An Experimental Analysis of Female Sterility in the Rabbit

Transcription

1 An Experimental Analysis of Female Sterility in the Rabbit M. C. Chang IMPORTANT contributing factors in female sterility in mammals are: ( 1) the probability of ovulation and the number of ova shed; (2) the possibility of fertilization, which depends on the ova, the fertility of the male, and the timing of mating; (3) the probability of normal embryonic and fetal development in utero. This last factor includes the role of lethal and sublethal genes, but is mainly dependent on the physiology of the uterus which in turn is influenced by the endocrinologic, constitutional, and nutritional status of the female. An experimental analysis of the relative importance of these factors when the fertility of the male is strictly controlled is of both academic and clinical interest. This paper deals with an examination of these factors in a group of rabbits whose heredity and breeding req)rds are known. ANIMALS AND METHODS The animals used were 45 rabbits of different breeds, with relatively known genetic constitution, from a colony of rabbits reared in the Hamilton Station, Jackson Memorial Laboratory, Bar Harbor, Maine. Their age ranged from 8 to 64 months and most of them had been bred at least four times From the Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and the Department of Biology, Boston University. This work was accomplished in the Laboratory of Dr. P. B. Sawin, Hamilton Station, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, when the writer was a summer investigator, Sincere gratitude is due to Dr. Sawin for his constant interest in the problem and the supply of animals and the breeding records. Thanks are also due to Mr. Sidney Cohen of Yale University for assistance. This investigation was supported by a grant from the American Cancer Society to Jackson Laboratory and grants from the Rockefeller Foundation and Planned Parenthood Federation to the Worcester Foundation. 251

2 252 CHANG [Fertility & Sterility previously. Irrespective of their races, about two-thirds of them were chosen either for their complete sterility or infertility and one-third for their relatively high fertility. They had been isolated for at least twenty-two days. The experiment was started on July 15 and finished on August 30, This time, in general, is considered a poor season for rabbit breeding. Only one buck rabbit (an F1 hybrid between New Zealand Red and New Zealand White) with proven fertility was used throughout this study. The semen was collected twice a week, Tuesday and Friday, by means of an artificial vagina, and suspended in Ringer's solution in a ratio of about 1 to 5. Half a milliliter of the sperm suspension containing about million spermatozoa was inseminated into the vagina of a rabbit. Immediately after insemination, 5 mg. (about 20 R. U.) of sheep pituitary extract suspended in saline were injected intravenously into each animal to induce ovulation. About 6 animals, 4 infertile, and 2 fertile, were thus treated at a time. Six days after insemination, the right uterus (in most cases) was removed from the animals which had been anesthetized by ether or nembutal. The excised uterus was Hushed two to three times with saline and the washing fluid was carefully received and examined under a stereoscopic microscope. The blastocysts in the fluid were measured and the degenerated ova were examined under a compound microscope. Twenty-two days after insemination, the animals were killed. The ovaries were examined, the corpora lutea counted and the uterus as well as its contents were examined. In the examination of ova or blastocysts the following categories were established: 1. Normal blastocysts: Spherical blastocyst larger than 2.5 mm. in diameter with a faint or distinctive germ disk. 2. Small blastocysts: Spherical blastocysts smaller than 2.5 mm. in diameter, without an obvious germ disk but healthy in appearance. 3. Degenemted ova: Small degenerated blastocysts with rather dark and irregular cellular structure and fragmented ova with sperm in the zona pellucida or in the perivitelline space. 4. Unfertilized ova: Uncleaved or fragmented ova without sperm in the zona pellucida or perivitelline space. 5. Missing ova: Number of corpora lutea exceeded the number of ova recovered.

3 Vol. 3, No. 3, 1952] STERILITY IN THE RABBIT 253 In the examination of the uterine contents the following criteria were taken: 1. Normal fetus: Living normal fetus. 2. Degeneration after implantation: This includes: (a) the presence of dead or mummified embryos in various sizes with placenta; (b) the presence of fetal placenta, a thick vascular structure, in the center of maternal placenta; and (c) the presence of a group of small decidual tissues or a relatively large piece of maternal placenta. This indicates the age of their degeneration. Since these degenerations were fairly evenly distributed throughout pregnancy, they were presented under one heading. 3. Degeneration before implantation: Number of corpora lutea more than the number of normal fetuses and degenerations observed in the uterus. RESULTS Table 1 presents the data of 14 animals who either failed to ovulate or in TABLE 1. Sterile Animals Due to Failure of Ovulation, and Due to Failure of Ova-Transportation or Implantation Rabbits Previous breeding record Experimental results Age Young Av. No. No. per litter No. No. Race (months) mating pregn. mating size ovulat. Remarks 1 Sandy Poor condition. Thin uterus. 2 Sandy Normal. 3 Sandy Normal. 4 III Poor condition. 5 III Infantile ovaries. 6 III Infantile ovaries. Fat animal, cysts on endometrium. 7 III II Normal. 8 FAx Poor condition. 9 III Poor condition. 10 III Infantile ovaries; pale thin uterus. II Ang. 23 Not Not Normal. bred bred 12 An g Thin uterus. Adhesion of ovary and tube in one side. 13 Ax Pyometra; bilateral adhesion of ovary and tube. 14 FORD Normal.

4 254 CHANG [Fertility & Sterility whom no ovum and no implantation was observed. The failure of ovulation was rather high: 8 out of a total of 45 animals ( 18 per cent), or 8 out of 27 infertile animals ( 30 per cent). Because gonadotropic hormone was administered the failure of ovulation must have been due to the unresponsiveness of the ovaries. No ovum or implantation was observed in 6 animals ( 13 per cent of total animals; 22 per cent of sterile animals) though ovulation had occurred. This may have been due to failure of ova-transportation, of fertilization and implantation, or due to the possible loss of ova at the time of recovery on the sixth day. Of these 14 animals, 4 were in poor condition, 3 had infantile ovaries, 2 were diseased, and 5 were apparently normal. Genetically, 11 of these 14 animals belong to Sandy, III, and Angora, indicating the low fertility of these races. Table 2 presents the data of another 13 sterile animals as determined by the present study. Normal development of embryos failed in 11 rabbits (24 per cent of a total 45 animals; 41 per cent of 27 sterile animals) and fertilization failed in 2 animals (Nos. 26 and 27), that is, 4.4 per cent of the total animals; 7.4 per cent of the sterile animals. Fifteen per cent of the total ova failed to be fertilized, showing that fertilization is influenced by the reproductive state of the female. Of these 13 animals, 2 Angoras had about 30 transparent cysts on.the endometrium and 2 animals had very thin, small uteri. There was, however, no obvious abnormality in the ovaries and uteri of the other 9 animals, except that the corpora lutea in most cases were smaller than normal pregnancy corpora lutea. Whether the degeneration of embryos caused the degeneration of the corpora lutea or vice versa is not known. The fertility of this group of animals, as shown in the breeding records, is in general low, except one animal (No. 26) which was highly fertile. The sterility of this animal was probably caused by the exhaustion of reproductive activity after 9 successful breedings. Genetically, 8 of these 13 animals belonged to race Sandy, Angora, TC. Table 3 presents the data on 18 fertile animals as determined in the present study. It is clear that even in those animals which had previously proven fertile, degeneration of embryos at various stages was observed. All these 18 animals were in relatively good condition, showing no abnormal or pathologic conditions. Furthermore, it is of interest to note that in these 18

5 TABLE 2. Sterile Animals, Mainly Due to Failure of Development of Embryos Other uterus examined on Rabbits Breeding record One uterus examined on the sixth day twenty-second day Young Av. Fert. De g. De g. No. Race Age No. No. per litter Ovula- Norm. Small but deg. Unfert. Missing Ovula- after before mating pregn. mating size tion blast. blast. ova ova ova tion impl. impl. 15 TC I 3 5 I I 0 16 TC I I 17 TC 14 7 I I 5 4 I 18 TC Not examined 9 8 I 19* An g I * An g I I 3 I 2 21 Sandy 25 4 I I t Sandy I III 25 9 I I Br 8 2 I Hyde? I 0 4 I 3 26 Ax I I t WH I 6 6 ToTAL *Cysts on endometrium f Thin uteri

6 TABLE 3. Fertile Animals Other uterus examined on Rabbits Breeding records Uterus examined on sixth day twenty-second day Young Av. Fert. De g. De g. No. No. per litter Ovula- Norm. Small but deg. Unfert. Missing Ovula- Norm. after before No. Race Age mating pregn. mating size tion blast. blast. ova ova ova tion fetus impl. impl. 28 III III ll TC TC '~ TC TC Os Os Os Os Mis Mis Br Br Br Born? Wek F TOTAL * Transferred into other uterus

7 Vol. 3, No. 3, 1952] STERILITY IN THE RABBIT 257 animals, only 1 out of 104 expected ova was unfertilized. Thus, failure of fertilization is very rare if the fertility of both male and female is high. According to the data presented in Tables 2 and 3, the number of blastocysts larger than 2.5 mm. in diameter recovered from the first uterus at the sixth day was 53 per cent of the expected ova, while the normal and degenerated embryos after implantation observed in the other uterus at the twenty-second day was 63 per cent. This shows that all the large blastocysts and a portion of small normal blastocysts were implanted. It is obvious, therefore, that implantation, at least the decidual formation, never fails if the blastocysts can grow large enough in a certain time after fertilization. Table 4 presents data wherein the number of corpora lutea is compared with the number of ova, and with the degenerated and normal embryos. In the column of degenerated ova, the small blastocysts as presented in Tables 2 and 3 were included because most of them were probably degenerated before implantation. The percentage of ova unaccounted for was surprisingly high, 25 per cent at the sixth day and 37 per cent at the twentysecond day in 31 animals. Since 22 per cent of ova were unfertilized or degenerated on the sixth day and since 37 per cent of ova were unaccounted for on the twenty-second day, one may estimate that about 15 per cent of the ova may have failed to reach the uterus. If this is so there may have been 10 per cent ( 25-15) of ova lost at recovery on the sixth day. The proportion of unfertilized ova (15:1) and that of degenerated ova (27:10) was much higher, while the percentage of normal blastocysts was much lower (30:66), in the sterile animals as compared with the fertile animals. This shows that the growth of fertilized ova and even the process of fertilization is influenced by the reproductive state of the female. Furthermore, the percentage of degeneration after implantation in sterile animals is also higher than that in fertile animals (36:22). A comparison of the percentage of degenerated ova with degenerated embryos (27:36 in sterile animals; 10:22 in fertile animals) shows that degeneration is slightly higher after implantation in both groups. The results of examination of the sterile and fertile animals have been added together, and presented also in Table 4. If these data are representative of the normal rabbit population, it seems that one-third of the ova (.'35 per cent) develops into normal young, one-third degenerates before or after implantation ( = 43 per cent) and about one-third (31-37 per

8 258 CHANG [Fertility & Sterility TABLE 4. Comparison of Number of Corpora Lutea with That of Ova or Embryos Sterile group Fertile group Both groups At At twenty- At At twenty- At At twentysixth day second day sixth day second day sixth day second day Corpora lutea (no.) Normal fetus (%) Degen. after impl. (%) Normal blastocysts (%) Degen. ova (%) Failure of fertiliz. (%ova) Unaccounted for (% ova) cent) may be lost because of the failure of transportation and failure of fertilization. The causes of sterility in the rabbits belonging to different races is summarized in Table 5. In spite of the small number of animals in each race, the data indicate the following points: The sterility of the race III rabbits TABLE 5. Fertility of Rabbits in Different Races No. of rabbits Failure of ovulation Av.No. Av. and Failure of corpus litter Race Total transpmt. develop. Fertile lutea size Angora l Ax t Sandy J III TC OS/ Br \ Others was merely due to the failure of ovulation and of ova-transport, while that of the Angora, Ax, and Sandy rabbits is not only due to these causes but also to failure of normal development. In the TC race the only cause of sterility is that of failure of normal development. The high proportion of fetal degeneration in this race is also indicated by the comparison of average number of corpora lutea with average litter size (9.1:3.6) in this race. A similar comparison indicates the small number of ova shed in the animals of the Angora, Ax, and Sandy races, and the proportion of ova degeneration

9 Vol. 3, No. 3, 1952] STERILITY IN THE RABBIT 259 in the animals of the other races. The higher fertility of the animals of the Os, Br, and other races is probably a reflection of their relative heterogeneity as compared with III, Angora, and Sandy. Table 6 presents the number of rabbits classified as sterile, infertile, or fertile according to breeding records along with the causes of their sterility as revealed by the present study. In general, one can predict their fertility by means of previous records. However, 7 out of 32 (22 per cent) infertile TABLE 6. Number of Rabbits Classified According to Breeding Records and Examination Infertile Infertile Fertile Sterile ( 1 successful ( 1 successful (2 successful (No succes;jul out of 3-9 out of out of 2; or 3 mating) matings) matings) out of 4) Fertility according to breeding records Fertility according to examination Failure of ovulation and transport Failure of develop Normal fetus observed animals have been shown fertile, while 2 out of 13 fertile animals have been shown to be sterile under experimental examination. DISCUSSION The prenatal mortality has been estimated to be 40 per cent or more 1 ' or 18 to 50 per cent 4 among mammals. In the rabbits, Corner 4 states that the prenatal loss is 18 to 33 per cent as reported by several investigators. Four different strains of rabbits studied by Hammond 7 gave a 36 per cent prenatal mortality as checked by the number of corpora lutea. In the wild rabbit, prenatal mortality was reported to be 64 per cent by Brambell and Mills. In the present investigation, a percentage of 42 was revealed even in the fertile rabbits. Since Hammond's rabbits were bred the whole year around and the present study was performed in a poor breeding season, this 6 per cent difference may be due to a seasonal variation. These percentages, however, are only a comparison between the number of corpora lutea and that of normal young. There are only scanty data on the fate of ova before implantation. After a thorough investigation of ferret ova

10 260 CHANG [Fertility & Sterility from fertilization to birth, Robinson reported that besides 16 per cent of ova that escaped into the peritoneum, 20 per cent died within thirteen days, and 30 per cent died after implantation. He suggested that the failure of fertilization and of development is due partly to the inability of the ova and spermatozoa of certain individuals to unite, and partly due to the production of abnormal embryos by the union of certain ova and the spermatozoa. From this account, it is obvious that the infertility of his male ferrets may have influenced his results. In the investigation of the early development of pigs, Comer 3 stated that lo per cent of the ova did not divide, 10 per cent developed into blastocysts and then degenerated, another 5 or 10 per cent became abnormal during the subsequent course of pregnancy, and only 70 per cent of the ova were represented at term by living pigs. In the present study the percentage of degenerated ova in the fertile group is in agreement with Comer's findings on pigs (10 per cent). The percentage of unfertilized ova is much lower (1 per cent) in the fertile rabbits but it is similar to the pigs in sterile rabbits (15 per cent). The percentage of degeneration after implantation in our fertile rabbits (22 per cent) is twice as great as in the pigs. Since the rabbit colony used in the present study consisted of inbred strains, the characteristics of degeneration after implantation may have been accumulated during inbreeding as in the case of race TC. After examining 22 pregnant pigs with a total of 396 corpora lutea, Hammond" reported 67.4 per cent of normal fetuses, 12.4 per cent of atrophic fetuses, and 20 per cent of ova unaccounted for. In the present study, coincidently, the percentage of ova unaccounted for was per cent in the fertile animals. The failure of fertilization, of development, and of implantation was ordinarily attributed to these unaccountable ova. But failure of transportation of ova from ovary to the uterus has not been pointed out by previous investigators. By comparison of the percentage of ova unaccounted for on the twenty-second day (20 per cent or 64 per cent) and the percentage of unfertilized and degenerated ova on the sixth day (11 per cent or 42 per cent), the present study reveals that about 9 to 22 per cent of the ova failed to reach the uterus after ovulation. Considering the anatomic connection between the ovary and the infundibulum of the tube, the loss of ova at ovulation is highly probable. Furthermore, physiologic blockade of Fallopian tubes can be caused by imbalance of progesterone and estrogen.2 The nature of reproductive failures of 104 infertile cows was examined by Tanabe and Casida. They reported that 3 per cent of cows failed to ovulate,

11 Vol. 3, No. 3, 1952] STERILITY IN THE RABBIT per cent showed failure of fertilization, and 39 per cent showed failure of normal fetal development before thirty-four days' pregnancy, while only 21 per cent of cows had a normal fetus at thirty-four days. In the present study on the rabbits, the failure of ovulation was much higher and the failure of fertilization was much lower as compared with cows. The failure of ovumtransport is probably one important factor that contributes to the high percentage of failure of fertilization in the case of cows. In a study of the inheritance of fertility in the rabbit, Hammond 6 stated that the number of ova shed appears to behave as a multiple factor character, a character dependent upon different amounts of gonadotropic hormone in the blood. Fetal atrophy in the rabbit, a recessive character, is inherited as a maternal character and not as a homozygous embryonic condition as in the case of yellow mice. In the present study, due to the small number of animals in each race, a genetic analysis of the data is not feasible. However, the degeneration of ova and embryos at different stages of development suggests that this degeneration is an inherited maternal character. Since a higher percentage of fetal degeneration occurs in normal controls than in those animals in which one Fallopian tube has been cut to decrease the number of embryos, it has been thought that the degeneration of ova or embryos may be caused by a deficiency of progesterone secreted by the corpora lutea. 8 In the present experiment, however, in spite of the fact that one uterus was removed the percentage of degeneration was not lowered, i.e., 11 out of 45 animals showed complete degeneration (Table 2). It seems, therefore, that although the inheritance of the number of ova shed and that of the number of embryos degenerated depends on the different amounts of pituitary or luteal hormone in the blood, other physiologic factors, such as the reactivity of the ovary and that of the endometrium, may be involved in the inheritance of fertility. SUMMARY Forty-five sterile, infertile, and fertile female rabbits were inseminated with the spermatozoa collected from one male and induced to ovulate by administration of gonadotropic hormone. One uterus was examined on the sixth day and the other on the twenty-second day in order to find the proportion of unfertilized ova, degenerated ova and degeneration after implantation. Failure of ovulation occurred in 8 animals (18 per cent) and no ovum and

12 262 CHANG [Fertility & Sterility no implantation following ovulation was observed in 6 animals (13 per cent). Most of these animals had poor breeding records, poor physical conditions, infantile ovaries, and diseased reproductive tracts. The failure of transportation of ova from ovary to uterus was estimated at about 15 per cent of the expected ova. When the fertility of the male was established the failure of fertilization was very rare, 2 out of 31 rabbits (6.4 per cent), 1 per cent of the total ova in the fertile group. The failure of decidual formation or perhaps implantation was practically nil if the blastocyst had grown large enough (2.5 mm. in diameter) by the sixth day. Development of embryos till birth failed in 11 out of 45 animals (24 per cent). The degeneration of embryos before or after implantation was higher in the sterile group (27 per cent or 36 per cent) than in the fertile group (10 per cent or 22 per cent), about 10 per cent lower before implantation than after. The degeneration or death of embryos was fairly evenly distributed throughout the pregnancy after implantation. The cause of infertility varies according to different races. Degeneration of embryos was predominant in one race (TC), failure of ovulation and of ova-transportation in another (III). These two factors both play a part in another two races (Angora and Sandy). Fertility was relatively high in heterogeneous races. It appears that the inheritance of these physiologic characters involves not only the amount of hormone in the blood but also the reactivity of target organs. The breeding records, in general, agree with the experimental findings in determining sterility but exceptional cases were rather high. REFERENCES 1. Brambell, F. W. R., and Mills, I. H.: Nature 153:558, Burdick, H. 0., Whitney, R., and Pincus, G.: Anat. Rec. 67:513, 19.' Corner, G. W.: Am.]. Anat. 31:523, Corner, G. W.: Ourselves Unborn. New Haven, Yale University Press, Hammond, J.: ]. Agr. Sc. 6: , Hammond, J.: The inheritance of fertility in the rabbit. The Harper Adams Utility Poultry ]ournal19:551, Hammond, J.: ]. Exper. Biol. 11:140, Hammond, J.: Biol. Rev. 16:165, Parkes, A. S.: Nature 153:245, Robinson, A.: Edinburgh M. ]. 26:137,209, Tanabe, T. Y., and Casida, L. E.: ]. Dairy Sc. 32:237, 1949.