Studies on Induced Ovulation in the Intact Immature Hamster. Charles W. Bodemer, Ph.D., Ruth E. Rumery, Ph.D., and Richard J. Blandau, Ph.D., M.D.

Transcription

1 Studies on Induced Ovulation in the Intact Immature Hamster Charles W. Bodemer, Ph.D., Ruth E. Rumery, Ph.D., and Richard J. Blandau, Ph.D., M.D. IT IS WELL KNOWN that gonadotropins are incapable of inducing ovulation in the newborn animal and that the sensitivity of the ovaries to anterior pituitary hormones develops only gradually.3 The response of the ovaries of the immature animal to gonadotropins is no doubt subject to modification by many factors. Some of these may include (1) an inherent, species-specific, maturation rate; the general environmental milieu; (2) the effects of other endocrine glands on them throughout their development; (3) the influence of endogenous gonadal hormones; and (4) various inductive mechanisms, particularly of nervous system origin. From the consistent results obtained in some preliminary investigation, the hamster appeared to be an ideal animal for exploring some of the factors which control and modify the reactivity of the gonads, particularly during the period which precedes sexual maturity. In recent years this animal has been used extensively in the laboratory, and there is considerable information regarding its reproductive cycle, breeding habits, and response to various hormones. 2, 4, 7 10, 18 There is, however, very little known concerning the effectiveness of gonadotropic hormones in inducing ovulation in the immature female. This investigation is part of a more inclusive study of the mechanism of ovulation in mammals and its modification by various hormonal and environ- From the Department of Anatomy, University of Washington, Seattle 5, Wash. This study was supported by funds from Research Grant 4241, U. S. Public Health Service, Department of Health, Education and Welfare. 350

2 Vol. 10, No.4, 1959 INDUCED OVULATION IN IMMATURE HAMSTERS 351', mental factors. This publication deals specifically with the responsiveness of the immature female ovary to gonadotropins and the optimal conditions for inducing ovulation in female hamsters between the twenty-first and thirtysixth day after birth. MATERIALS AND METHODS Four hundred and sixty-four intact, immature female hamsters were used for this investigation. The animals were raised in the laboratory and fed on Purina fox chow supplemented with greens twice a week. Water was supplied ad libitum. The date of birth of each litter was timed by observing all pregnant females for delivery between 8 A.M. and lo A.M. each day. In order to establish the relationship of postpartum age and the response of the ovaries to gonadotropins, the females were injected at ages ranging from 21 to 36 days. * Single subcutaneous injections of pregnant mare's serum, (PMS, Ayerst-Equinex) at concentrations of lo, 20, 30, or 40 I.U. were followed 54 hours later by a single subcutaneous injection of chorionic gonadotropin (ICSH). In order to determine the effects of high dosages of PMS and varying ICSH dosages upon the ovary, several combinations of these two hormones were administered to hamsters over 30 days of age. Groups of 9 and 5 animals each were injected with 60 I.U. PMS and 80 I.U. PMS, respectively, the ICSH remaining constant at 20 I.U. Five 31-day hamsters were injected with 40 I.U. PMS and 54 hours later received 40 I.U. ICSH. Five additional 33-day hamsters received 20 I.U. PMS alone. All injections were made into the nape of the neck with a #27 hypodermic needle. Care was exercised in rotating the needle before withdrawal in order to prevent loss of the injected materials. Within 48 hours after the last injection the animals were decapitated and the ovaries and oviducts removed immediately for examination. Each oviduct was severed at the uterotubal junction and the periovarial sac membrane was cut away from the ampullar loops. The oviducts were placed in a Maximow slide containing Locke's solution. A fine pipet was inserted into each fimbriated end and the oviduct flushed in such a manner that the ova could be observed escaping from the cut end. We are confident that in this way the total number of eggs ovulated was obtained for counting. Representative ovaries from all groups were fixed in Bouin's fluid, sectioned at 10 /L, and stained with hematoxylin and eosin. A hamster designated as 24 days old received the first injection on the twenty-fourth day after its birth.

3 352 BODEMER ET AL. Fertility & Sterility OBSERVA nons AND RESULTS Postpartum Age and the Response of the Ovaries to Gonadotropins Without exception, no ovulations occurred in the 28 hamsters receiving the first injection of pregnant mare's serum on the twenty-first day postpartum. The failure of ovulation, however, was not paralleled in all animals by a uniform ovarian response. In some of the 21-day-old hamsters, the ovaries were essentially refractory to the combination and dosages of gonadotropins injected. Histologically, the ovaries appeared similar to the untreated controls in that there was no evidence of follicular development. In others, there was some follicular stimulation as revealed by the presence of either a limited population of small vesicular follicles, or a moderate number of small and medium-sized follicles with antra. Certain of the ovaries from this group occasionally contained several sizable hemorrhagic follicles. A review of the sectioned material suggests that dosages of U. and U. of PMS elicit a slightly greater over-all follicular stimulation. Age of the female and gonadotropin dosage, however, may not be the only factors determining the amount of stimulation, since there is some preliminary evidence that there may be a correlation between body weight and the intensity of the TABLE 1. Relationship of Age and PMS Dosage to Ovulation Response in the Hamster 10 i.u. PMS 20i.u. PMS 30i.u. PMS 40i.u PMS Age of Hamster 20 i.u. I.C.S.H. 20 Lu. I.C.S.H. 20 Lu.LCS.H 20i.u.ICS.H No. of % No. of % No. of % No. of % Animals Ovulating Animals Ovulating Animals Ovulating Animals Ovulating II \

4 Vol. 10, No.4, 1959 INDUCED OVULATION IN IMMATURE HAMSTERS 353 ovarian reaction. The mean weight of the animals in this age group was 41 Gm., with a range of Gm. In most cases, those hamsters revealing the greater follicular stimulation were in the higher weight range. By the twenty-second day postpartum, only an occasional animal ovulated in response to gonadotropic injections (see Table 1). Although in some of the ovaries in this group there was no evidence of follicular stimulation, most of them contained a limited population of small and medium-sized follicles with antra and an occasional large, cystic follicle. The ovaries of 25-day-old hamsters receiving 30 I.U. or 40 I.U. of PMS consistently contained a greater number of hemorrhagic and cystic follicles. With advancing age of the hamster there was an accentuated ovarian response to injected gonadotropins (Table 1). A limited number of follicles ovulated in approximately half of the animals injected on the twentyfourth day postpartum (Figs. 1, 2, and 3). It is interesting that in this group there was no significant increase in either the number of animals ovulating or the mean number of eggs recovered as the dosage of PMS was raised. Examination of the serial sections of the ovaries of those hamsters which failed to ovulate again revealed only a negligible follicular development. During the next 24 hours the ovarian sensitivity to gonadotropic hormones increased rapidly so that approximately 80 per cent of the animals injected on the twenty-fifth day ovulated. At this age the ovaries were significantly enlarged as the result of the development of numerous small and intermediate-sized follicles with antra, and the presence of 48-hour-old corpora lutea. It should be emphasized that the mean number of eggs recovered in. this group was somewhat larger than in the 24-day series, but again, the number of follicles ovulating was not enhanced by increasing the amount of hormone injected. As noted earlier, there also appears to be some correlation between body weight and ovulation response in the 25-day-old animals. With the exception of a small group of hamsters receiving 10 I.U. of PMS, ovulation occurred in practically every animal injected on the twenty-sixth day postpartum. Again, on the twenty-sixth day, the animals that failed to ovulate were the lightest of the group. Histologically, all of the ovaries revealed widespread follicular stimulation and numerous normal-appearing corpora lutea in animals that had ovulated. One notable characteristic in the ovaries of this group was the significant increase in the interstitial tissue. On the twenty-seventh day postpartum, ovulation was induced in all

5 354 BODEMER ET At. Fertility & Sterility

6 Vol. 10, No.4, 1959 INDUCED OVULATION IN IMMATURE HAMSTERS 355 hamsters injected with gonadotropins in all dosages (see Table 1). As on the preceding day, over-all follicular stimulation was pronounced. In this group as well, there was a significant increase in the amount of interstitial tissue and the number and size of follicles with antra (Fig. 4). One may conclude from the data in Table 1 that the degree of ovarian stimulation evoked by the PMS-ICSH injection schedules appears to increase with advancing age of the hamster. Thus the ovaries of animals receiving gonadotropins on the twenty-ninth day showed a greater sensitivity and were even larger than those of the younger animals. This increase in size, again, was the result of both a larger number of follicles responding to the hormones and an increase in the amount of interstitial tissue. Although thecal luteinization was uncommon following injection of 10 LU. of PMS, it was frequently observed in animals subjected to 30 I.V. or 40 LV. of PMS. In animals in which there were many ovulations, the corpora lutea obviously comprised the major element of the ovary, although even here many small follicles with antra were present. It is noteworthy that no large ovulatory follicles remained, although an occasional cystic follicle was present. Ovulation occurred in all hamsters receiving the first hormone injection on the thirty-first, thirty-third, or thirty-sixth day postpartum (Figs. 5 and 6). The first spontaneous ovulation, said to be coincident with the first estrus,8. 9 was found to occur in the hamsters of our colony between the thirty-second day and thirty-sixth day postpartum. The mean number of induced ovulations in animals at this age group was significantly above normal at all dosage levels. The preceding data reveal, then, that the number of immature hamsters ovulating in response to gonadotropic hormones increases as the animal approaches its first estrus. The postpartum age of the hamster is indeed an important factor in determining the ovarian response to gonadotropic hormones. Figs Sections through the ovaries of hamsters removed 48 hours after treatment with different dosages of PMS and a constant dosage of ICSH which illustrate the range of ovarian stimulation obtained on the twenty-fourth day postpartum. Fig. 1. Minimal ovarian stimulation. Injections of 10 LV. PMS and 20 LV. ICSH (X 26). Fig. 2. Optimal ovarian stimulation. Injections of 20 LV. PMS and 20 LV. ICSH (X 26). Fig. 3. Maximal ovarian stimulation. Injections of 40 LV. PMS and 20 LV. ICSH ( X 25). Fig. 4. A section through the ovary of a 27 -day-old hamster treated with 20 LV. PMS and 20 LV. ICSH. Note the absence of large ovarian follicles and the welldeveloped interstitial tissue (X 25).

7 356 BODEMER ET Al. Fertility & Sterility Fig. 5. A section through the ovary of a 36-day-old hamster treated with 20 I.U. PMS and 20 I.U. ICSH illustrating optimal ovarian stimulation. Relation of Postpartum Age and PMS Dosage to the Number of Ova Ovulated In addition to the correlation between postpartum age and the response of the ovaries to gonadotropins, there appears to be a comparable relation between chronologie age and the number of ova ovulated. It will be noted Fig. 6. A section through the ovary of a 36-day-old hamster treated with 40 I.U. PMS and 20 I.U. ICSH illustrating maximal ovarian stimulation.

8 Vol. 10, No.4, 1959 INDUCED OVULATION IN IMMATURE HAMSTERS 357 TABLE 2. Relationship of Age and PMS Dosage to Number of Eggs Ovulated in the Hamster Age of Hamster Lu. PMS 20i.u. PMS 30Lu. PMS 40i.u. PMS Lu. I.C.SH. 20 i.u. I.C.S.H. 20 i.u. I.C,S,H. 20 LU I.C S,H, Mean Mean Mean Mean No. Ova Range No. Ova Range No. Ova Range No, Ova Range 5 5 I I \ \ from the data summarized in Table 2, that, regardless of PMS dosage, there is a gradual increase in the mean number of ova ovulated as the hamsters approach puberty (32-36 days). Thus, postpartum age represents a factor of considerable importance in determining the magnitude of the ovulatory response to gonadotropins. It is also noteworthy that after the twenty-fifth postpartum day the amount of PMS administered seems to exert, within boundaries, a definite influence upon the total number of ova ovulated. On the contrary, in the younger hamsters in which only a percentage of the total treated animals ovulated, there was no apparent correlation between the number of ova found in the oviducts and PMS dosage. This response again emphasizes the gradual development of sensitivity of the ovaries to hormonal stimulation. As suggested earlier, the effectiveness of PMS dosage in increasing the number of eggs ovulated is limited. High PMS dosages fail to increase Significantly the number of ova ovulated. As shown in Table 2, an average of 48 ova per animal was removed from 36-day-old hamsters injected with 40 LU. of PMS. Hamsters of identical age injected with 60 LU. of PMS and 80 LU. of PMS ovulated an average of 40 and 52 ova, respectively. The number of ovulations was not significantly increased in 36-day-old hamsters receiving either 40 LV. or 60 LU. of PMS followed 54 hours later with 40 LV. of ICSH.

9 358 BODEMER ET AL. Fertility & Sterility DISCUSSION This investigation has demonstrated that single consecutive injections of PMS and ICSH may induce ovulation in about 10 per cent of prepuberal hamsters as early as the twenty-second day postpartum and in all hamsters 26 days of age or older. The onset of the first estrus and ovulation apparently varies with the individual colonies and has been reported as occurring between the twenty-eighth and thirty-second day after birth.5, 8,9,14,18 In the hamsters used for this study, the first heat period and ovulation usually occurred between the thirty-second and the thirty-sixth day postpartum. Thus, by appropriate injections of PMS and ICSH, ovulation may be induced from 6 to lo days prior to the first spontaneous ovulation. Experiments with a limited number of animals suggests that the injection of PMS or ICSH alone is not effective in inducing ovulation in the hamster. This agrees with the report by Ortiz that she found no corpora lutea in the ovaries of animals which had received PMS injections only on either the twenty-sixth day or the thirty-sixth day postpartum. Despite the fact that PMS represents a sine qua non for the induction of ovulation in the hamster, in animals 22 days of age or younger the amount of this hormone injected does not exert an appreciable influence upon the ovary. In older prepuberal hamsters, however, there is an apparent correlation between PMS dosage and the magnitude of ovarian stimulation, vis-a-vis ovulation. Ten LU. of PMS, for example, sets the stage for a small number of ovulations, and hemorrhagic and cystic follicles only infrequently develop at this dosage. As the PMS dosages are raised from 20 LV. to 40 LV. there is a significant increase in the number of cystic and hemorrhagic follicles, and dosages of 60 LU. to 80 LU. of PMS elicit the development of numerous hemorrhagic and cystic follicles, thus indicating significant overstimulation. In view of the relation of PMS dosage and the magnitude of ovarian response in older prepuberal hamsters, it is not too surprising to observe a similar correlation between PMS dosage and the number of ova ovulated. Although only a limited number of animals were subjected to varying amounts of ICSH with the PMS dosage held constant at 20 LV., the data suggest that the interstitial cell-stimulating hormone does not exert a determining influence upon the number of ovulations. Conditions in the hamster thus parallel those described for the rat,13 and it seems likely that the number of ovarian follicles brought to maturity in both animals is a function of the FSH present in the PMS. The inability of ICSH alone to induce ovulation is consistent with this concept.

10 Vol. 10, No.4, 1959 INDUCED OVULATION IN IMMATURE HAMSTERS 359 The role of postpartum age in determining the response of the reproductive tissues to hormones has been the subject of considerable investigation. Many investigators have described an early refractory period followed by a gradual increase in responsivity of the ovary to gonadotropins in the rat/' mouse/ 1 and rabbit. 6 In the hamster Ortiz noted an increase in ovarian weight in response to PMS as early as the tenth day postpartum. It was not due, however, to follicular development. Our results reveal a uniform group response to gonadotropins on the twenty-sixth day and the maximal ovarian response between the thirty-third and the thirty-sixth day postpartum. There is little reason to doubt that age is of importance in determining the effects of hormones on the ovary, although the precise significance of this observation remains obscure. It has been proposed 12 that, in the rat, ovarian reactivity to gonadotropins is a function of the population of large ovarian follicles at the time of hormone administration. Ortiz suggests that the hamster ovary becomes responsive to PMS coincident with the occurrence of an appreciable number of antral follicles in that organ. The very limited response of the hamster ovary before the twenty-fourth day after birth and its variable response before the twenty-sixth day suggest that this organ undergoes a gradual development of sensitivity before it responds fully to gonadotropic influences. This maturation may be expressed by the number of antral follicles contained within the ovary, but the nature of the maturation mechanism needs to be explored. SUMMARY The optimal conditions for the induction of ovulation was studied in 464 immature hamsters of 21 to 36 days of age. Ovulations could be induced 6 to 10 days before sexual maturity by single consecutive subcutaneous injections of 20 I.U. of pregnant mare's serum followed 54 to 56 hours later by U. interstitial cell-stimulating hormones. The effectiveness of these hormones is age-dependent. Ovulations were inconsistent before the twenty-sixth day, but after this time ovulations were induced in all treated hamsters. The maximal ovarian reaction to PMS and ICSH occurs at days, the approximate time at which the hamster attains sexual maturity. In older prepuberal hamsters there is an apparent relation between the amount of administered PMS and the number of ova ovulated. possible factors accounting for the gradual increase in ovarian reactivity have been discussed.

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