The Eggs of Mammals (1936) 7

Chapter VII Fertilization and Cleavage

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Pincus G. The Eggs of Mammals. (1936) The Macmillan Company, New York.

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The Eggs of Mammals

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

The events occurring at fertilization in the fallopian tubes have been subject to detailed examination chiefly in polyovular mammals, e.g., the rabbit, rat, mouse, ferret, etc. In all cases the sperm surround the ova embedded in the mass of follicle cells, and penetrate to the ova causing the follicle cells to fall away at the same time. That the sperm swarm present in the tubes is actively responsible for the falling away of the follicle cell mass is abundantly evident from numerous recent observations of fertilization in the rabbit (Pincus, 1930; Yamane, 1930, 1935; Pincus and Enzmann, 1932, 1935). As described previously rabbit ova in does mated to sterile bucks begin to separate out of the follicle cell mass by 16

hours after copulation at the earliest, and the process is normally completed between the 17th and 19th hours. In fertile matings free ova have been observed as early as 113/^ hours after coitus, and all ova are invariably free by the 14th hour. Furthermore, when freshly o\ailated ova from sterile matings are placed in vitro with sperm suspensions there is a rapid dispersion of the surrounding follicle cells which does not occur in control cultures of ova in sperm-free media. Similar phenomena have been observed by Gilchrist and Pincus (1932) in the rat (Figures 23 to 25) and by Pincus (unpublished observations) in the mouse.

Fig. 23, Rat ovum recovered from the tubes at 16 hours after a sterile mating. Note surrounding folUcle cells. (From the Anatomical Record.)

Yamane (1930) has ascribed the phenomenon of follicle cell dispersion to the presence of a proteolytic enzyme in the spermatozoa. He was able to secure a similar dispersal of

follicle cells from sperm suspensions heated to 60° C. and from preparations of pancreatin containing trypsin. Yamane (1930) believes that this J "^ '^^^^P^^^' J^^H proteolytic enzyme is also ref f^&^S^X€jli^S sponsible for the activation

of the egg since he observed polar" bodies formed in rabbit ova exposed to the suspensions of dead sperm and to the enzyme preparations.

Pincus and Enzmann (1935) have examined this situation in some detail. Sperm suspensions free of seminal fluid were obtained from the vas deferens of adult rabbit males. Dilutions were made with a buffered RingerLocke solution at pH 7.3 — 7.5. The ova were taken at 12 H to 153^ hours after copulation from rabbit does mated to sterile (vasectomized) males ; these ova were invariably well embedded in the massed follicle cells. The procedure followed was to place the massed ova in the sperm suspension and incubate for at least two hours. All ova were examined at two hours after semination and in some instances where no obvious signs of fertilization were observed incubated for 12 hours.

Fig. 24. Rat ovum of Fig. 23 after 2 hours with Hving sperm. Note absence of follicle cells and protrusion resembling a polar body. (From the Aiiatomical Record.)

Fig. 25. R.it ovum recovered 11 hours after sterile mating and incubated with living sperm for 2 hours. Note shrunken vitellus and two polar bodies. (From the Anatomical Record.)

In most instances the ova were fixed in Bouin's solution and sectioned in order to determine the nuclear condition. The

TABLE IX

The Effect of Various Concentrations of Live Sperm upon Freshly Ovulated Rabbit Ova. (From the Journal of Experimental Zoology)

Concentration OF

Effect on Cumulus

Date

Sperm per

MM. 3

Cell Mass

Effect on Eggs

6/II/34

(undiluted)

Destroyed in 2 to

2 polar bodies; ova completely

185,000

3 minutes

dissolved after 24 hours

6/II/34

92,500

Destroyed in several minutes

2 polar bodies

20/1/34

(undiluted)

Destroyed very

1 polar body after two hours;

90,000

rapidly

polyspermy probable because of very active sperm suspension

10/III/34

80,000

Destroyed

2 polyspermic ova, one polar body; one monospermic with 2 polar bodies

16/1/34

(undiluted) 62,500

2 polar bodies; fertilized

20/1/34

55,000

}f

1 polar body

20/1/34

40,000

"

1 polar body

26/1/34

38,400

}j

2 polar bodies; fertihzed

10/III/34

30,000

Destroyed in 20

1 egg with 3 sperm attached and

minutes

2 polar bodies; 2 eggs with single sperm attached and 2 polar bodies; not incubated

6/II/34

32,200

Destroyed

2 polar bodies; fertilized

6/II/34

30,000

>)

1 polar body; no sperm entry

26/T/34

25,000

}f

2 polar bodies; fertihzed

26/1/34

14,300

Partly destroyed

1 polar body; not fertilized

26/1/34

10,700

" "

6/II/34

10,100

" "

6/II/34

8,000

n ;>

26/1/34

7,200

)} ft

6/II/34

4,000

" "

26/1/34

3,600

>> }f

2/II/34

6,000

Destroyed almost

1 polar body; \

at once

no fertilization)

2/II/34

3,000

Destroyed in

1 polar body; ( rat

2 minutes

no fertilization/ sperm

2/II/34

1,000

Destroyed in

1 polar body; \

33^ minutes

no fertilization/

sectioned ova of the experiments listed in Table IX invariably showed true polar bodies; no achromatic extrusions were observed. Furthermore, all ova with two polar bodies contained either attached sperm or male pronuclei, whereas all ova with single polar bodies showed no signs of sperm entry with the exception of two heavily polyspermic ova. Polyspermy may prevent the second polar division, but probably only when extremely active and dense sperm suspensions are used. The presence of two polar bodies may therefore ordinarily be taken as a sign of activation.

It is evident from the data of Table IX that both the degree and speed of dispersion of the follicle cell mass is roughly proportional to the concentration of the sperm suspensions used and that those sperm concentrations which fail to effect a complete dispersion of the follicle cell mass also fail to cause second polar body formation. But rat sperm as well as rabbit sperm can effect complete dispersal of the follicle cells about rabbit ova and yet no polar body formation occurs. This seems to indicate that the activation of the o\aim and follicle cell dispersion involve distinct and separate reactions.

The data of Table X substantiate this conclusion for they show that sperm-free fluid from the vas deferens and sperm suspensions heated to 60° C. for a few minutes cause typical follicle cell dispersion but no polar body formation. That a heat-labile substance is involved in the follicle cell dispersion is evidenced by the data on ova exposed to boiled sperm suspensions. This substance is probably carried by the sperm since similar follicle cell dispersion in vivo is brought about by sperm that have travelled the length of the oviducts.

Yamane (1930) found that both rat and horse spermatozoa caused second polar body formation in rabbit ova, and since his pancreatin solutions also caused the same result he concluded that a non-species-specific sperm-borne tryptase was involved. As shown in Table IX above rat sperm were ineffective in causing second polar body formation, but they were more potent than rabbit sperm suspensions in causing follicle cell dispersion. Accordingly Pincus and Enzmann (1936a) undertook the experiments with trypsin preparations presented in Table XL

TABLE X

The Effect of Dead Sperm Preparations and Sperm-Free Seminal

Fluid upon Freshly Ovulated Rabbit Ova.

Experimental Zoology)

(From the Journal of

Treatment of Sperm

Effect on

TTimrr^'T' m>j T^^nnci

Dilution of

Date

Suspensions

Cumulus Mass

Hjr r liiK^x KJj^t xjouo

Preparation

10/1/34

Heated to 60° C.

Destroyed in

1 polar body;

Undiluted

all sperm dead

10 minutes

no fertilization

30/III/34

Completely dessi

Destroyed in

1 polar body

Made up to

cated at room

2 minutes

original vol

temperature; all

ume

sperm dead

30/III/34

t)

Destroyed in 5 minutes

))

Made up to original volume and diluted 3^

30/III/34

))

Destroyed in 11 minutes

>f

Made up to original volume and diluted 34

30/III/34

>»

Destroyed in 21 minutes

>>

Made up to original volume and diluted Vs

12/1/34

Centrifuged at 3000 R.P.M. for 5 minutes; heated to 60° C; supernatant fluid used

Destroyed

>j

Undiluted

17/111/34

Centrifuged at 3000 R.P.M. for 40 minutes; heated to 60° C; supernatant fluid used

Destroyed in 3 minutes

))

Diluted 1/40

17/III/34

}}

Destroyed in 43/2 minutes

}f

Diluted 1/80

17/111/34

n

Destroyed in 73^2 minutes

Diluted 1/120

17/III/34

j>

Destroyed in 8 minutes

))

Diluted 1/160

12/1/34

Centrifuged at 3000 R.P.M. for 5 min

Destroyed

3 eggs out of 9 with sec

Undiluted

utes; not heated;

ond polar

supernatant fluid

body and

used; a few sperm

sperm

present

17/III/34

Centrifuged at 3000 R.P.M. for50minutes; not heated; no sperm present

Destroyed in 13^ minutes

1 polar body

Diluted 1/20

20/IX/35

Boiled for 12 minutes; all sperm dead

Left intact after 1 hour

)>

Diluted H

TABLE XI

The Effects of Exposing Freshly Ovulated Rabbit Ova to Various Solutions of Trypsin. (From the Journal of Experimental Zoology)

Trypsin Con

Date

centration

(Dry Trypsin

PER 100 c.c.

Effect on

Cumulus Cell

.Mass

Effect on Eggs

Ringer-Locke

Solution)

10/11/34

0.50

Destroyed

3 "polar" bodies in 10 minutes

10/11/34

0.25

M

Egg shrunken

10/11/34

0.125

"

>)

10/11/34

0.062

Partly destroyed

M

10/11/34

0.032

»»

"

6/II/34

25.00

Destroyed almost

6 to 10 "polar" bodies followed

immediately

by partial digestion of ova

6/II/34

21.00

"

"

17/11/34

1.00

Destroyed in 1 minute

Egg partly digested

17/11/34

0.50

Destro3^ed in 13^ minutes

1st polar body digested

17/11/34

0.25

Destroyed in 3 minutes

1 polar body, egg shrunken *

17/11/34

0.125

Destroyed in 6 minutes

>) M :tc

17/11/34

0.062

Destroyed in 14 minutes

)) i1 *

17/11/34

0.032

Destroyed in 31 minutes

>> n *

• All these ova showed irregular masses of webbed tissue in the perivitelline space.

The data of these experiments show typical folUcle cell dispersion and also ^^ polar body" formation (Figure 26). These are, however, not true polar bodies but rounded cytoplasmic masses caused by the action of the enzyme preparation upon the egg surface. Sections of the ova of these experiments showed the polar bodies" to be chromatin free. The polar bodies observed by Yamane in his pancreatin experiments were probably of this nature. The polar bodies formed in his experiments with rat and horse sperm may have also have been false polar bodies due to the strongly digestive action of the heterologous sperm suspensions, for, as we have seen, rat sperm suspensions are extremely effective as follicle cell dispersing agents even in very low concentrations. Krasovskaja (19356) believes that actual penetration and pronucleus formation occurred in his attempts to fertilize rabbit eggs with rat sperm. No figures showing actual sperm penetration are given in this paper. The nuclear configurations shown may, in fact, occur in ova cultured in vitro with no sperm added (see Chapter VIII).

Fig. 26. Rabbit ovum from sterile mating treated with trypsin solution. Note many "polar" bodies. See text.

The inamediate effect of semination (and fertilization) upon mammalian ova is a definite shrinkage of the vitellus (Pincus and Enzmann, 1932). Quantitative estimates of this shrinkage in rat eggs have been made by Gilchrist and Pincus (1932). In Table XII are presented their data on folhcular and tubal ova. They show that a 14 per cent reduction in volume occurs in fertilized tubal ova. Furthermore, when unfertilized ova are exposed to sperm suspensions a similar shrinkage occurs (Table XIII). This shrinkage is not due to polar body extrusion since it occurs in vitro within 5 to 10 minutes, and polar bodies are normally extruded at 45 minutes to 1 hour after semination in vitro (Long, 1912). The ova apparently increase somewhat in volume after this initial shrinkage. Krasovskaja (1935a) has observed an exactly similar initial shrinkage followed by a return to normal in rabbit ova seminated in vitro.

TABLE XII

The Volume of Rat Eggs in Three Stages of Development. Gilchrist and Pincus, 1932)

(From

Stage

Follicular Tubal, unfertilized 1-cell

Average Volume of Round Eggs, cu. mm.

0.000333 (1)

0.000251 0.000202

0.000023 0.000009

Average Volume of Elongated Eggs,

0.000339 ± 0.000017

0.000226 0.000200

0.000013 0.000010

Average Volume of All Eggs, cu. mm.

0.000337 ^ 0.000010

0.000234 0.000201

0.000018 0.000010

TABLE XIII

The Size of Rat Eggs under Various Conditions of Culture. (From Gilchrist and Pincus, 1932)

Treatment

Incubated in Ringer's solution alone

Incubated with live sperm

Incubated with dead sperm

Number

OF

Eggs

Average

Diameter

Immediately

AFTER

Putting Eggs on

Slide, Microns

74.4 ± 1.4 77.9 ± 1.4 72.8 ± 1.1

Average Volume Calculated, cu. mm.

0.0002 IG 0.000248 0.000204

Average Diameter Some Time

AFTER

Incubation, Microns

76.3 ± 0.4 72.7 ± 1.4 69.7 ± 1.3

Average Volume,

Calculated

cu. mm.

0.000232 0.000205 0.000179

Shrink age. Per Cent

17 12

Sperm penetration into living ova has been observed only once (Pincus, 1930); a modified fertilization cone appears to form at the point of contact. This cone very quickly subsides as is apparent also from fixed preparations of mammalian ova in the tubes {e.g., Lams and Doorme, 1908; Sobotta and Burkhard, 1911 ; Lams, 1913; and others).

The length of time that the mammalian ovum remains capable of fertilization has been largely a matter of speculation. Exact experimental inquiry has, however, been undertaken in the rabbit (Hammond and Marshall, 1925; Hammond, 1928 and 1934) and in the ferret (Hammond and Walton, 1934). Taking advantage of the fact that the

Litter Size and Fertility

TABLE XIV

IN Timed Matings of Rabbit Does. (From Hammond, 1934)

No. OF

Matings

Matings at

Hours

after

Sterile

Coitus

Hours

before ( + )

or after (— )

Ovulation

Average

Litter

Size

Matings Fertile, Per Cent

No. OF YOUNQ

PER Mating Made

(a) All strains together (52 different does used)

323

Normal

+ 10

6.4

79.6

5.3

6

5

+ 5

6.4

82.3

5.3

65

6

+ 4

4.7

64.6

3.0

55

7

+ 3

4.4

58.2

2.5

81

8

+ 2

4.2

42.0

1.8

85

9

+ 1

3.6

37.6

1.4

68

10

4.5

22.1

1.0

57

11

- 1

3.4

12.3

0.4

63

12

- 2

3.2

6.3

0.2

(b) C strain (17 different does used)

131

Normal

+ 10

7.4

75.0

5.0

25

6

+ 4

5.4

52.0

2.8

18

7

+ 3

3.7

55.6

2.1

22

8

+ 2

2.8

27.3

0.8

21

9

+ 1

4.3

28.6

1.2

20

10

4.5

10.0

0.4

18

11

- 1

19

12

_ 2

(c) E strain (21 different does used)

90

Normal

+ 10

8.1

80.0

6.5

3

5

+ 5

7.0

100.0

7.0

19

6

+ 4

5.8

63.2

3.7

23

7

+ 3

5.6

65.2

3.8

37

8

+ 2

4.9

48.6

2.4

48

9

+ 1

3.6

41.7

1.5

25

10

5.4

36.0

2.0

21

11

- 1

4.2

23.8

1.0

21

12

- 2

4.0

9.5

0.4

(d) F strain (14 different does used)

102

Normal

+ 10

4.0

84.3

3.4

3

o

+ 5

5.5

66.6

3.7

21

6

+ 4

3.4

81.0

2.7

14

7

+ 3

2.7

50.0

1.4

22

8

+ 2

3.8

5.5

1.7

16

9

+ 1

2.7

47.5

1.0

23

10

2.2

37.4

0.4

18

11

- 1

1.5

11.1

0.2

23

12

- 2

2.5

18.7

0.2

rabbit OMilates at 10 hours after copulation and the ferret at about 30 hours, Hammond and his coworkers undertook a series of matings using an initial sterile mating to initiate the o\ailation stimulus and then fertile mating to permit sperm access to ova at successively later intervals. In the

80

- 8

- , —

y

70

- 7

_

\

W 1-1

geo

-|6

\ \

u

K

'. \

u.

2

___.

- — — > \ \

^50

-h5

_

\ \ \

a

Ed

\ '--A A

H

9

\ "V / \

<40

-^4

—

\ X/ \

S

>

\ X V

Ui

<

O30

- 3

—

\

^

\ \

20

- 2

~

\\

10

- 1

'

s \

\ \

fi

It

I

1 1 1 1 1 1 1 1

+10

+ 5 +4 +3 +2 +1 -1 -2 OVULATION

AVERAGE UTTER SIZE

^c OF MATINGS WHICH WERE FERTILE NUMBER OF YOUNG PER MATING

HOURS INTERVAL BEFORE (I-) OR AFTER (-) OVULATION

Fig. 27. Fertility of matings made at different intervals of time before or after mating (all strains). (From the Journal of Experimental Biology.)

most extensive series of rabbit matings (Hammond, 1934) employed three inbred strains of rabbits in order that homogeneous conditions of fertility might exist in his experiments. The data of his experiments are given in Table XIV, and a graphical representation in Figure 27.

It is at once obvious from these data that matings to fertile bucks made after the 5th hour following a sterile mating show a decline both in absolute (per cent of fertile matings) and relative fertihty (number of young produced). WTien matings are made to fertile bucks at twelve hours after the sterile copulation, i.e., at two hours after o\ailation minimum fertility is attained.

In order to make quite certain that the cause of the smaller litters produced after the experimental matings made late in relation to ovulation was due to the ova not being fertilized and not to any interference with the process of ovulation or other causes, a few does so mated were killed during pregnancy and the number of corpora lutea {i.e., ova shed) compared with the number of foetuses present. The results are given in Table XV, and demonstrate that there is a decrease in the number of ova fertilized in the later matings. This implies that the sperm reach the portion of the tubes containing the ova at a time when these ova are for some reason no longer fertilizable.

TABLE XV

The Percentages of Rabbit Ova Fertilized in Matings Made at Various Times before and after Ovulation. (From Hammond, 1934)

Matings at

Does

Number of

Ova Not

Hours

before ( + )

or after

(-) Ovulation

Hours

after Sterile Coitas

Number

Strains

Ova Shed

Normal Foetuses

Atrophic Foetuses

Ova Not Fertilized

LIZED,

Per

Cent

6 7 8 9 11

+ 4 + 3 + 2 + 1 - 1

2

2 3 2 2

E E,F

E

E E, F

25 23

41 25 19

15

10

18

6

4

2 3

5

8

7

18 19 15

32 35 44

76 79

On the basis of Heape's (1905) observations that rabbit sperm reach the tops of the tubes in about 4 hours after coitus, Hammond concludes that rabbit ova can remain fertilizable for at most 6 hours after o^Tllation, by allowing a 2-hour postovulatory interval in the matings made at 12 hours after the ovulation-inducing mating. This period coincides approximately with the time {i.e., 7 hours) that it takes for the ova of sterile matings to begin to separate from the follicle cell mass and start their free travel down the tubes. Hammond concludes therefore that the presence of the plug of massed ova is necessary for fertiUzation. He reasons as follows:

'^The plug, of liquor folliculi and detritus, containing the ova dams up the top of the Fallopian tube and remains there for some 4 (in fertile matings) to 7 (in infertile matings) hours, during which time the ascending sperms are collecting in its lower layers (see Figure 28). The accumulation of sperms so effected ensures that sufficient shall be available to fertilise the ova as they emerge from the plug. As the sperms are put in progressively later than normal in relation to the time of ovulation, the accumulation of sperms becomes progressively less and the chances of all the ova

FALLOPIAN TUBE

PLUG CONTAINING OVA

Fig. 28. Diagram illustrating how the chances of the ova becoming fertilized are reduced as the interval between mating and ovulation is reduced, a = amount of sperm swarm which would accumulate if mating were made at the ordinary time — 10 hours before ovulation, b = amount of sperm swarm which would accumulate if mating were made 4 hours before ovulation. (From the Journal of Ex-perimental Biology.)

becoming fertilised are reduced in proportion to the time the fertile mating is delayed with reference to the time of o\ailation.

^^The ascent of the sperms can be represented as a curve (see Figure 28 and Hammond and Asdell, 1926) or as a swarm (in the statistical sense). The apex of the sperm swarm (shown, in order to assist visualisation of the problem, very diagrammatically in Figure 28) reaches the top of the tube just at the time the plug is formed, i.e., at ovulation, and so during the time that the plug exists (about 4 hours) it dams up but few sperms as compared with a normal mating made 10 hours before ovulation when the sperm swarm has ascended further (to the point a in Figure 28).'^

While Hammond's deductions are entirely reasonable, it is possible that the 6 hours of fertilizable life allotted to rabbit ova is possibly too short since in normal matings 13^ to 3 hours are required by the sperm to reach the ova. This would make the critical period some 73^ to 9 hours long. Furthermore it is not the arrival of the first sperm that is effective, since as we have previously seen (pages 77 to 78) a definite minimal sperm concentration is necessary for both folUcle cell dispersion and fertilization. If the critical period were thereby further lengthened by 1 to 2 hours it would coincide almost exactly with the time when the ova separating out of the tubal plug begin to acquire a coating of albumen. This coating is impervious to sperm (Pincus, 1930).

Similar experiments of Hammond and Walton (1934) with the ferret show that fertile matings made as late as 30 hours after ovulation result in the production of young. The reasons for the maintenance of the fertilizing capacity of ferret ova for as long as 30 hours are not deducible in detail since the exact tubal history of ferret ova is not known. Hammond and Walton attribute the greater length of fertilizable life in this case to the longer time it takes for the ova to traverse the oviduct, e.g., 5 to 6 days in. the ferret compared with 3H days in the rabbit and the presumably correlated slower dissolution of the plug of massed ova.

In the spontaneously o\ailating mammals the fertilizable life of the ova is also of short duration, but exact data are not available since it is ordinarily difficult to ascertain the specific time of ovulation. Hartman (1924) has shown that opossum ova traverse the tubal portion of the oviduct in 24 hours and that upon entry into the uterus unfertilized ova are definitely degenerated. Charlton (1917) found clear signs of degeneration in unfertilized tubal mouse ova by two days after parturition. Since post-partum ovulation occurs in the mouse at about 14 hours after parturition (Long and Mark, 1911) mouse ova may be said to retain cytological normality for about 35 hours. In the rat ova present in the first third of the oviduct appear cytologically normal (Mann, 1924). According to the data of Long and Evans (1922) the ova remain in this portion of the oviduct for about 33 hours. Hartman's (1932a) data on timed matings in Macacus show that fertile matings occur only between the 9th and 18th days of the menstrual cycle with maximum between days 11 and 16. This, of course, does not imply that the ova are fertilizable for several days, but presumably that ovulation may occur at any time during the critical 9 day period. Matings time in relation to the onset of oestrus in the sheep (Quinlan, Mare and Roux, 1932) and the pig (Lewis, 1911) indicate a maximum period of fertility of 48 hours.

It is unnecessary in this monograph to discuss the cytological details of fertilization and cleavage in mammalian ova, since these are now textbook commonplaces. Our interest is primarily in the physiological mechanisms underlying these events and their relation to the dynamics of growth and development. We shall again discuss certain aspects of the fertilization process in the chapter dealing with the activation of unfertilized eggs. Now we shall turn our attention to the relatively scant data that deal with the mechanism of cleavage in tubal ova.

Until fairly recently no very accurate data on the rate of cleavage in tubal ova have been available. This has been due in part to the difficulty of timing ovulation. Even now it is possible to construct only approximate growth curves for a limited number of species. These curves are presented in Figure 29. It will be noted that rabbit ova cleave much more rapidly than those of the other species (see Plate VII). It is a matter of some interest to ascertain whether this difference in the cleavage rate is the result of an especially stimulating tubal environment in rabbits, or whether the cleavage rate is an inherent property of the ova. The data on the monkey were, in fact, deduced from Lewis and Hartman's (1933) observations of cleavage in vitro, and may be taken to indicate that segregation from the tubes results in no great acceleration of cleavage since the growth rate remains at about the level of the other slow-cleaving species. The writer has transplanted mouse ova into the fallopian tubes of the rabbit and has noted no increase in the cleavage rate over a period of 72 hours.

RABBIT

MONKEY

GUINEA PIG

MOUSE

DAT'

20 40 60 80

Fig. 29. Showing the cleavage rates of tubal ova in various species of mammals. Abscissa: time in hours after copulation. Ordinate: number of cells. The rabbit = data of Gregory, 1930, and Pincus, 1930. The monkey = data of Lewis and Hartman, 1933. The guinea pig = data of Squier, 1932. The mouse = data of Lewis and Wright, 1935. The rat = data of Gilchrist and Pincus, 1932. The pig = data of Heuser and Streeter, 1929.

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

Fig. 13

Fig. 14

Fig. 15

Fig. 16

Fig. 17

Fig. 18

Fig. 19

Fig. 20

Fig. 21

Plate VII (Caption on facing page.)

Fig. 22

Castle and Gregory (1929; also Gregory and Castle, 1931) have, in fact, found certain definite congenital differences in cleavage rate between different races of rabbits. A resume of their data is given in Table XVI. The animals of their large race (A) attain an average adult weight of about 5500 grams in females and 5400 grams in males. The corresponding adult weights in the small race (B) are 1500 grams for females and 1400 grams for males. The various hybrid combinations show roughly intermediate adult weights. Their data show clearly that certainly beyond the 32nd hour after copulation the cleavage rate is fastest in the large race animals and the expected sort of intermediate rates occurs in the various hybrid combinations. It is entirely possible that even the earliest cleavages do actually occur sooner in large race animals since large does ovulate later than small does and therefore their ova should be fertilized later. The number of mitoses in cleaving eggs of the large races also exceeds those in the small race, as the data in the columns labelled prospective" indicate. Since this difference is consistently present in reciprocal hybrids between the races the implication is that the sperm nuclei also participate in the control of the cleavage rate.

In spite of the inherent differences in the speed of segmentation the processes of differentiation occur at the same time in the large and small size rabbits. Thus the blast o Plate VII. All photographs on this plate were made from the living rabbit eggs in Locke's solution, as soon as possible after removal from oviduct or uterus at an enlargement of 180 diameters (apochromatic objective 16 mm., compensating ocular 8). They are arranged in order of development and show the principal features of cleavage and formation of segmentation cavity. It will be noted that the trophoblast is precocious in its differentiation as compared with the remainder of the egg, and as soon as the trophoblast becomes histologically different one sees fluid begin to accumulate within the egg, thereby forming the segmentation cavity.

Figs. 4 to 9, Litter C 43, 25 hours after coitus. Fig. 4, one-cell stage with two polar bodies; Fig. 5, one cell, with coarse granules, perhaps abnormal; Figs. G to 9, showing two primary blastomeres, one tending to be larger than other. Figs. 10 and 11, Litter C 36, 28^:^ hours after coitus. Four-cell stage with crossed arrangement of blastomeres. Figs. 12 to 14, Litter C 45, 32 hours after coitus. 5, 6 and 8-cell stages. In Fig. 13 the cell at top is just dividing. Fig. 15, Litter C 35. 16-cell stage. Fig. 16, Litter C 41, 55 hours. Morula of about 32 cells. Fig. 17, Litter C 32, Q9% hours. Smooth surfaced morula. Fig. 18, Litter C 38, 71^ hours. Differentiated trophoblast cells on surface. Fig. 19, Litter C 33, 76^ hours. Fluid beginning to collect in cleft between trophoblast and inner-cell mass. At this time the albumen coat is at its maximum. Fig. 20, Litter C 33, 76^ hours. Subtrophoblastic lakelets of fluid determining early appearance of segmentation cavity. Fig. 21, Litter C 34, 90 hours. Definite segmentation cavity. Note demarcation between trophoblast and inner-cell mass. Fig. 22, Litter C 42, 92 hours. Zona much stretched and layer of albumen much thinned out. Inner-cell mass flattening into typical germ-disc. From Gregory, 1930.

TABLE XVI

The Mean Number of Blastomeres per Ovum at Various Times after Copulation in Large and Small Rabbits and in Certain Hybrids BETWEEN Them. (From Castle and Gregory, 1929, and Gregory and Castle, 1931)

Hours

AITEK

Number

Number

Mean Number OF

Probable

Copula

Race

OF

Does

OF

Eggs

Blasto

Error

tion

meres

32M

A (actual)

3

31

4.06

—

>>

A (prospective)

3

31

4.29

—

tf

B (actual and

prospective)

3

12

4.41

—

40

A (actual)

4

45

9.94

±0.24

A (prospective)

4

45

10.82

—

B (actual)

8

27

8.29

±0.19

B (prospective)

8

27

8.37

—

AB (actual)

1

9

8.44

—

AB (prospective)

1

9

8.60

—

41

A (actual)

3

22

11.64

±0.44

A (prospective)

3

22

12.68

—

B (actual)

()

21

8.62

±0.47

B (prospective)

G

21

9.09

—

BD (actual)

2

11

8.63

—

BD (prospective) B and BD combined

2

11

9.18

—

(actual)

8

32

8.62

—

AD (actual)

3

20

9.25

—

AD (prospective)

3

20

9.55

±0.36

48

F (actual)

4

28

21.75

—

F (prospective)

4

28

22.80

—

B (actual)

4

15

14.00

—

B (prospective)

4

15

14.50

—

A = large race.

B = small race.

AB = Fi hybrid.

BD = seven-eights small (D = AB XB).

F = three-quarters large (AB X A).

actual = number of blastomeres observed.

prospective = number of blastomeres observed plus the number of mitoses.

dermic vesicle forms at the end of the 3d day (Plate VII, Figs. 18-20), and the embryonic disc by the 168th hour after coitus. Castle and Gregory therefore attribute large size to an inherent mitotic intensity independent of differentiation potentials.

The ova of the rabbit begin their differentiation early in comparison with the eggs of other species. Thus Gregory (1930) detected the beginning of the formation of the inner cell mass just after the 16-cell stage at about 47 hours after coitus (37 hours after ovulation) and the cavity of the blastodermic vesicle may begin to form while the ova are still in the tubes. Guinea pig (Squier, 1932) ova enter the uterus in the 8-cell stage at the end of the 3d day after copulation and the blastodermic vesicles form only in the uterus at about 43^2 days after coitus. In the rat (Huber, 1915) the ova enter the uterus during the 4th day after coitus in about 12 cells and start to form the blastodermic vesicle during the 4th to 5th days post coitum, and in the mouse (Enzmann, Saphir and Pincus, 1932; Lewis and Wright, 1935) blastocyst formation occurs in the uterus during the 4th day after copulation.

The physiological factors governing the cleavage of mammalian ova have been scarcely examined. It has already been stated that the whole course of cleavage of rabbit eggs may proceed normally in vitro and in heterologous as well as homologous blood plasma (Pincus, 1930). This would seem to imply that no special environmental factors supervene in the tubes. On the other hand the ova of mice, rats and guinea pigs do not cleave under the ordinary (or a variety of) tissue culture conditions. The reasons for this species difference are not known though the superior vitality of rabbit ova has been attributed to their unique albumen coating; but Lewis and Hartman (1933) have over a period of approximately 24 hours, observed the regular cleavage in vitro of a monkey o\aim which lacks an albumen coating.

In the case of those ova which have not undergone cleavage in vitro one can only deduce that some limiting factor obtaining in vivo has not been duplicated. Since it is known that the secretory activity of the tubal epithelium is under hormonal control of the ovary (c/. Snyder, 1923) it is possible that a special contribution to the economy of cleaving ova is made by a hormonally induced secretion. The cleaving ova of all mammals journey through the tubes during the early life of the corpus luteum. The secretory activity of the tubal epithelium changes markedly during the transition from the oestral to the luteal phase. Furthermore, it is possible that the ovarian hormones themselves may directly affect the cleavage process. Oestrin, for example, definitely stimulates the mitotic activity of the vaginal epithelium, progestin inhibits uterine mitoses, etc.

Accordingly Burdick and Pincus (1935; also Pincus and Kirsch, 1936) have investigated the effect of ovarian hormones upon the development of rabbit and mouse ova. They found that the injection of large amounts of oestrin in no way affected the cleavage process although ova in the early uterine stages degenerate and die when only moderate amounts of this hormone are injected (see Tables XXIII to XXV, pages 118-120, 122). That the hormone injected definitely affected the tubal tissue was evidenced by the fact that in both mice and rabbits an effective closure of the tubo-uterine junction was attained, and in rabbits both the contractile activity and the histological appearance of the tubal tissue were definitely altered to the oestrus type. In addition (Pincus and Kirsch, 1936) it was found that rabbit ova grow^n in cultures containing appreciable amounts of oestrin continued to cleave at the normal rate. Finally fertilized rabbit ova in 1- and 2-cell stages were injected into the fallopian tubes of does on heat (and therefore lacking corpora lutea), and these were found to develop normally up to the early blastocyst stage. Corner (1928) had already shown that in bilaterally ovariectomized rabbit does egg development stops at the early blastocyst stage. The segmentation processes appear, therefore, to be independent of the secretory activity of the ovaries, and of any effect that the ovarian condition may have upon tubal secretion. Rabbit ova will, indeed, go through the morula stage in a carefully balanced buffered Ringer-Locke solution, indicating a fairly complete lack of dependence upon any special organic nutrition. It has, of course, been repeatedly noted by observers of living material {e.g., van Beneden, 1875; Gregory, 1930; Gilchrist and Pincus, 1932; Squier, 1932) and by those who have examined fixed speci mens (Sobotta, 1895; Huber, 1915; and others) that mammalian ova show no appreciable increase in size until the blastocyst stage. The most convenient approach to the study of the physiological processes underlying segmentation has involved the study of the respiratory processes (Warburg, 1908-14 ; J. Loeb and Wasteneys, 1912-15; J. Loeb, 1913; Runnstrom, 1930; Whitaker, 1933; and others). Mammalian ova are available in such small numbers that exact quantitative measurements of respiratory activity are difficult to make and have not been made. Nonetheless some indication of the nature of the underlying processes may be had by the use of specific poisons known to combine with and inhibit the reactions of definite components of the chain of reactions involved in respiration. Thus HCN is known to combine with ironcontaining enzyme phaeohemin which is the initial activator in the aerobic phaeohemin-cytochrome chain (Warburg, 1932) and so to inhibit the respiration involving phaeohemin activity. Cyanide also inhibits the cleavage of ova of non-mammalian forms (Lyon, 1902; J. Loeb, 1906; see Needham, 1932), as does an oxygen-free medium (J. Loeb, 1895). Runnstrom (1935) has demonstrated that the mitotic process at segmentation in sea-urchin eggs is not dependent upon the level of respiration since the addition of pyocyanine to cyanide-inhibited egg suspensions restored oxygen consumption to normal levels but no division ensued.

Rabbit ova presumably develop in a medium relatively low in oxygen, since the oxygen tension of the abdominal cavity, and by inference that of the tubes (which have free access to abdominal fluids), is 40 mni. Hg (Campbell, 1924) as compared with 150 mm. Hg, the oxygen tension of the air. It is of interest to inquire whether the segmentation of rabbit ova is Hnked with the aerobic phaeohemin system. Pincus and Enzmann (19366) have added KCN in appropriate concentration to cultures of cleaving rabbit eggs and the segmentation has ceased. Cinematographs of these ova indicated that the eggs were not killed" by the poison since they exhibited the cyclosis (cytoplasmic movements) typical of living ova. Similar experiments with iodoacetamide added to the cultures showed normal cytoplasmic activity of the ova but a limited amount of cleavage. lodoacetamide presumably combines with the coenzyme concerned in the reduction of pyruvic to lactic acid (Meyerhof and Kiesling, 1933) so that the inhibition of both the oxygenactivating system and its presumable substrate system results in the arrest of cleavage. While the exact coupling of the respiratory system with the mitotic mechanism has yet to be delineated these data do demonstrate that the fundamental processes are aUke in manomalian and nonmammahan ova.

We have seen that rabbit ova may be fertilized and cultured in vitro. It is a matter of some importance' to determine whether such ova may give rise to normal rabbits. Accordingly the writer (see Pincus and Enzmann, 1934) undertook the transplantation of such ova into the oviducts of pseudopregnant rabbit does and found that ova fertilized in vitro and also normally fertilized ova kept in culture during the cleavage period apparently resumed normal development after transplantation as evidenced by the production of normal young at term. It is a matter of some interest to note that one set of ova had failed to cleave during 20 hours in culture but nonetheless young were obtained.

The development of a technique for the transplantation of mammalian ova into the oviducts makes possible the testing of a number of problems of development hitherto inaccessible. As we shall see later (Chapter IX) it is necessary that a progestational uterus be available for ensuring differentiation of uterine stages. Thus Biedl, Peters and Hof stater (1922) transplanted rabbit ova into non-pregnant uteri in some 70 experiments and in only one doubtful case were young recovered. Nicholas (19336) transplanted the isolated blastomeres of the 2-cell stage in the rat under the kidney capsule and observed varying degrees of development of the three germ layers and their various derivatives. The writer has transplanted single blastomeres of 2-cell rabbit embryos into the tubes and obtained normally differentiating, but small sized blastodermic vesicles from the pseudopregnant uteri of the recipient does. The physiological processes occurring in such embryos are of extraordinary interest and certainly deserve further investigation.

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Pincus G. The Eggs of Mammals. (1936) The Macmillan Company, New York.

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The Eggs of Mammals

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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