The purpose of present study was to evaluate the effects of in vivo and in vitro treatment of GnRH agonist on morphological and physiological changes in the ovaries of intact cyclic mice. Several in vivo and in vitro studies performed in rats have described mostly the antigonadal effect of GnRH analogs . The majority of in vivo study was performed on hypophysectomized rat. Major findings of this study are that in vivo treatment of GnRH-Ag caused both stimulatory and inhibitory effects on ovarian follicular development, ovulation and luteinization in intact cyclic mice. The short term (8 days) treatment with 5 μg per day dose of GnRH-Ag caused stimulatory effects on ovarian steroidogenesis and follicular development. On the other hand, 25 μg per day dose of GnRH-Ag treatment caused inhibitory effects on follicular development and ovulation. The ovaries treated with GnRH-Ag alone in vitro, showed significant decline in progesterone secretion and steroidogenic markers. But when ovaries were treated with GnRH-Ag along with LH, there is increase in progesterone synthesis. This increase in progesterone synthesis is due to increase responsiveness of LH in the presence of GnRH-Ag.
The mice treated with the different doses of GnRH-Ag showed a marked variation in the circulating steroids concentration, luteal morphology and ovarian expression of LH receptor, StAR and 3β-HSD proteins. Treatment with high (5 μg/day) dose of GnRH-Ag showed only a few large antral follicles but showed many newly formed functional corpus luteum in the ovary. These mice also showed significantly high circulating progesterone level, increase expression of LH receptor, StAR and 3β-HSD proteins in the ovary. This can be correlated with healthy luteal morphology suggesting recent ovulation. These observations suggest that treatment with high dose of GnRH-Ag caused stimulatory effects on the ovary, perhaps due to increased gonadotropin release.
The treatment with pharmacological dose of GnRH-Ag showed subnormal luteal morphology and only a marginal increase in the ovarian expression of LH receptor and StAR proteins while no change in 3β-HSD protein expression and circulating progesterone level compared with the control. These observations suggest that the mice treated with pharmacological doses of GnRH-Ag lack functional corpus luteum in the ovary, perhaps due to decreased gonadotropin release. However, the inhibitory action of GnRH analogs on progesterone synthesis has earlier been demonstrated in the ovaries of rat and human [22–24]. It has been reported that short-term GnRH-Ag treatment impairs the progesterone synthesis by directly acting on ovary . Our study showed that the treatment with GnRH-Ag showed both stimulatory and inhibitory effects on steroidogenesis in the ovary. The changes in ovarian steroidogenesis by treatment with GnRH-Ag may be due to their indirect effect on gonadotropin release or direct effects on steroidogenic factors involved in cholesterol transport and/or on the enzyme involved in the steroidogenic pathway.
The mice treated with pharmacological (25 μg/day) dose of GnRH-Ag also showed inhibitory effects on follicular development and ovulation. This was assessed by decreased number of follicles at different stages of development and confirmed by the sharp decline in the circulating E2 level and ERα expression in the ovary. It is well known that estrogens are essential for normal follicular growth and are crucial for the survival of ovarian follicles [26, 27]. These mice showed morphological sign of regression particularly in the granulosa cells of antral follicles, since GnRH receptor is mainly localized on these cells [3, 4]. GnRH-Ag treatment also caused reduction in thecal layer of the antral follicles. This may be probably due to pharmacological dose of GnRH-Ag induced decrease in gonadotropin support to the theca cells and/or indirect suppression of paracrine factors produced by the granulosa cells, which affects thecal development . It is likely that pharmacological dose of GnRH-Ag desensitize pituitary gonadotropes resulting in decreased release of LH and FSH and a decline in ovarian follicular development and steroidogensis . This desensitization phenomenon is extensively applied in clinical medicine for the treatment of wide range of diseases .
Although serum gonadotropin levels could not be measured in this study, it is known that moderate dose of GnRH agonist treatment increases LH secretion but FSH remains unaffected . In that case ovulation may be normal but folliculogenesis and selection of dominant follicle is hampered. In our experiment we have found both stimulatory and inhibitory effects of GnRH agonist which dependents on dose of treatment. Treatment with 5 μg dose of GnRH-Ag caused stimulatory effect with stimulatory role in ovulation, while 25 μg dose of treatment failed to induce ovulation or formation of functional CL in the ovary. Both the doses decreased folliculogenesis with decreased number of late antral follicles suggesting low FSH.
Treatment with pharmacological dose of GnRH-Ag significantly suppressed the circulating estradiol concentration when compared with the control. The inhibition of folliculogenesis and the increase in ovarian follicular apoptosis observed in the mice treated in vivo with GnRH-Ag may suggest suppression of gonadotropin secretion resulting in decline in ovarian expression of estrogen receptor and serum estradiol production [28, 31, 32]. These mice showed presence of several antral follicles, instead of being selected these follicles undergo atresia and produce reduced level of estradiol suggesting decreased gonadotropin secretion [30–32]. It is well known that increase level of gonadotropin stimulation is required for proliferation (growth) and cellular differentiation (steroid synthesis) of the late antral follicles.
The treatment with increasing dose of GnRH-Ag in vivo also caused a gradual increase in the number of atretic follicles in the ovaries of cyclic mice. Since follicular atresia is mediated by apoptosis, the effect of GnRH-Ag on apoptotic factors was analyzed in the mice ovary. The results of this study demonstrated that treatment with GnRH-Ag (high and pharmacological dose) in vivo showed a significant increase in the ovarian expression of caspase-3 and cleaved PARP proteins in mice. Increase in the expression of caspase-3 and cleaved PARP proteins noticed in the mice treated in vivo with GnRH-Ag also showed increase in the number of atretic follicles in the ovary. In an earlier study in rat, GnRH treatment, in vivo, has shown to significantly reduce the mitotic activity of granulosa cells and increase the number of pyknotic cells in the ovarian follicles of hypophysectomized female rats, thus providing evidence for the direct effects of GnRH in follicular atresia . This finding thus support earlier studies that suggest GnRH analogs may enhance follicular atresia and it is by directly stimulating apoptotic factors in the ovary.
The present in vitro study showed suppressive effect of GnRH-Ag alone on ovarian progesterone synthesis. This decrease in progesterone synthesis is associated with decreased LH receptor and 3β-HSD proteins, thus showed inhibitory action of GnRH-Ag on the ovary. However, both the doses of GnRH-Ag together with LH showed increase progesterone synthesis and LH receptor in the ovary. It appears that ovarian GnRH plays a physiological role via local GnRH-gonadotropin axis. GnRH and its receptors have been well demonstrated in the ovary and previous findings showed the physiological role of ovarian GnRH during reproductive cycle of the animal [34, 35]. Recently, local production of gonadotropin hormones has also been demonstrated in the ovary of rat . Therefore, the present in vitro study further showed that the ovarian response to GnRH-Ag depends upon the dose of treatment and presence or absence of LH. This finding supports the in vivo study showing high dose of GnRH-Ag caused stimulatory effect on the ovary indirectly by increasing gonadotropin release.
This study further showed that the treatment with GnRH-Ag alone decreases ovarian expression of estrogen receptor protein, but together with LH significantly increases the expression of estrogen and progesterone receptor proteins in the ovary. Since estrogen receptors are required for differentiation of granulosa cells and maturation of dominant follicle and progesterone receptor is required for ovulation [37–40], it may be suggested that GnRH-Ag alone may be responsible for inhibition of follicular development, but in presence of LH, GnRH agonist may be stimulating follicular development and maturation. These effects of GnRH analogs may be modulated by the presence of gonadotropins.