Studies on gonadotropins gonodotropic regulation of follicular maturation in cyclic hamsters using antisera to FSH & LH

Abstract

The dependence on FSH and LH in the process of follicular maturation, as assessed by physiological and biochemical indices in adult cyclic hamsters, has been described in the present thesis. Anti-gonadotropin sera have been used as tools in studying the role of FSH and LH in this process. Antisera produced against ovine FSH and LH in the monkey and the rabbit, respectively, were thoroughly characterized for specificity employing a variety of criteria. The antisera used were: (a) highly specific, as indicated by their ability to neutralize only the corresponding hormone, without affecting the circulatory level of the other gonadotropin; (b) highly potent, as evident from the minimal effective doses, which were very small; and (c) able to neutralize the gonadotropin of the hamster, as evident from specific bioassays as well as by the effect the antisera produced on ovarian events when given to hamsters. By using such antisera, it was possible to selectively deprive one gonadotropin at a time in an intact animal and study the effect of withdrawal of trophic hormone support on ovarian events, with particular attention to follicular development. Neutralization of the proestrous surge of FSH had no effect on several periovulatory processes like oocyte maturation and ovulation occurring on the next day. The former was assessed by the ability of the expelled ova to undergo fertilization and implantation normally. It also had no effect on the process of luteinization and luteal function, as indicated by the normal levels of plasma and ovarian progesterone from the evening of proestrus till diestrus-1. Confirmatory evidence for FSH being not required as a trophic factor for luteal function was obtained in the pregnant hamster as well, when it was found that acute neutralization of FSH during pregnancy had no effect on luteal progesterone concentration. In the same system, however, lack of FSH affected follicular development, as indicated by the estrogen concentration in the non-luteal portion of the ovary and the inability to ovulate in response to hCG. In contrast, neutralization of LH on proestrus produced dramatic effects on all the above processes, indicating a specific need for the trophic support of LH in all the above-mentioned periovulatory processes. Of the several periovulatory events considered, neutralization of the proestrous surge of FSH, however, affected the process of follicular maturation during the cycle. In the initial experiments, effects on gross parameters like vaginal cyclicity, ovarian and uterine weights after long-term exposure to FSH antiserum were observed. In later studies, attention was focused on examining the effect on follicular development during the cycle when treatment was initiated. These experiments indicated that both FSH and LH are required for the process of follicular development during the cycle. Administration of a single dose of antiserum to FSH or LH on proestrus caused a disruption in follicular development, as assessed by the following criteria: (a) a blockade of ovulation expected to occur on the next estrus, ovulation being considered an index of completion of follicular development; (b) absence of ovulable follicles on the next proestrus, as evident from histological observations; (c) a reduction in ovarian estrogen concentration, the latter being a product of the follicles and considered to indicate the state of follicular growth; (d) lack of the surges of FSH and LH on the next proestrus as a consequence of reduced estrogen on diestrus-2; and (e) a reduction in ovarian progesterone and estradiol concentrations on the next proestrus. Administration of a single injection of FSH antiserum on proestrus was found to cause virtually continued neutralization of circulatory FSH for the next 4 days, and hence the above effects observed could have been due to a total lack of FSH during the cycle. Withdrawal of FSH by injecting FSH antiserum at any time of the cycle until the evening of diestrus-2 caused a blockade of follicular maturation, as indicated by the lack of ovulation at the ensuing estrus. However, a similar treatment with FSH antiserum on the morning of proestrus was ineffective in preventing ovulation occurring on the next day. These experiments indicated that there is a continuous need for FSH in follicular development during the cycle, and this need ceases by the morning of proestrus, by which time the follicles reach the final stages of maturation and require the trophic support of only LH. Further evidence for the above contention, that there is a differential need for FSH during the final stages of maturation, was obtained when it was found that while FSH antiserum administered on diestrus-2 affected the estrogen concentration of the ovary on the next day, the same dose of antiserum given on proestrus had no effect on the estrogen concentration on the evening of the same day. Since the role of the surge of FSH or LH in the initiation of follicular development was not clear from the above studies, experiments were specifically devised to examine this aspect. In these, after administration of antisera at the beginning of the surge, the excess antibody found at the end of the surge was inactivated by injecting an optimal amount of the corresponding hormone, thus achieving neutralization of only the surge of FSH or LH. Such specific neutralization of only the surge of either FSH or LH affected the overall process of follicular maturation as indicated by the lack of ovulation at the end of the cycle. Biochemical markers for indicating initiation of follicular maturation were then sought, and the effect of antisera on these was studied. 1. An examination of the pattern of incorporation of H-thymidine into ovarian DNA in vitro, throughout the cycle, indicated that the highest rate of incorporation occurred on estrus. This correlated well with the reported increase in the number of large preantral follicles at the beginning of the cycle and hence could be considered indicative of the initiation of follicular growth during the cycle. The stimulus for this increased incorporation was, indeed, found to be the proestrous surges of FSH and LH occurring 19 h prior to the maximum rate of incorporation observed. The presence of FSH for a few hours was sufficient to maximally stimulate this event, since neutralization of FSH at the end of the proestrous surge did not significantly affect the rate of incorporation of H-thymidine into DNA on estrus. It was interesting to observe that simultaneous neutralization of surges of both FSH and LH on proestrus brought about a reduction in H-thymidine incorporation to the same extent as found with either of the antisera. This indicated that both gonadotropins could be affecting the same cell type. Further, the effect of LH in this process appeared to be a direct one, as it could not be reversed by simultaneous injection of estrogen. Similarly, exogenous LH and FSH could not substitute for the lack of endogenous FSH and LH, respectively, thus indicating the specificity of the observed effect. It was interestingly observed that in the antiserum-treated animals, the pool size of the nucleoside, as indicated by the radioactivity in the acid-soluble fraction, did not change, thus making the observed reduction in the incorporation of H-thymidine more valid. Using an immature hamster model system and PMSG as the gonadotropin, it was observed that maximal stimulation of H-thymidine incorporation occurred 18 h after administration of a single dose of PMSG, and here again, 8-10 h later, the activity of PMSG could be neutralized without significantly affecting the rate of incorporation of labeled thymidine into ovarian DNA in vitro 18 h after PMSG. 2. Another parameter studied, with a view to use as an index of the growth process, was the activity of ornithine decarboxylase (ODC), the enzyme involved in polyamine biosynthesis. The only time during the cycle when its activity could be found was on the evening of proestrus, concomitant with the surges of gonadotropins. Its activity was associated both with the preovulatory Graafian follicles as well as with the smaller "growing" follicles. The increase in its activity was abolished with neutralization of only the surge of LH, FSH neutralization having no effect on this event. It could be inferred from these results that LH, thus, affects both polyamine and DNA synthesis, while the effect of FSH was discernible only on DNA synthesis, having apparently no effect on polyamine biosynthesis as indicated by the activity of ODC. This suggested a dissociation of these two events, i.e., DNA synthesis and polyamine synthesis, normally expected to be closely related events. An attempt has been made in the present study to measure the concentration of FSH in the ovarian tissue and correlate the same to follicular growth. For this purpose, a specific radioimmunoassay, carried out in its entirety at 37°C, was employed, and the efficacy of the method to measure all the hormone present in the tissue was ensured. It has been assumed that FSH measured in the ovary represents that bound to follicular cells, since no other cell type has been known to contain receptors for FSH. The tissue concentration of FSH did not exhibit correlation to serum levels of FSH at all periods of the cycle. On the other hand, it correlated well with follicular growth. Indeed, the tissue concentration of FSH was significantly higher at stages such as diestrus-2 and proestrus, when large follicles could be found in the ovary. On proestrus, FSH could be found in the Graafian follicles, particularly in the follicular cells and not in the follicular fluid. The small "growing" follicles also had considerable amounts of FSH, and this appeared to be important in follicular maturation, since neutralization of FSH caused a drastic reduction in tissue FSH level, and hence in FSH receptors on diestrus-2 and proestrus, at a time when normally it shows high levels during the cycle. This gave another indication of the importance of FSH in the follicular maturation process. On diestrus-2, the tissue concentration of FSH, which showed high values despite the serum concentration being low at this period, was found to be uninfluenced by either estrogen or LH, the two hormones which could be expected to have some interaction with ovarian cells. However, estrogen in another model system using immature rats could cause an increase in binding of endogenous FSH to the tissue and also elicited a response in terms of incorporation of H-thymidine. The latter effect, however, was negated when antisera to FSH or LH was given along with estrogen. It appears from these results that there is a complex interplay of FSH, LH, and estrogen in bringing about this response in the intact immature rat. A bioassay for FSH could, perhaps, be devised using this parameter. Since estrogen is a specific product of the follicle, the role of FSH and LH in estrogen synthesis was studied. Long-term deprivation of FSH or LH affected the in vitro estrogen synthesis by the ovary in response to LH. Since this could have been due to the inhibition of growth of the follicular apparatus per se, the effect of acute withdrawal of FSH or LH on estrogen biosynthesis was studied. Administration of antiserum to FSH on diestrus-2, within a few hours, caused a reduction in the endogenous estradiol concentration and also decreased the ability of the ovary to synthesize estrogen from exogenous testosterone. Only the addition of FSH, complemented with testosterone, resulted in the production of estrogen in amounts similar to that made by untreated control ovaries incubated with testosterone alone. On the other hand, the effects of LH antiserum were totally different. Within a few hours of its administration, the endogenous estradiol concentration was drastically reduced. Such ovaries, when incubated in vitro with testosterone, could, however, synthesize estradiol in amounts comparable to that of ovaries from untreated animals. This result suggested that testosterone was the component affected by the lack of LH in vivo. This was, in fact, found to be so, since endogenous levels of testosterone in the ovary were significantly affected by LH antiserum treatment. FSH antiserum, however, did not reduce ovarian testosterone, but specifically affected its aromatization to estradiol. However, it was interesting to find that without either endogenous FSH or LH, testosterone added in vitro itself could stimulate estradiol synthesis and thus apparently regulate its own conversion. Surprisingly enough, a similar administration of FSH antiserum on the morning of proestrus was ineffective in affecting the in vitro conversion of testosterone to estradiol on the evening of proestrus, while LH antiserum exerted its inhibitory effect whenever given, thus providing further evidence for a differential need for FSH during follicular growth, as mentioned earlier. The effect of neutralization of FSH or LH for short or long durations on the ovarian cholesterol and triglyceride was also examined. These studies were undertaken following qualitative histochemical observations. A decrease in free and esterified cholesterol as well as triglycerides was observed on short-term or long-term neutralization of endogenous FSH. LH antiserum caused an opposite effect with respect to cholesterol ester and triglyceride, but resulted in a reduction of free cholesterol. The involvement of FSH in sterol and lipid metabolism in the ovary of cyclic animals has been entirely unknown so far and needs further investigation. In conclusion, the above studies have shown that it is possible to utilize antisera in a finely regulated manner to gain maximum information about the physiological role of endogenous hormones in regulating key physiological events such as follicular development. Although in general the need for FSH and LH in this process has been known, the present study has brought to light several interesting features such as the differential or simultaneous requirement for the gonadotropins at particular times of the cycle in stimulating specific events during follicular development. Using this approach, there appears to be further scope to extend these studies to examine the mechanism of action of FSH and LH at the molecular level in exerting a mitogenic action on follicular cells.