stage of posterior thickening. XL, medium primitive streak. XK, pre-somite. XB, three somite, p.st., primitive streak; c.pl, chorda plate; p.kt., primitive knot; p. pi., prochordal plate; p.m.s., pre-mesodermal somite; s., somite. (From the Journal of Experimental Biology.)


Nicholas and Rudnick (1934) similarly were unable to obtain any adequate development of rat blastocysts in stages earlier than the pre-somite or 5-7 somite. But vesicles in the latter stages developed markedly in a medium consisting of equal parts of rat plasma and 14-15 day rat embryo extract. They report that growth occurs during the first twenty-four hours in vitro gradually slowing and ceasing by the 36th hour. At 48 hours or earlier, differentiation in the embryo has reached a maximum, at which it may be maintained for another 24 hours.


During this period the embryos in the best cases have differentiated from 2 to 16 somites. The allantoic bud has grown from a small lump of tissue at the angle between the amnion and the posterior part of the embryo to join with the superior surface of the ectoplacental cone. The heart, unformed at the time of implantation, has differentiated a two chambered structure and has initiated its beat, the blood islands have developed in the yolk sac epithelium, and circulation has commenced, both in the yolk sac and in the embryo. The nervous system has differentiated considerably; eyes and ears have differentiated and the embryo as a whole has gone through a primary torsion, separating it from the embryonic membranes in the region of the intestinal portal and contributing to its apparent reversal of posture.


"The total growth attained in the 48 hour period is less than half that attained by the normal embryo during the same period. The maximum differentiation is nearly threequarters of that undergone by the normal. The factors limiting growth are affected earlier than those limiting differentiation.

'^ Apparently respiratory interchange is the most important functional necessity at this stage. The efficiency of this mechanism is not only lowered by the total absence of maternal circulation but even further prevented by the growth of a new enveloping membrane in the nature of a decidua from the marginal cells of the ectoplacental cone. The accumulation of break-down products due to metabolic activity is another checking factor. A few preliminary experiments have shown that these can be removed by washing the entire culture in sterile Ringer's solution and adding fresh embryonic extract. By using this method embryos have been kept alive for 96 hours although growth and differentiation occur only at a low rate during the last 24 hours."


Nicholas (1934) has also observed a few cases of the development of rat embryos from ova dropped into the uterine cavity, and extra-uterine pregnancies in man are of course well known. In the rat the removal of the entire gestation sac from the uterus into the peritoneal cavity may be performed without hindering fairly advanced embryo development in the extra-uterine environment (Selye, Collip and Thomson, 19356).^ It therefore appears that some somatic influence carries the ova through the critical early blastocyst stages and that this influence does not operate in the ordinary tissue culture media.


It will be recalled that this critical stage occurs at the time of the disappearance of the egg envelopes and Hall (1935) has recently presented data offering a possible clue to the critical events. He found that the zona pellucida of rat and mouse ova placed in fluids of low acidity quickly disappeared (at pH 3.7 or below). In a few cases the zona pellucida was dissolved in Ringer's solution with a pH as high as 5.4. Deciduomata of the rat have shown pH values as low as 5.7, which are, however, not below the critical levels of the in vitro experiments. Pincus and Enzmann (unpublished data) have taken a number of measurements of the pH of pseudopregnant and pregnant endometria and have never observed pH values below 6.5. Nonetheless it is possible that in the small decidual crypts into which the ova fall the critical acidity may be attained.



Fig. 31. Left, normal rabbit blastocysts of the 5th day of pregnancy. Right, blastocysts of the 5th day of pregnancy from rabbit doe ovariectomized 18 hours after mating. (From the American Journal of Physiology.)


Burdick and Pincus (1935) and Pincus and Kirsch (1936) have examined this critical stage of development from a somewhat different angle. Corner (1928) had noted that in rabbit does in which both ovaries or all the corpora lutea were removed shortly after fertilization the uterine ova remained in the early blastocyst stage (see Figure 31 and Tables XX to XXII), whereas in control rabbits with corpora lutea normal development occurred. The degenerating blastocysts were associated with an oestrus type of endometrium, and normal growth of a progestational endometrium with implantation of embryos occurred when corpus luteum extracts were injected daily after ovariectomy (Allen and Corner, 1929). Burdick and Pincus (1935) observed that the daily injection of oestrone begun one or two days after copulation in unoperated rabbits (100-150 rat units per day) and mice (5 rat units per day) resulted in the degeneration of rabbit ova in the early blastocyst stages and of mouse ova in late morula stages, i.e., at the stages during which uterine entry occurs. Pincus and Kirsch (1936) extended these observations in order to fix the critical time of action of the hormone. Injections of oestrone were made at various periods both before and after ovulation, and in the case of the post-0 vulatory injections the uteri were examined at the 10th to 12th days to determine the extent of implantation.


Their data presented in Tables XXIII and XXIV indicate clearly that the minimum sterilizing dosage can be given on days 3 to 4 post coitum. These days cover the period of early blastocyst development. The minimum daily sterilizing dosage for days 3 and 4 is 150 rat units of oestrone-in-oil. When lesser dosages are injected a partially sterilizing effect is observed. This partially steriUzing effect is measured by observing the ratio between the number of corpora lutea and the number of implantations. The relation of the implantation ratio to the hormone dosage is given in Figure 32. It will be seen that even relatively low hormone dosages have a lethal effect upon a number of the embryos. This effect may be due either to prevention of implantation of vesicles developing normally till implantation time or to a degeneration before implantation. The latter alternative seems most likely when one observes the degenerated condition of the preimplantation blastocysts. In addition practically all the embryos that do become implanted are normal in appearance, and, in fact, give rise to normal young at term (rabbit no. 47).


TABLE XX

GROUP I BOTH OVARIES REMOVED AT 14-lH HR8. |

NO.

AUTOPSIED

STATE OF EMBRYOS

PROLIF.

1

4Hd

DEGENERATED 0.2 MM. DIAM.



18

4Kd

0.2 '<



34

4^d

0.15-0.2"



3

5Hd

0.2 "



2

7Hd

0.4 «<



4

7%d

0.3 ..



38

5%d

0,45 "


TABLE XXI

UKoUf U CONTROL OPERATIONS AT 1 j-18 V- HKS.

NO.

OP.

AUTOPSIED

STATE OF EMBRYOS

PROLIF.

33

oj

ei^

7 NORMAL 0.5 MM.

+

24

01

eid

7 NORMAL O.C MM. 4 DEQEN.

■¥

27

m

5 rid

1 ABNORMAL 1 MM.

+

37

00

5Kd

7 NORMAL 2 MM.

+

23

08

G%d

3 NORMAL, SHIELD STA3E

+

5

so

l%d

5 NORMAL, iX SOMITES

+

21

SQ

md

1 NORMAL,

SOMITE 8TA0E

+

TABLE XXII

GROUP lU ALL CORPORA LUTEA EXCISED AT 15-20 HR3. |

NO.

OP.

AUTOPStED

STATE OF EMBRYOS

PROUF.

16

R. L.

m

4Hd

4 EARLY DEGEN. 0.4 MM.



\9

Qe

A%d

4UN8EQ.0VA IN TUBE



30

fi§

h%d

NO EMB. (OVULATION -(-)



'31

se

5Kd

..



32

so

h%d

..



10

08

V/2d

4 DEG. BLASTOCYSTS 0.2 MM. IN TUBE



OUND

DBES

OR

ERUS

HH


Numb

OF CoRPO

Lute


TABLE XXIV

The Effect of Various Types of Oestrone Injections during the Preimplantation Period upon the Implantation Ratio. (From Pincus and Kirsch, 1936)


Days

Rat

Total

Number

Number


Animal

AFTER

Units

Number

OF

OF Im


Number

Mating

Injected

OF Rat

Corpora

planta

Remarks


Injected

Daily

Units

Lutea

tions


16

1

200*

200

9

9

Implantations normal

17

1-2

200*

400

9

2

" "

18

1-3

200*

600

7

1

y) >>

20

1-3

200*

600

5

2

j> >>

19

1-4

200*

800

7

1

M >>

25

1-5

200 *

1000

10






24

4

200*

200

10

8

Implantations normal

38

4

400

400

7

1

)y >>

21

4-5

200

400

7






41

4-5

100

200

9

8

3 dying; 5 normal

26

4-6

200

600

8






44

5-6

200

400

12

7

Implantations subnormal in size

48

5-6

200

400

To term

No litter

47

3-4

100

200

"

"

Litter of four

37

3-4

200

400

12






45

3-4

150

300

10




1 100%


71

3-4

150 t

300

11




dead


69

3-4

150 §

300

8

6

Implantations normal

40

3-4

100

200

14

5

Implantations normal

60.3% dead

52

3-4

100

200

13

6

2 embryos subnormal


60

3-4

75

150

8

1

Implantations normal

87.5% dead

56

3-4

373^

75

11

1

Implantations normal


61 66

3-4 3-4

37H 371^

75 75

6 5

5


Implantations normal

72.7% dead



58

3-4

30

60

10

3

Implantations normal

33.3%

65

3-4

30

60

11

11

Average diameter of egg chambers 1.43

dead

59

3-4

25

50

9

7

X 1.07 Implantations normal


62

3-4

25

50

10

10

Implantations normal

11.1% dead

64

3-4

25

50

8

7

Implantations normal


13

1-5

3c.c. .005%

15C.C.

6

5

Implantations normal


54

No inj

NaOH

ections


10

10

1 subnormal in size

9.8% dead

55a

"

"


5

5

Implantations normal


55b

n

n


11

8

,.


63


9

9

"



  • Oestrone in aqueous solution (Parke-Davis Theelin).

t Crystalline oestrone in oily solution. § Crystalline oestrone in aqueous solution.



Fig. 32. Abscissa: oestrone dosage in R.U. per day. Ordinates: A, per cent of embryos unimplanted; B, number of unimplanted embryos per female. (From the American Journal of Physiology.)


When eggs in the blastocyst stage are placed in culture they will develop normally for 24 to 36 hours (Brachet, 1913; Pincus, 1930). Cleaving ova will, as we have seen, develop for several days and collapse when the blastocyst stage is reached and presomite stages continue development for 3 to 9 days. This implies that the explanted blastocyst either carries with it from the uterine environment a limited supply of necessary nutrition or that it rapidly exhausts the necessary materials from the ordinary culture medium.


If oestrone in some way directly interferes with the assimilation or metabolism of this critical nutrition then blastocysts cultured with this hormone should show inhibited development compared to that of controls in a normal medium. Pincus and Kirsch (1936) cultured early blastocysts taken from the uterus of rabbit does with varying amounts of oestriol (12.5 to 25.2 y per culture) and found that control blastocysts developed at the same time as those in the oestriol-containing media. Oestriol was used instead of oestrone because the former is much more soluble in aqueous media and it also has a lethal effect upon developing blastocysts when injected in vivo (see Table XXV). These experiments show that the lethal effect of the hormone is not due to the direct action of the hormone upon the developing blastocyst. The sterilizing effects of oestriol and dihydrooestrone (Table XXV) indicate that the lethal effect is not oestrone specific, and point again to the disturbance of a needed nutritive condition.

TABLE XXV

The Effect of Various Injections of Oestriol and Dihydrooestrone UPON the Implantation Ratio. (From Pincus and Kirsch, 1936)


Animal

Number

Days

AFTER

Mating Injected

Amount

Injected

Daily

(IN

Gamma)

Total Amount

(in Gamma)

Number

OF

Corpora Lutea

Number

of Implantations

Remarks

46 70

78

3-4 3-4 3-4 3-4 3-4 4-5

4-5 4-5 3-4 3-4

3-4 3-4

16.7* 16.7t 18.0t 22.2

  • 22.2t 11.1
  • 5.5
  • 11.1
  • 66.0§

100.0§

150.0§ 225.0§

33.3 33.3 36.0 44.4 44.4 22.2

11.0

22.2

132.0

200.0

300.0 450.0

9

9

8 12 10 16

10

Tot 6

7

9

8

8

6


16

10 erm 6 3

2


Implantations normal

51


7."^


42

43 49 68 74

76

77

Implantations subnormal

in size Implantations normal No litter

Implantations normal Average diameters of egg

chambers 1.90 X 1.43

cm. Egg chambers = .8 X 1.0

and .9 X 1.1


  • Dihydrooestrone in aqueous solution, t Dihydrooestrone in oily solution.


§ Oestriol in oily solution.


Just what special conditions are needed for carrying the blastodermic vesicle over this critical stage cannot be explicitly stated. It is obvious that corpus luteum activity is necessary for the establishment of these conditions, and the oestrone effect is due to an inhibition of this activity. Thus it is possible to overcome the partially sterilizing effect of low oestrone dosages by the simultaneous injection of a corpus luteum hormone preparation (Pincus and Kirsch, unpublished data). Other substances {e.g., vitamins A and C) are ineffective as inhibitors of complete sterilization. There seem to be tw^o alternatives: either (1) progesterone or some corpus luteum product act directly upon the blastocysts or (2) corpus luteum secretions induce a special uterine environment through their action upon the endometrium. Pincus and Enzmann (unpubhshed data) have made crude extracts of the endometrium of pseudopregnant rabbit does, and have cultured blastocysts in media containing these extracts. No marked effect was obtained with the particular preparations employed, but further investigation may disclose the presence of an active substance. It is certain that blastocyst death due to oestrone action occurs in a uterus the endometrium of which still shows at least partial pseudo^ pregnant proliferation. The minimum sterilizing dosage employed by Pincus and Kirsch is insufficient to abolish pseudopregnant growth completely (Leonard, Hisaw and Fevold, 1931; Courrier and Raynaud, 1933). Courrier and Raynaud (1934) have also found that dosages sufficient to prevent implantation are below the level necessary for the abolition of pseudopregnant growth. The data presented here on sub-sterilizing dosages demonstrate explicitly that a certain number of vesicles fail to develop in a uterus in which others proceed normally. We may consider therefore that there is necessary at least a threshold amount of a necessary active substance, or an optimum-hydrogen ion concentration alterations of which differentially affect the various blastocysts, or a rate of uterine contraction which causes the proper lodging of the blastocysts in the endometrium thus preventing their injury. The fact that blastocysts in culture also show unusual sensitivity leaves the first two of these alternatives.


The behavior and differentiation of the blastodermic vesicle at the time of implantation have been the object of extensive investigation by mammalian embryologists since the publication of Bischoff's (1852) classical memoir on the subject. These investigations have been concerned chiefly with presenting exact descriptions of the mode of implantation in various classes of mammals (see Robinson, 1904; Grosser, 1909; Bonnet, 1903; Spee, 1915; Wilson, 1928; Sansom and Hill, 1930) and the accompanying differentiation of the vesicle. The physiological processes underlying these phenomena have been scarcely investigated.


The writer has been interested in the phenomenon of the delayed pregnancy which seems to offer an opportunity to exploit the processes occurring at implantation. Delayed pregnancy, or late parturition, occurs notably in the lactating mouse or rat which is carrying a set of fertilized eggs during lactation. This is a result of the fact that mice and rats have an oestrus period within 48 hours of parturition in which normal mating and fertilization take place. Enzmann, Saphir and Pincus (1932) have analyzed all the available data in the literature and found that in mice and rats each suckling young on the average prolonged pregnancy by about 21 hours (see Figure 33), though this time of prolongation seemed to vary somewhat from strain to strain. An examination of mated mice in a series of timed matings disclosed the fact that the preimplantation vesicle in suckling females failed to implant at the normal time but some time later depending upon the number of young suckling (see Kirkham, 1916, 1918). Once implantation occurs the growth of the embryo proceeds at the rate characteristic of normal embryos (Enzmann, 1935). Obviously the lactation process results in the establishment of conditions in uUro which inhibit implantation, and the rather exact relationship between the degree of delay of pregnancy and the number of young suckling suggests that definite quantities of necessary substances are withdrawn from the uterus as the result of mammary gland activity.


Fig. 33. Showing the relationship between the degree of delay of pregnancy and the number of suckling young. (From the Anatomical Record.)


Teel (1926) found that the daily injection of a NaOH extract of the anterior hypophysis delayed implantation in rats when injections were begun on the day of mating. Injections on days 1 to 6 caused delayed implantation with parturition occurring in normal fashion but several days after term; injections on days 1 to 12 also caused delayed implantation but a definite interference in the birth mechanism so that only one of a series of females produced normal hving young in a late parturition; injections over a longer period resulted not only in delayed implantation but also in stillbirths 5 to 7 days after normal term. The impairment of the birth mechanism can therefore be avoided by early injection and is presumably a phenomenon distinct from that of delayed implantation. The inhibition of parturition can be caused not only by alkaline pituitary extracts (Evans and Simpson, 19296; Snyder, 1934) but also by corpus luteum extracts (Nelson, PMner and Haterius, 1930). Since the pituitary extracts employed by Teel caused marked luteinization of the ovaries of injected animals it is possible that the delay in implantation may be due to excessive corpus luteum secretion. Selye, CoUip and Thomson (1935??) have ingeniously demonstrated that the rat ovary during lactation presumably produces little or no oestrin, so that the hormone-producing tissue of the ovary during lactation is predominantly the luteal tissue. One need not postulate hypersecretion by the corpus luteum during lactation but merely an unbalance in which corpus luteum hormone predominates (thus Selye, Collip and Thomson actually obtain larger corpora lutea in lactating mice when oestrin is injected).


Wislocki and Goodman (1934) injected a preparation of progestin (after Allen, 1930), for 8 days after mating into two rabbits but no delay of pregnancy ensued. Antuitrin-S and antuitrin-G injected in fairly large amounts during early pregnancy were also ineffective although these preparations induced a fresh ovulation and new corpus luteum formation. The ineffectiveness of progestin in the two experiments of Wislocki and Goodman may have been due to an insufficient dosage. On the other hand it is possible that delayed pregnancy is due to an insufficiency of corpus luteum secretion, so that the immediate effect of Teel's extract may be considered the stimulation of oestrin production with inhibition of luteal secretion followed by corpus luteum activity which induced or completed the implantation process. Hamlett (1935) is in fact of the opinion that delayed implantation is due to hyposecretion of the corpus luteum. He has found (1932) that copulation and cleavage occur in the nine-banded armadillo of Texas during July, and the unimplanted vesicle lies free in the uterine lumen until early November when implantation takes place. Correlated with the quiescent period is a large corpus luteum the cells of which contain few or no secretory droplets or granules. Shortly before implantation vacuolization and lipoidal secretion occurs in the luteal cell cytoplasm, and the removal of such corpora lutea leads to abortion whereas removal during the free vesicle period has no discernible effect upon the uterus or ovum. Hamlett (1935) quotes a number of instances of naturally-occurring delayed implantation of a presumably similar nature.


This possibility has been tested by injecting oest rone-free corpus luteum extracts into lactating pregnant mice during the early part of pregnancy (unpublished data). Injections of approximately l/20th of a Corner- Allen rabbit unit were made over a 5 to 8 day period. A number of the mice failed to produce any young but seven females gave birth to normal litters. These were born not at term but much later; in fact, the average date of birth was 4 days later than would occur in delayed pregnancy if the expected delay is calculated on the basis of 21 hours per suckling young.


The implication is clear that excessive corpus luteum secretion caused a delay of pregnancy in mice. Since Teel (1926) found that deciduomata formation could be readily induced in the uteri of unmated females treated with his extracts corpus luteum activity undoubtedly occurred as a result of luteinizing hormone injection. The act of suckling then, by prolonging corpus luteum activity (which it does — see Parkes, 1929; Turner, 1932), results in a delay of implantation. Selye and McKeown (1934a) have in fact shown that suckling in rats prolongs pseudopregnancy and that the effects of suckling do not occur in the absence of the ovary (Selye and McKeown, 19346).


The fact that Teel obtained definite deciduomata in animals subjected to a treatment that produces delayed pregnancy indicates either: (1) that mechanical irritation is more effective than ovum contact and that therefore the corpus luteum effect is really subnormal or (2) that excessive corpus luteum activity in some way inhibits the actual process of implantation of the blastocysts. The problem is an interesting one and is receiving further investigation.



Historic Disclaimer - information about historic embryology pages 
Mark Hill.jpg
Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)
The Eggs of Mammals (1936): Introduction | The Origin of the Definitive Ova | The Growth of the Ovum | The Development and Atresia of Full-Grown Ova and the Problem of Ovarian Parthenogenesis | Methods Employed in the Experimental Manipulation of Mammalian Ova | The Tubal History of Unfertilized Eggs | Fertilization and Cleavage | The Activation of Unfertilized Eggs | The Growth and Implantation of the Blastodermic Vesicle | Summary and Recapitulation | Bibliography | Figures | Historic Disclaimer

Cite this page: Hill, M.A. 2017 Embryology The Eggs of Mammals (1936) 9. Retrieved October 22, 2017, from https://embryology.med.unsw.edu.au/embryology/index.php/The_Eggs_of_Mammals_(1936)_9

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