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Fertility and Sterility as Revealed in the Study of Fertilization and Development of Rabbit Eggs
r Fertility and Sterility as Revealed in the Study of Fertilization and Development of Rabbit Eggs M. C. Chang, Ph.D. (Cambridge)
in mammals is primarily dependent upon the viability of the spermatozoa and eggs, the facility as well as the timing of their union (fertilization), and the normal development of fertilized eggs until birth. The process of fertilization certainly is the most important phase in reproduction that affects fertility. Nevertheless, our knowledge of fertilization of mammals is mainly based upon the descriptions of earlier workers. 26 • 39 The first part of this paper discusses some of the controlling factors of fertilization in the light of original observations and recent contributions by other workers. The second part deals with the factors that affect the normal development of fertilized eggs since it has been estimated that in the mammal about 40 per cent of eggs fail to develop into young. 33
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FERTILIZATION OF RABBIT EGGS Since ovulation in the rabbit is not spontaneous but occurs about 10 hours after copulation or after intravenous injections of gonadotrophic hormones, the time sequence of fertilization can be estimated with more accuracy than in other mammals. The rapid growth of follicles begins about six hours From the Worcester Foundation for Experimental Biology and Tufts College Medical School. Ortho Award Essay, presented at the Sixth Annual Conference of the American Society for the Study of Sterility, San Francisco, June 24, 1950. The author wishes to acknowledge his gratitude to Dr. C. Pincus for reading the manuscript and to Miss Jacqueline Choiniere for the preparation of histological sections. This work is supported by a grant from the Committee on Human Reproduction, National Research Council, acting on behalf of the National Committee on Maternal Health. 205
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after copulation andthe thin follicular stigma dilates into a pimple-like cone a few minutes before ovulation. The rupture of the cone, which is not explosive, involves only 5 to 7 seconds. 25 • 44 The ciliated infundibulum of the fallopian tube embraces the ovary at the time of ovulation. The transportation of eggs from the ovary to the ampulla is due to the activity of the muscular tissues connecting the ovary, the tube, and the uterus and to the contraction of the tube itsel£.45 The transportation of the eggs from the ampulla through the isthmus to the uterus is perhaps mainly dependent upon the muscular contraction of the tube rather than the ciliary activity according to observations in the rat and mouse. 1 • 9 The transportation of spermatozoa from the vagina to the upper third of the tube, where fertilization occurs, takes about 4 hours. 24 In the uterus, the progress of sperm is mainly due to their own motility/7 in the tube, it is perhaps mainly due to the contraction of the tube. 32 Since ovulation occurs lO hours after mating, the spermatozoa must wait 6 hours for the arrival of the eggs. This interval may allow time for an adequate number of spermatozoa to accumulate or physiologic changes of sperm in the female tract. Since the first cleavage of fertilized rabbit eggs occurs 23 to 24 hours after mating,2, 18 the procr"s of fertilization from ovulation to the first cleavage takes 13 to 14 hours. In the present study, the rabbits were superovulated36 in order to obtain a large number of eggs for observation. In one series,. fresh eggs were Hushed from the tube with serum diluted with an equal volume of saline at various intervals after insemination. They were then examined and photographed under a phase microscope. In another series, in which animals were inseminated with about the same number of spermatozoa, single tubes of animals were removed separately at different times and fixed immediately. Altogether 6 tubes from 3 animals were sectioned and stained with Masson's trichromatic stain. Just after ovulation, the eggs reach the ampulla of the tube surrounded with follicular cells or cumulus oophorus. The follicular cells, immediately attached to the individual eggs, constitute the corona radiata, here called PLATE I 1. A living rabbit egg recovered just after ovulation. In cumulus mass, corona cells with long processes. Slightly pressed. Phase microscope. 8 mm. objective used. FIGURE 2. Two living eggs recovered 2 hours after ovulation. Two polar bodies visible in one egg. 8 mm. objective. FIGURE 3. One section of an egg in situ 2 hours after ovulation. In cumulus mass. One spermatozoan in the zona pellucida, at 6 o'clock position. 4 mm. objective. (255 X) FIGURE
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corona cells. Usually several eggs are massed together in a mucus clot with loosely suspended follicular cells, referred to here as the cumulus mass. The rabbit eggs recovered from the ampulla of the tube just after ovulation (lm~ hours after artificial insemination and ovulation-inducing injection) were still in the cumulus mass. The corona cells were thick, with long processes apparently penetrating to the zona pellucida while the cells of the outer layer were long and sharp (Fig. 1). Two hours after ovulation, the cumulus mass was more or less dispersed both by the contraction of the tube and by the liquefying action of hyaluronidase, liberated by the spermatozoa present, on the mucus cumulus mass. The corona cells, however, were still closely attached to the zona pellucida though they were not so thick and the cells of the outer layer were now round and smooth (Fig. 2). The tearing off of the corona cells had started. One or two spermatozoa could be seen in the perivitelline space though they are not clear in the photograph of the intact eggs (Fig. 2). Figs. 3, 4, and 5 are sections of 3 eggs in situ 2 or 3 hours after ovulation. Special attention is called to the presence of spermatozoa in the egg or in the zona pellucida in spite of the fact that some eggs were still in the cumulus mass and that all of them had corona cells attached. By 4 hours after ovulation the outer layer of the corona cells was tom off. Several moving spermatozoa were seen in the perivitelline space during photography under the microscope. The second polar body was formed in all the eggs recovered at this time (Fig. 6). The male pronucleus was well formed while the female nuclear material was still in the form of chromoPLATE II FIGURE 4. One section of an egg in situ, 2 hours after ovulation. One spermatozoan in the ooplasm at 2 o'clock position. One spermatozoan in the zona peIIucida at 4 o'clock position. 4mm. objective. (255 X) FIGURE 5. One section of an egg in situ 3 hours after ovulation. In cumulus mass with two polar bodies indicating the penetration of a spermatozoan. 4 mm. objective. (255 X) FIGURE 6. A living egg recovered 4 hours after ovulation. Thin corona radiata. 8 mm. objective. FIGURE 7. One section of an egg in situ 4 hours after ovulation, showing male pronucleus (right) and female nuclear material (left). 4 mm. objective. (255 X) FIGURE 8. One section of an egg in situ 4 hours after ovulation. Male pronucleus visible. 4 mm. objective. (255 X) FIGURE 9. A living egg recovered 5 hours after ovulation. Very few corona cells left. 4 mm. objective.
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somes as shown in the sections (Figs. 7 and 8). Fig. 9 shows a living egg recovered 5 hours after ovulation; only a few corona cells were still attached to the zona pellucida. Six hours after ovulation, the denudation (tearing off of the corona cells) was complete in most cases and the two pronuclei were close together (Fig. lO). The sections of eggs in situ at this time are shown in Fig. 11 with the male and female pronuclei approaching each other. Eight hours after ovulation, the two pronuclei were already fused in a few cases, and there were many sperm in the zona pellucida and perivitelline space, as shown in the sections (Figs. 12 and 13). In a group of fertilized eggs recovered from the tube 14 hours after ovulation, some were cleaved and others were uncleaved. The albumin coat had started to form around the zona pellucida (Fig. 14). Until recently it was commonly believed that the spermatozoa could not penetrate the egg before the dispersal of follicular cells and that the enzymatic action of hyaluronidase was the main factor in this dispersal,16, 29, 37 However, the presence of spermatozoa in the eggs having corona cells still attached was observed in the mouse 28 and in the rat. 3 , 27, 31 The contemporary view does not stress the dispersal of follicular cells by hyaluronidase as an essential part of the mechanism of fertilization. The present study dearly shows that the penetration of spermatozoa definitely precedes the complete denudation of eggs and also precedes the dispersal of the mucous cumulus mass in some cases (Figs. 3 and 5). It is quite possible, however, that the enzymatic action of hyaluronidase may assist the spermatozoa to pass through the mucous cumulus mass, the intercellular space of corona cells, and the zona pellucida. In the presence of spermatozoa, as revealed in the present investigation, PLATE III 10. A living egg 6 hours after ovulation. Phase microscope. 8 mm. objective. 11. One section of an egg in situ 6 hours after ovulation. Two pronuclei superimposed. 4 mm. objective. (255 X) FIGURE 12. One section of an egg in situ 8 hours after ovulation. Spermatozoa in the perivitelline space and in the zona pellucida. 4 mm. objective. (255 X) FIGURE 13. One section of an egg in situ 8 hours after ovulation. 4 mm. objective. (255 X) FIGURE 14. A group of living eggs recovered from the tubes 14 hours after ovulation (24 hours after insemination and ovulation-inducing injection). 16 mm. objective. (93 X) FIGURE 15. Section of eggs 14 hours after ovulation. 16 mm. objective. FIGURE FIGURE
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8 of 35 eggs (23 per cent) were in the cumulus mass 2 to 3 hours after ovulation and 13 of 16 eggs (81 per cent) were completely denudated 6 hours after ovulation. The dispersal of follicular cells in 'the absence of spermatozoa is rather slow. 34. 47 When 163 eggs recovered from 10 superovulated does were examined 7~~ to 8 hours after ovulation without insemination, the author observed that 32 per cent of the eggs were in the cumulus mass with corona radiata, 47 per cent were free from the cumulus mass but with corona radiata, and 21 per cent were completely denudated. When freshly ovulated eggs were kept in saline or saline diluted with an equal part of serum at 38° C. for 14 hours, no dispersal of the cumulus mass or denudation was observed. It has been reported that the denudation of rabbit eggs is a function of the tube rather than an action of hyaluronidase. 43 It is obvious from these observations that the dispersal of the cumulus mass and denudation of eggs are mainly a function of the tube, but that the presence of spermatozoa (the enzymic activity of hyaluronidase) facilitates the process. As for the spermatozoa, the number which reach the tubes is very small indeed. In order to secure tubal spermatozoa each of 2 does was allowed to mate with 3 fertile bucks and 12 hours later a count showed about an estimated two thousand spermatozoa. By microscopic examination of complete serial sections of tubes, the spermatozoa in the tubes were surprisingly scarce. Spermatozoa were easily located in the egg but only on very rare occasions were they observed between the corona cells and hardly any were seen on the lining of the tubes. This observation confirms the results reported by Austin4 in the rabbit and by Blandau and Odor7 in the rat. The very small number of spermatozoa from one ejaculate, 1 out of 100,000, that reach the site of fertilization indicates the slight chance and extreme difficulty of a spermatozoan ascending the tube and the severe selection that occurs in the sperm population. Therefore the assumption that the astronomical number of spermatozoa in an ejaculate is for the purpose of dispersing the follicular cel1s or "egg plug" is rather speculative. The spermatozoa in the eggs fixed at different intervals after ovulation were counted (Table 1). It was found that the number of sperm increased remarkably from the s~cond to the fourth hour during which time the majority of the corona cells were tearing off. From the fourth to the sixth hour the number of sperm increased moderately, and the last remaining corona cells were torn off. From the sixth to the eighth hour and subsequently, no significant increase in the nurriber of sperm could be observed. It is quite
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obvious, therefore, that the penetration of sperm occurs at the time of tearing off of the corona cells. Although at the second hour no tearing off of the corona cells can be seen, probably because of the large number present, the penetration of sperm at that time is most probably due to the fact that the denudation has already begun. Once the denudation of the eggs is entirely completed (6 hours after ovulation), the penetration of spermatozoa is rendered impossible despite the fact that more sperm may be present in the lower part of the tube. The TABLE 1.
Penetration of Spermatozoa During the Denudation of Eggs*
Eggs Doe No.
Hours after ovulation
It
2 3 4 6 8 11 14
2f 1 3f 3 2 4,5+
Average No. of Sperm in the Egg Corona cells
Thick Thick Thin Very little No No Thin albumin coat
Ooplasm
.3 .8 Pronucleus Pronucleus Pronucleus Metaphase Cleaved
Perivitelline space
Zona pellucida
Total
1.2 2.1 9.2 15.2 19.1 14.5 19.5
1.2 2.1 10.2 12.3 12.0 10.5 6.0
2.7 5.0 19.4 27.5 31.1 25.0 25.5
• The spermatozoa from 10-20 eggs were counted. f Animals inseminated with 168-210 million spermatozoa. t Animals were mated, data from previous experiments.
outer layer of the zona pellucida at the fourth and sixth hour was irregular as shown in the section and was viscid as observed in the recovered living eggs. At about the eighth hour, several layers were formed in the zona pellucida and its outer layer was rather smooth and no longer viscid. This observation also indicates the improbability of sperm penetration after complete denudation. The loss of fertilizability of eggs 6 to 9 hours after ovulation,21.35 is coincident with the complete denudation of the eggs rather than with the deposition of an albumin coat which occurs a few hours later. Furthermore, there is no deposition of an albumin coat on rat eggs or guinea pig eggs and yet they cannot be fertilized 10 or 20 hours after ovulation. 6 • 8 Nevertheless, the loss of fertilizability of eggs in these two species is closely related to the complete denudation of the egg (Cf. Odor and Blandau, and Squier). The denudation of ferret eggs takes much longer-48 or 66 hours after ovulation12 -and they are capable of fertilization 30 hours or longer after ovulation. 23
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The denudation of ungulate eggs takes place rather rapidly.19 Therefore one may predict the early loss of fertilizability of eggs in these species. This is true so far as we know in the mare, because inseminations 2 to 24 hours after ovulation were all sterile.15 In the rabbit as observed in the present study, approximately 10 to 20 spermatozoa entered the perivitelline space from the second to the eighth hour after ovulation (Table 1). The spermatozoan actually effecting fertilization is probably one which arrives early at the upper end of the tube, penetrating the egg as soon as pOSSible. This must be so since the pronuclear behavior of the egg is far advanced at the fourth and at the sixth hour after ovulation. The transportation of eggs in the first 6 hours is very rapid (Table 2). The TABLE 2.
Position of Rabbit Eggs in the Tube During Fertilization Hours after Ovulation 8-11 4-6 2-3
Total No. of eggs Percentage of eggs in the tube Upper half Lower half Anterior section Middle section Posterior section
15
167
35
34
37
43
29
16
20
49 9 0
47 23 0
54 30 0
53 27
o
accumulated results show that during the first 3 hours after ovulation a relatively high percentage of eggs was in the upper half of the tube, but by the eighth hour they were in the lower half of the tube and remained there until the fourteenth hour or longer. In the present histologic preparations, the ciliary cells were stained purple while the secretory cells in the tube were stained green. It was noticed that there are more ciliary cells in the upper half of the tube whereas there are more secretory cells in the lower part. Judging from the size of the egg and of the cilia it seems that the ciliary activity is too weak to move the egg. However, it is reasonable to assume that the function of the cilia is to brush off the corona cells. As the egg is transported to the lower part of the tube the tearing off of the corona cells by the action of cilia has been completed and the physiologic condition of the zona pellucida changes due to its direct contact with the secretory cells, which renders the penetration of sperm
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impossible. Thereafter the secretory cells deposit the albumin coat on the zona pellucida. The tubal factors in the process of fertilization have been underestimated by previous workers. The denudation of the egg and the dispersal of the cumulus mass both require the participation of the tube. That the penetration of spermatozoa is closely correlated to the tearing off of the corona cells as described here is an obvious relation between fertilization and tubal function. Extensive long-term unsuccessful attempts to fertilize rabbit eggs in vitro have convinced the author of the necessity for stressing the importance of tubal factors in the process of fertilization. The failure of fertilization in pseudopregnant rabbits 30 was ascribed by Austin 5 to the small number of spermatozoa present in the tube. We know that the transportation of eggs is accelerated on the fourth and fifth day of pseudopregnancy,46 which may also contribute to the failure of fertilization. Thus the mechanism which transports spermatozoa and eggs together at the right time, the muscular and ciliary activity of the tube exerting a stimulation to the eggs and spermatozoa, and the chemical composition of the tubal fluid at different reproductive phases may all affect the process of fertilization. FACTORS REGULATING THE DEVELOPMENT OF FERTILIZED RABBIT EGGS The segmentation and development of fertilized rabbit eggs take place very rapidly. Most of the fertilized eggs cleave into 2 cells one day after mating or artificial insemination; for convenience, they are here called I-day eggs (Fig. 14). Two days after mating they are at the morula stage with a rather thick albumin coat (Fig. 16). Most of the eggs are still in the tubes on the third day; a few of them are at the early blastocyst stage with an inner cell mass (Fig. 17). By the fourth day, they are at the blastocyst stage and nearly all have reached the uterus. They are here referred to as 4-day eggs (Fig. 18). Six days after mating, large blastocysts, 3-4 mm. in diameter with a well-formed germ disc, can be easily flushed out of the uterus (Fig. 19). Thereafter the blastocysts cannot be flushed out because their rapid expansion wedges them in the uterus and because of the beginning of implantation (seventh to eighth day). The length of gestation in the rabbit is 32 days. The physiologic conditions of the tube and uterus as they affect the trans-
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portation of eggs, their development, and the implantation of blastocysts were investigated. The method used was to transfer rabbit eggs at various stages of development to the tubes or uteri of a foster mother at different stages of ovarian activity (i.e. before or after ovulation as induced by intravenous injection of gonadotrophic hormone). The detailed results will be published elsewhere. 11 The main features of this study are graphically presented in Figure 20. After studying Figure 20, it is clear that the highest percentage of eggs developing into normal young were transferred to the tubes or uteri of a foster mother at the corresponding stage of luteal development. For example, the I-day eggs have the best chance of developing if transferred to the tubes of foster mothers 1 day after ovulation-inducing injection and 4-day eggs if transferred to the uterus at the fourth day of pseudopregnancy. Two days before, and 1 or 2 days after the corresponding luteal stage, the development of the transferred eggs completely failed. It should be noticed here that the low percentage of development in the case of transferred 2-day eggs and the short interval in which it is possible for their development (Fig. 20) is due to the fact that, in this particular experiment, the 2-day eggs were recovered from the tubes but transferred to the uterus. It is of importance to note here that I-day eggs recovered from fallopian tubes but transferred to the uterus degenerated at a very early stage regardless of the luteal stage of the foster mothers.lO, 11 In the case of the transplant of tubal eggs to the uterus, the 2-day eggs (all from the tubes) had a low percentage of development (21 per cent) while the 3-day eggs (most of them from the tubes) had a higher percentage (31 per cent). This shows clearly that the uterus at any luteal stage is not suitable for the development of very early stage eggs. As the development of the eggs progresses their chance of survival in the uterus increases. The fate of eggs transferred to a foster mother whose luteal stage was PLATE IV 16. Two living eggs recovered from the tube 2 days albumin coat. 16 mm. objective. (93 X) FIGURE 17. Two living eggs recovered from the tube 3 days cell mass in one egg. 16 mm. objective. (93 X) FIGURE 18. Three living rabbit blastocysts recovered from insemination. One shrunken. 16 mm. objective. (93 X) FIGURE 19. A group of living blastocysts recovered from 6 days after insemination. (3 X) FIGURE
after insemination. Thick after insemination. Inner the uterus 4 days after the uteri of one animal
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3 days earlier or 2 days later than the egg development was further studied. The transferred eggs as well as the genital tracts of the foster mother at various intervals after transplantation were examined. One-day eggs were transferred to the tubes of foster mothers at the third day of pseudopregnancy and examined 2 or 3 days later. If the eggs were deposited at the bottom end of the ampulla, they passed to the uterus much earlier than normally and degenerated at an early stage. This is a condition similar to I-day eggs transferred directly to the uterus. If they were deposited
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at the top end of the ampulla, they developed normally in the tubes. If these normal eggs chanced to reach the uterus, they eventually degenerated as in the case of 4-day eggs transferred to the uterus of a rabbit at the sixth day of pseudopregnancy (see below). Very few of the I-day eggs that were transferred to the tubes of foster mothers in estrus (i.e., 2 days before ovulation-inducing injection) were recovered. Those recovered from the tubes were at a normal stage of development while those recovered from the uterus were degenerated early blastocysts. This indicates that 2~~ days before ovulation the tubes lack the
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mechanism to receive and to transport the eggs but do not hinder the early stage of development. But if by any chance these normally developing eggs reach the uterus, they would degenerate due to the lack of proliteration of the endometrium. When 4-day eggs were transferred to the uterus of a foster mother on the first day of pseudopregnancy, a relatively low percentage of eggs were recovered, probably because the contraction of the uterus at this time 38 expelled them. Most of the recovered eggs, however, were degenerated blastocysts at a stage similar to or slightly more advanced than at the time of transfer. This is certainly due to the lack of an adequate supply of progesterone for the growth of the blastocyst, as reported by Corner. When the 4-day eggs were transferred to the uteri of foster mothers at the sixth day of pseudopregnancy, they grew for a short time but degenerated before implantation due to the inadequate formation of decidua tissue. Decidua formation in the rabbit as determined by mechanical stimulation of endometrium has been observed to occur only on the fifth to eighth days of pseudopregnancyY' 20 Thus the 4-day eggs in the uterus of a rabbit at the sixth day of pseudopregnancy may not mature enough to stimulate decidua formation at first. Should they grow for another 3 days, the endometrium, now 9 days old, would then be unable to form deciduomata. When the 6-day eggs were transferred to the uteri of foster mothers at the third day of pseudopregnancy, most of the transferred eggs degenerated before decidua formation. This indicates that 6-day eggs require a rather advanced proliferation of the endometrium for their nutrition and implantation. When they were transferred to foster mothers at the ninth day of pseudopregnancy, most of the eggs were implanted but eventually degenerated, perhaps due to the inadequate formation of deciduomata or maternal placenta. When transferred to foster mothers at the tenth day of pseudopregnancy, all of them degenerated because deciduomata cannot be formed at this time. It is clear from the foregOing description that during the process of transformation from follicular phase to functional luteal phase, a series of physiologiC activities or conditions is set up in the tubes and uteri in a definite sequence which is essential for the survival, development, and implantation of eggs. Thus, any disturbance of the physiologic conditions in the ovary, the tube, and the uterus, or even a disturbance in the magnitude of these
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physiologic activities will lead to the degeneration of the eggs before or even after implantation. In a previous study,l° it was shown that a much higher percentage of eggs which had been subjected to low temperature or culture failed to implant or degenerated after implantation as compared with untreated eggs. It was repeatedly observed that a few small blastocysts would appear in a group of large ones recovered from an animal and that these small blastocysts would shrink in the serum used as flushing medium after a short time (Fig. 19). These observations demonstrate the importance of the physiologic condition of the eggs which also affects their future development. The failure of development after fertilization as described above was considered only in connection with the physiologic condition of the female tract at various reproductive phases. The pathologic, immunologic and nutritional causes for the prenatal mortality, though of importance, are out of the scope of this paper. The genetical constitution of the mother and that of fertilized eggs certainly also plays an important part in their development. 22 It should be mentioned here that one of the factors that affects the normal development of fertilized eggs is the age of the eggs rather than the age of spermatozoa at the time of fertilization. When rats or guinea pigs were inseminated before the time of ovulation, the percentage of fertility decreased as the time lengthened, but there was no effect on the development and gestation. However, when they were inseminated at various times after ovulation the percentage of fertility decreased and the percentage of abnormal pregnancy (reabsorption and abortion) increased. 4o ,41
SUMMARY The fertilization and early development of rabbit eggs were studied in order to reveal the factors controlling fertility and sterility in mammals. The process of fertilization from the time of ovulation to the first cleavage, which takes 14 hours, was studied with living eggs and with sections of eggs in situ. The number of spermatozoa which reach the fallopian tubes is very small indeed. The penetration of sperm precedes the complete dispersal of follicular cells. The dispersal of the cumulus mass and the denudation of eggs are a function of the tube but the presence of sperm was shown to facilitate the process. The penetration of spermatozoa occurred at the time of tearing off of the corona cells attached to the zona pellucida. Although
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more spermatozoa penetrated the eggs from the second to the sixth hour after ovulation, when the intensive denudation occurred, the spermatozoan that fertilizes the egg is the one which first enters. The transportation of eggs during the first six hours is very rapid. The ciliary cells in the upper half of the tube function to brush off the corona cells while the secretory cells in the lower half deposit an albumin coat on the zona pellucida. Once the denudation is completed the penetration of spermatozoa is not possible. The tubal factors in the process of fertilization are stressed . The development, transportation, and implantation of eggs as affected by the physiologic condition of the female tract was investigated by transferring eggs at different stages to the tubes or uteri of foster mothers before or after ovulation. The physiologic condition of the uterus is unsuitable for the development of early stage eggs. The reception and transportation of eggs in the tubes, the reception and nourishment of blastocysts in the uterus, the optimal time for decidual formation, and the maturation of eggs to stimulate decidua formation all require the participation of ovarian activity at a definite stage. Thus, the transferred eggs failed to develop when transference was performed 3 days earlier or 2 to 3 days later than the corresponding stage of the corpora lutea of the foster mothers.
REFERENCES 1. Alden, R. H.: Anat. Rec. 84:137, 1942. 2. Assheton, R.: Quart. J. Micro. Science 37:113, 1895. 3. Austin, C. R.: Nature 162:63, 1948. 4. Austin, C. R.: Nature 162:534, 1948. 5. Austin, C. R.: J. Endocrinology 6:63, 1949. 6. Blandau, R. J., and Jordan, E. S.: Am. J. Anat. 68:275, 1941. 7. Blandau, R. J., and Odor, D. L.: Anat. Rec. 103:93, 1949. 8. Blandau, R. J., and Young, W. C.: Am. J. Anat. 64:303, 1939. 9. Burdick, H. 0., Whitney, R., and Emerson, B.: Endocrinology 31:100, 1942. 10. Chang, M. C.: Proc. Soc. Exper. BioI. & Med. 68:680, 1948. 11. Chang, M. C.: J. Exper. Zoo!. 114:197, 1950. 12. Chang, M. C.: Anat. Rec. 108:31, 1950. 13. Corner, G. W.: Am. J. PhysioI. 86:74, 1928. 14. Courrier, R., and Kehl, R.: Compt. rend. Soc. de bioI. 104:1180. 1930. 15. Day, F. T.: J. Agric. Science 30:244,1940. 16. Fekete, E., and Duran-Reynals, F.: Proc. Soc. Exper. BioI. & Med. 52:119, 1943. 17. Florey, H., and Walton, A.: J. Physio!. 74:5,1932. 18. Gregory, P. W.: Contrib. Embryo!. 21:143, 1930. 19. Hamilton, W. J., and Laing, J. A.: J. Anat. 80:194, 1946. 20. Hammond, J.: J. Exper. BioI. 4:349, 1927. 21. Hammond, J.: J. Exper. Bio!. 11:140, 1934. 22. Hammond, J.: BioI. Rev. 16:141, 1941.
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Hammond, J., and Walton, A.: J. Exper. BioI. 11:307, 1934. Heape, W.: Proc. Roy. Soc., London, s.B. 76:260, 1905. Hill, R. T., Allen, E., and Kramer, T. C.: Anat. Rec. 63:239, 1935. Lams, H., and Doorme, J.: Arch. bioI., Paris, 23:259,1908. Leonard, S. L., Perlman, P. L., and Kurzrok, R.: Proc. Soc. Exper. BioI. & Med. 66:517, 1947. 28. Lewis, W. H., and Wright, E. S.: Contrib. to EmbryoI. 25:115, 1935. 29. McLean, D., and Rowlands, I. W.: Nature, London 150:627, 1942. 30. Murphree, R. L., Warwick, E. J., Casida, L. E., and McShan, W. H.: Endocrinology 41:308, 1947. . 31. Odor, D. L., and Blandau, R. T.: Anat. Rec. 104:9, 1949. 32. Parker, C. H.: Phil. Trans. Roy. Soc. London 219:381, 1931. 33. Parkes, A. S.: Nature, London 153:245,1944. 34. Pincus, C.: Proc. Roy. Soc., London, s.B. 107:132, 1930. 35. Pincus, C.: The Eggs of Mammals. New York, Macmillan, 1936. 36. Pincus, C.: Anat. Rec. 77:1, 1940. 37. Pincus, C., and Enzmann, E. V.: J. Exper. ZooI. 73:195, 1936. 38. Reynolds, S. R. M., and Friedman, M. H.: Am. J. PhysioI. 94:696, 1930. 39. Sobotta, J.: Arch. f. mikr. Anat. 45:15, 1895. 40. Soderwall, A. L., and Blandau, R. J.: J. Exper. ZooI. 88:55, 1941. 41. Soderwall, A. L., and Young, W. C.: Anat. Rec. 78:19, 1940. 42. Squier, R. R.: Contrib. EmbryoI. 23:225, 1932. 43. Swyer, C. I. M.: Nature, London 159:873, 1947. 44. Walton, A., and Hammond, J.: J. Exper. BioI. 6:190, 1928. 45. Westman, A.: Sup. ad. acta obst. et gynec. Scandinav. 5, Fasc., 1926. 46. Wislocki, C. B., and Snyder, F. F.: Bull. Johns Hopkins Hosp. 52:379, 1933. 47. Yamane, J.: Cytologia 6:233, 1935. 23. 24. 25. 26. 27.
in mammals is primarily dependent upon the viability of the spermatozoa and eggs, the facility as well as the timing of their union (fertilization), and the normal development of fertilized eggs until birth. The process of fertilization certainly is the most important phase in reproduction that affects fertility. Nevertheless, our knowledge of fertilization of mammals is mainly based upon the descriptions of earlier workers. 26 • 39 The first part of this paper discusses some of the controlling factors of fertilization in the light of original observations and recent contributions by other workers. The second part deals with the factors that affect the normal development of fertilized eggs since it has been estimated that in the mammal about 40 per cent of eggs fail to develop into young. 33
RRTILITY
FERTILIZATION OF RABBIT EGGS Since ovulation in the rabbit is not spontaneous but occurs about 10 hours after copulation or after intravenous injections of gonadotrophic hormones, the time sequence of fertilization can be estimated with more accuracy than in other mammals. The rapid growth of follicles begins about six hours From the Worcester Foundation for Experimental Biology and Tufts College Medical School. Ortho Award Essay, presented at the Sixth Annual Conference of the American Society for the Study of Sterility, San Francisco, June 24, 1950. The author wishes to acknowledge his gratitude to Dr. C. Pincus for reading the manuscript and to Miss Jacqueline Choiniere for the preparation of histological sections. This work is supported by a grant from the Committee on Human Reproduction, National Research Council, acting on behalf of the National Committee on Maternal Health. 205
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after copulation andthe thin follicular stigma dilates into a pimple-like cone a few minutes before ovulation. The rupture of the cone, which is not explosive, involves only 5 to 7 seconds. 25 • 44 The ciliated infundibulum of the fallopian tube embraces the ovary at the time of ovulation. The transportation of eggs from the ovary to the ampulla is due to the activity of the muscular tissues connecting the ovary, the tube, and the uterus and to the contraction of the tube itsel£.45 The transportation of the eggs from the ampulla through the isthmus to the uterus is perhaps mainly dependent upon the muscular contraction of the tube rather than the ciliary activity according to observations in the rat and mouse. 1 • 9 The transportation of spermatozoa from the vagina to the upper third of the tube, where fertilization occurs, takes about 4 hours. 24 In the uterus, the progress of sperm is mainly due to their own motility/7 in the tube, it is perhaps mainly due to the contraction of the tube. 32 Since ovulation occurs lO hours after mating, the spermatozoa must wait 6 hours for the arrival of the eggs. This interval may allow time for an adequate number of spermatozoa to accumulate or physiologic changes of sperm in the female tract. Since the first cleavage of fertilized rabbit eggs occurs 23 to 24 hours after mating,2, 18 the procr"s of fertilization from ovulation to the first cleavage takes 13 to 14 hours. In the present study, the rabbits were superovulated36 in order to obtain a large number of eggs for observation. In one series,. fresh eggs were Hushed from the tube with serum diluted with an equal volume of saline at various intervals after insemination. They were then examined and photographed under a phase microscope. In another series, in which animals were inseminated with about the same number of spermatozoa, single tubes of animals were removed separately at different times and fixed immediately. Altogether 6 tubes from 3 animals were sectioned and stained with Masson's trichromatic stain. Just after ovulation, the eggs reach the ampulla of the tube surrounded with follicular cells or cumulus oophorus. The follicular cells, immediately attached to the individual eggs, constitute the corona radiata, here called PLATE I 1. A living rabbit egg recovered just after ovulation. In cumulus mass, corona cells with long processes. Slightly pressed. Phase microscope. 8 mm. objective used. FIGURE 2. Two living eggs recovered 2 hours after ovulation. Two polar bodies visible in one egg. 8 mm. objective. FIGURE 3. One section of an egg in situ 2 hours after ovulation. In cumulus mass. One spermatozoan in the zona pellucida, at 6 o'clock position. 4 mm. objective. (255 X) FIGURE
l
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corona cells. Usually several eggs are massed together in a mucus clot with loosely suspended follicular cells, referred to here as the cumulus mass. The rabbit eggs recovered from the ampulla of the tube just after ovulation (lm~ hours after artificial insemination and ovulation-inducing injection) were still in the cumulus mass. The corona cells were thick, with long processes apparently penetrating to the zona pellucida while the cells of the outer layer were long and sharp (Fig. 1). Two hours after ovulation, the cumulus mass was more or less dispersed both by the contraction of the tube and by the liquefying action of hyaluronidase, liberated by the spermatozoa present, on the mucus cumulus mass. The corona cells, however, were still closely attached to the zona pellucida though they were not so thick and the cells of the outer layer were now round and smooth (Fig. 2). The tearing off of the corona cells had started. One or two spermatozoa could be seen in the perivitelline space though they are not clear in the photograph of the intact eggs (Fig. 2). Figs. 3, 4, and 5 are sections of 3 eggs in situ 2 or 3 hours after ovulation. Special attention is called to the presence of spermatozoa in the egg or in the zona pellucida in spite of the fact that some eggs were still in the cumulus mass and that all of them had corona cells attached. By 4 hours after ovulation the outer layer of the corona cells was tom off. Several moving spermatozoa were seen in the perivitelline space during photography under the microscope. The second polar body was formed in all the eggs recovered at this time (Fig. 6). The male pronucleus was well formed while the female nuclear material was still in the form of chromoPLATE II FIGURE 4. One section of an egg in situ, 2 hours after ovulation. One spermatozoan in the ooplasm at 2 o'clock position. One spermatozoan in the zona peIIucida at 4 o'clock position. 4mm. objective. (255 X) FIGURE 5. One section of an egg in situ 3 hours after ovulation. In cumulus mass with two polar bodies indicating the penetration of a spermatozoan. 4 mm. objective. (255 X) FIGURE 6. A living egg recovered 4 hours after ovulation. Thin corona radiata. 8 mm. objective. FIGURE 7. One section of an egg in situ 4 hours after ovulation, showing male pronucleus (right) and female nuclear material (left). 4 mm. objective. (255 X) FIGURE 8. One section of an egg in situ 4 hours after ovulation. Male pronucleus visible. 4 mm. objective. (255 X) FIGURE 9. A living egg recovered 5 hours after ovulation. Very few corona cells left. 4 mm. objective.
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somes as shown in the sections (Figs. 7 and 8). Fig. 9 shows a living egg recovered 5 hours after ovulation; only a few corona cells were still attached to the zona pellucida. Six hours after ovulation, the denudation (tearing off of the corona cells) was complete in most cases and the two pronuclei were close together (Fig. lO). The sections of eggs in situ at this time are shown in Fig. 11 with the male and female pronuclei approaching each other. Eight hours after ovulation, the two pronuclei were already fused in a few cases, and there were many sperm in the zona pellucida and perivitelline space, as shown in the sections (Figs. 12 and 13). In a group of fertilized eggs recovered from the tube 14 hours after ovulation, some were cleaved and others were uncleaved. The albumin coat had started to form around the zona pellucida (Fig. 14). Until recently it was commonly believed that the spermatozoa could not penetrate the egg before the dispersal of follicular cells and that the enzymatic action of hyaluronidase was the main factor in this dispersal,16, 29, 37 However, the presence of spermatozoa in the eggs having corona cells still attached was observed in the mouse 28 and in the rat. 3 , 27, 31 The contemporary view does not stress the dispersal of follicular cells by hyaluronidase as an essential part of the mechanism of fertilization. The present study dearly shows that the penetration of spermatozoa definitely precedes the complete denudation of eggs and also precedes the dispersal of the mucous cumulus mass in some cases (Figs. 3 and 5). It is quite possible, however, that the enzymatic action of hyaluronidase may assist the spermatozoa to pass through the mucous cumulus mass, the intercellular space of corona cells, and the zona pellucida. In the presence of spermatozoa, as revealed in the present investigation, PLATE III 10. A living egg 6 hours after ovulation. Phase microscope. 8 mm. objective. 11. One section of an egg in situ 6 hours after ovulation. Two pronuclei superimposed. 4 mm. objective. (255 X) FIGURE 12. One section of an egg in situ 8 hours after ovulation. Spermatozoa in the perivitelline space and in the zona pellucida. 4 mm. objective. (255 X) FIGURE 13. One section of an egg in situ 8 hours after ovulation. 4 mm. objective. (255 X) FIGURE 14. A group of living eggs recovered from the tubes 14 hours after ovulation (24 hours after insemination and ovulation-inducing injection). 16 mm. objective. (93 X) FIGURE 15. Section of eggs 14 hours after ovulation. 16 mm. objective. FIGURE FIGURE
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8 of 35 eggs (23 per cent) were in the cumulus mass 2 to 3 hours after ovulation and 13 of 16 eggs (81 per cent) were completely denudated 6 hours after ovulation. The dispersal of follicular cells in 'the absence of spermatozoa is rather slow. 34. 47 When 163 eggs recovered from 10 superovulated does were examined 7~~ to 8 hours after ovulation without insemination, the author observed that 32 per cent of the eggs were in the cumulus mass with corona radiata, 47 per cent were free from the cumulus mass but with corona radiata, and 21 per cent were completely denudated. When freshly ovulated eggs were kept in saline or saline diluted with an equal part of serum at 38° C. for 14 hours, no dispersal of the cumulus mass or denudation was observed. It has been reported that the denudation of rabbit eggs is a function of the tube rather than an action of hyaluronidase. 43 It is obvious from these observations that the dispersal of the cumulus mass and denudation of eggs are mainly a function of the tube, but that the presence of spermatozoa (the enzymic activity of hyaluronidase) facilitates the process. As for the spermatozoa, the number which reach the tubes is very small indeed. In order to secure tubal spermatozoa each of 2 does was allowed to mate with 3 fertile bucks and 12 hours later a count showed about an estimated two thousand spermatozoa. By microscopic examination of complete serial sections of tubes, the spermatozoa in the tubes were surprisingly scarce. Spermatozoa were easily located in the egg but only on very rare occasions were they observed between the corona cells and hardly any were seen on the lining of the tubes. This observation confirms the results reported by Austin4 in the rabbit and by Blandau and Odor7 in the rat. The very small number of spermatozoa from one ejaculate, 1 out of 100,000, that reach the site of fertilization indicates the slight chance and extreme difficulty of a spermatozoan ascending the tube and the severe selection that occurs in the sperm population. Therefore the assumption that the astronomical number of spermatozoa in an ejaculate is for the purpose of dispersing the follicular cel1s or "egg plug" is rather speculative. The spermatozoa in the eggs fixed at different intervals after ovulation were counted (Table 1). It was found that the number of sperm increased remarkably from the s~cond to the fourth hour during which time the majority of the corona cells were tearing off. From the fourth to the sixth hour the number of sperm increased moderately, and the last remaining corona cells were torn off. From the sixth to the eighth hour and subsequently, no significant increase in the nurriber of sperm could be observed. It is quite
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obvious, therefore, that the penetration of sperm occurs at the time of tearing off of the corona cells. Although at the second hour no tearing off of the corona cells can be seen, probably because of the large number present, the penetration of sperm at that time is most probably due to the fact that the denudation has already begun. Once the denudation of the eggs is entirely completed (6 hours after ovulation), the penetration of spermatozoa is rendered impossible despite the fact that more sperm may be present in the lower part of the tube. The TABLE 1.
Penetration of Spermatozoa During the Denudation of Eggs*
Eggs Doe No.
Hours after ovulation
It
2 3 4 6 8 11 14
2f 1 3f 3 2 4,5+
Average No. of Sperm in the Egg Corona cells
Thick Thick Thin Very little No No Thin albumin coat
Ooplasm
.3 .8 Pronucleus Pronucleus Pronucleus Metaphase Cleaved
Perivitelline space
Zona pellucida
Total
1.2 2.1 9.2 15.2 19.1 14.5 19.5
1.2 2.1 10.2 12.3 12.0 10.5 6.0
2.7 5.0 19.4 27.5 31.1 25.0 25.5
• The spermatozoa from 10-20 eggs were counted. f Animals inseminated with 168-210 million spermatozoa. t Animals were mated, data from previous experiments.
outer layer of the zona pellucida at the fourth and sixth hour was irregular as shown in the section and was viscid as observed in the recovered living eggs. At about the eighth hour, several layers were formed in the zona pellucida and its outer layer was rather smooth and no longer viscid. This observation also indicates the improbability of sperm penetration after complete denudation. The loss of fertilizability of eggs 6 to 9 hours after ovulation,21.35 is coincident with the complete denudation of the eggs rather than with the deposition of an albumin coat which occurs a few hours later. Furthermore, there is no deposition of an albumin coat on rat eggs or guinea pig eggs and yet they cannot be fertilized 10 or 20 hours after ovulation. 6 • 8 Nevertheless, the loss of fertilizability of eggs in these two species is closely related to the complete denudation of the egg (Cf. Odor and Blandau, and Squier). The denudation of ferret eggs takes much longer-48 or 66 hours after ovulation12 -and they are capable of fertilization 30 hours or longer after ovulation. 23
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The denudation of ungulate eggs takes place rather rapidly.19 Therefore one may predict the early loss of fertilizability of eggs in these species. This is true so far as we know in the mare, because inseminations 2 to 24 hours after ovulation were all sterile.15 In the rabbit as observed in the present study, approximately 10 to 20 spermatozoa entered the perivitelline space from the second to the eighth hour after ovulation (Table 1). The spermatozoan actually effecting fertilization is probably one which arrives early at the upper end of the tube, penetrating the egg as soon as pOSSible. This must be so since the pronuclear behavior of the egg is far advanced at the fourth and at the sixth hour after ovulation. The transportation of eggs in the first 6 hours is very rapid (Table 2). The TABLE 2.
Position of Rabbit Eggs in the Tube During Fertilization Hours after Ovulation 8-11 4-6 2-3
Total No. of eggs Percentage of eggs in the tube Upper half Lower half Anterior section Middle section Posterior section
15
167
35
34
37
43
29
16
20
49 9 0
47 23 0
54 30 0
53 27
o
accumulated results show that during the first 3 hours after ovulation a relatively high percentage of eggs was in the upper half of the tube, but by the eighth hour they were in the lower half of the tube and remained there until the fourteenth hour or longer. In the present histologic preparations, the ciliary cells were stained purple while the secretory cells in the tube were stained green. It was noticed that there are more ciliary cells in the upper half of the tube whereas there are more secretory cells in the lower part. Judging from the size of the egg and of the cilia it seems that the ciliary activity is too weak to move the egg. However, it is reasonable to assume that the function of the cilia is to brush off the corona cells. As the egg is transported to the lower part of the tube the tearing off of the corona cells by the action of cilia has been completed and the physiologic condition of the zona pellucida changes due to its direct contact with the secretory cells, which renders the penetration of sperm
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impossible. Thereafter the secretory cells deposit the albumin coat on the zona pellucida. The tubal factors in the process of fertilization have been underestimated by previous workers. The denudation of the egg and the dispersal of the cumulus mass both require the participation of the tube. That the penetration of spermatozoa is closely correlated to the tearing off of the corona cells as described here is an obvious relation between fertilization and tubal function. Extensive long-term unsuccessful attempts to fertilize rabbit eggs in vitro have convinced the author of the necessity for stressing the importance of tubal factors in the process of fertilization. The failure of fertilization in pseudopregnant rabbits 30 was ascribed by Austin 5 to the small number of spermatozoa present in the tube. We know that the transportation of eggs is accelerated on the fourth and fifth day of pseudopregnancy,46 which may also contribute to the failure of fertilization. Thus the mechanism which transports spermatozoa and eggs together at the right time, the muscular and ciliary activity of the tube exerting a stimulation to the eggs and spermatozoa, and the chemical composition of the tubal fluid at different reproductive phases may all affect the process of fertilization. FACTORS REGULATING THE DEVELOPMENT OF FERTILIZED RABBIT EGGS The segmentation and development of fertilized rabbit eggs take place very rapidly. Most of the fertilized eggs cleave into 2 cells one day after mating or artificial insemination; for convenience, they are here called I-day eggs (Fig. 14). Two days after mating they are at the morula stage with a rather thick albumin coat (Fig. 16). Most of the eggs are still in the tubes on the third day; a few of them are at the early blastocyst stage with an inner cell mass (Fig. 17). By the fourth day, they are at the blastocyst stage and nearly all have reached the uterus. They are here referred to as 4-day eggs (Fig. 18). Six days after mating, large blastocysts, 3-4 mm. in diameter with a well-formed germ disc, can be easily flushed out of the uterus (Fig. 19). Thereafter the blastocysts cannot be flushed out because their rapid expansion wedges them in the uterus and because of the beginning of implantation (seventh to eighth day). The length of gestation in the rabbit is 32 days. The physiologic conditions of the tube and uterus as they affect the trans-
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portation of eggs, their development, and the implantation of blastocysts were investigated. The method used was to transfer rabbit eggs at various stages of development to the tubes or uteri of a foster mother at different stages of ovarian activity (i.e. before or after ovulation as induced by intravenous injection of gonadotrophic hormone). The detailed results will be published elsewhere. 11 The main features of this study are graphically presented in Figure 20. After studying Figure 20, it is clear that the highest percentage of eggs developing into normal young were transferred to the tubes or uteri of a foster mother at the corresponding stage of luteal development. For example, the I-day eggs have the best chance of developing if transferred to the tubes of foster mothers 1 day after ovulation-inducing injection and 4-day eggs if transferred to the uterus at the fourth day of pseudopregnancy. Two days before, and 1 or 2 days after the corresponding luteal stage, the development of the transferred eggs completely failed. It should be noticed here that the low percentage of development in the case of transferred 2-day eggs and the short interval in which it is possible for their development (Fig. 20) is due to the fact that, in this particular experiment, the 2-day eggs were recovered from the tubes but transferred to the uterus. It is of importance to note here that I-day eggs recovered from fallopian tubes but transferred to the uterus degenerated at a very early stage regardless of the luteal stage of the foster mothers.lO, 11 In the case of the transplant of tubal eggs to the uterus, the 2-day eggs (all from the tubes) had a low percentage of development (21 per cent) while the 3-day eggs (most of them from the tubes) had a higher percentage (31 per cent). This shows clearly that the uterus at any luteal stage is not suitable for the development of very early stage eggs. As the development of the eggs progresses their chance of survival in the uterus increases. The fate of eggs transferred to a foster mother whose luteal stage was PLATE IV 16. Two living eggs recovered from the tube 2 days albumin coat. 16 mm. objective. (93 X) FIGURE 17. Two living eggs recovered from the tube 3 days cell mass in one egg. 16 mm. objective. (93 X) FIGURE 18. Three living rabbit blastocysts recovered from insemination. One shrunken. 16 mm. objective. (93 X) FIGURE 19. A group of living blastocysts recovered from 6 days after insemination. (3 X) FIGURE
after insemination. Thick after insemination. Inner the uterus 4 days after the uteri of one animal
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FERTILIZATION OF RABBIT EGGS
•
For legends see facing page.
217
'. [Fertility & Sterility
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218
3 days earlier or 2 days later than the egg development was further studied. The transferred eggs as well as the genital tracts of the foster mother at various intervals after transplantation were examined. One-day eggs were transferred to the tubes of foster mothers at the third day of pseudopregnancy and examined 2 or 3 days later. If the eggs were deposited at the bottom end of the ampulla, they passed to the uterus much earlier than normally and degenerated at an early stage. This is a condition similar to I-day eggs transferred directly to the uterus. If they were deposited
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at the top end of the ampulla, they developed normally in the tubes. If these normal eggs chanced to reach the uterus, they eventually degenerated as in the case of 4-day eggs transferred to the uterus of a rabbit at the sixth day of pseudopregnancy (see below). Very few of the I-day eggs that were transferred to the tubes of foster mothers in estrus (i.e., 2 days before ovulation-inducing injection) were recovered. Those recovered from the tubes were at a normal stage of development while those recovered from the uterus were degenerated early blastocysts. This indicates that 2~~ days before ovulation the tubes lack the
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Vol. 2. No.3. 1951]
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mechanism to receive and to transport the eggs but do not hinder the early stage of development. But if by any chance these normally developing eggs reach the uterus, they would degenerate due to the lack of proliteration of the endometrium. When 4-day eggs were transferred to the uterus of a foster mother on the first day of pseudopregnancy, a relatively low percentage of eggs were recovered, probably because the contraction of the uterus at this time 38 expelled them. Most of the recovered eggs, however, were degenerated blastocysts at a stage similar to or slightly more advanced than at the time of transfer. This is certainly due to the lack of an adequate supply of progesterone for the growth of the blastocyst, as reported by Corner. When the 4-day eggs were transferred to the uteri of foster mothers at the sixth day of pseudopregnancy, they grew for a short time but degenerated before implantation due to the inadequate formation of decidua tissue. Decidua formation in the rabbit as determined by mechanical stimulation of endometrium has been observed to occur only on the fifth to eighth days of pseudopregnancyY' 20 Thus the 4-day eggs in the uterus of a rabbit at the sixth day of pseudopregnancy may not mature enough to stimulate decidua formation at first. Should they grow for another 3 days, the endometrium, now 9 days old, would then be unable to form deciduomata. When the 6-day eggs were transferred to the uteri of foster mothers at the third day of pseudopregnancy, most of the transferred eggs degenerated before decidua formation. This indicates that 6-day eggs require a rather advanced proliferation of the endometrium for their nutrition and implantation. When they were transferred to foster mothers at the ninth day of pseudopregnancy, most of the eggs were implanted but eventually degenerated, perhaps due to the inadequate formation of deciduomata or maternal placenta. When transferred to foster mothers at the tenth day of pseudopregnancy, all of them degenerated because deciduomata cannot be formed at this time. It is clear from the foregOing description that during the process of transformation from follicular phase to functional luteal phase, a series of physiologiC activities or conditions is set up in the tubes and uteri in a definite sequence which is essential for the survival, development, and implantation of eggs. Thus, any disturbance of the physiologic conditions in the ovary, the tube, and the uterus, or even a disturbance in the magnitude of these
220
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physiologic activities will lead to the degeneration of the eggs before or even after implantation. In a previous study,l° it was shown that a much higher percentage of eggs which had been subjected to low temperature or culture failed to implant or degenerated after implantation as compared with untreated eggs. It was repeatedly observed that a few small blastocysts would appear in a group of large ones recovered from an animal and that these small blastocysts would shrink in the serum used as flushing medium after a short time (Fig. 19). These observations demonstrate the importance of the physiologic condition of the eggs which also affects their future development. The failure of development after fertilization as described above was considered only in connection with the physiologic condition of the female tract at various reproductive phases. The pathologic, immunologic and nutritional causes for the prenatal mortality, though of importance, are out of the scope of this paper. The genetical constitution of the mother and that of fertilized eggs certainly also plays an important part in their development. 22 It should be mentioned here that one of the factors that affects the normal development of fertilized eggs is the age of the eggs rather than the age of spermatozoa at the time of fertilization. When rats or guinea pigs were inseminated before the time of ovulation, the percentage of fertility decreased as the time lengthened, but there was no effect on the development and gestation. However, when they were inseminated at various times after ovulation the percentage of fertility decreased and the percentage of abnormal pregnancy (reabsorption and abortion) increased. 4o ,41
SUMMARY The fertilization and early development of rabbit eggs were studied in order to reveal the factors controlling fertility and sterility in mammals. The process of fertilization from the time of ovulation to the first cleavage, which takes 14 hours, was studied with living eggs and with sections of eggs in situ. The number of spermatozoa which reach the fallopian tubes is very small indeed. The penetration of sperm precedes the complete dispersal of follicular cells. The dispersal of the cumulus mass and the denudation of eggs are a function of the tube but the presence of sperm was shown to facilitate the process. The penetration of spermatozoa occurred at the time of tearing off of the corona cells attached to the zona pellucida. Although
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Vol. 2, No.3, 1951]
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more spermatozoa penetrated the eggs from the second to the sixth hour after ovulation, when the intensive denudation occurred, the spermatozoan that fertilizes the egg is the one which first enters. The transportation of eggs during the first six hours is very rapid. The ciliary cells in the upper half of the tube function to brush off the corona cells while the secretory cells in the lower half deposit an albumin coat on the zona pellucida. Once the denudation is completed the penetration of spermatozoa is not possible. The tubal factors in the process of fertilization are stressed . The development, transportation, and implantation of eggs as affected by the physiologic condition of the female tract was investigated by transferring eggs at different stages to the tubes or uteri of foster mothers before or after ovulation. The physiologic condition of the uterus is unsuitable for the development of early stage eggs. The reception and transportation of eggs in the tubes, the reception and nourishment of blastocysts in the uterus, the optimal time for decidual formation, and the maturation of eggs to stimulate decidua formation all require the participation of ovarian activity at a definite stage. Thus, the transferred eggs failed to develop when transference was performed 3 days earlier or 2 to 3 days later than the corresponding stage of the corpora lutea of the foster mothers.
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