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Several studies suggest that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) modulate testicular function. In this study, we examined the expression of cGMP-dependent protein kinase G-I (PKG-I), and cyclic nucleotide-gated channel 1 (CNG-1), 2 known mediators of cGMP action, and the expression of soluble guanylyl cyclase (sGC) subunits in the rat testis. Immunohistochemical analysis revealed that the alpha subunit of sGC was expressed in the blood vessels and Leydig cells of adult rat testes. In addition, the sGC alpha subunit was observed in the acrosomal structures of spermatids undergoing the middle and later stages of spermiogenesis, but not in mature spermatozoa. Similar localization and expression patterns were seen for the sGC beta subunit, indicating coexpression of the sGC subunits. PKG-I was expressed in blood vessels and in the acrosomal region of spermatids during the early and middle stages of spermiogenesis but was not observed in Leydig cells or in mature spermatozoa. In contrast to sGC and PKG-I, CNG-1 was expressed only in cytoplasm and the residual bodies of late-stage (17-19) spermatids, with no staining observed in blood vessels and Leydig cells. These results demonstrate that sGC, PKG-I, and CNG-1 are expressed in a stage- and cell-specific manner in the rat testis. The distinct temporal patterns of expression of these components of cGMP signaling pathways suggest different physiological roles for sGC, PKG-I, and CNG-1 in spermiogenesis and steroidogenesis.

Objective: To demonstrate the expression of soluble guanylyl cyclase (sGC) alpha and beta subunits in rat granulosa cells and determine the effects sGC activation on levels of cyclic GMP (cGMP), E2, and cAMP., Design: Basic research study., Setting: University research laboratory., Animal(s): Estrogen-treated immature Sprague-Dawley female rats from which primary cell culture of granulosa cells was obtained., Intervention(s): Functionally immature rat granulosa cells were incubated for 48 hours with media alone, FSH, or FSH plus YC-1, a specific activator of sGC., Main Outcome Measure(s): Expression of sGC alpha and beta subunits was determined by immunoblot analysis. Media concentrations of E2, cAMP, and cGMP were measured by radioimmunoassays., Result(s): Immunoblot analysis of granulosa cells revealed the expression of sGC alpha and beta subunits. While cGMP accumulation was low in cells incubated with media alone or with FSH, cotreatment with FSH plus YC-1 increased cGMP levels approximately five-fold. Incubation of cells with FSH stimulated E2 production in a dose-dependent manner. However, cotreatment of cells with FSH plus YC-1 significantly decreased E2 concentrations at all doses of FSH tested. Similarly, while FSH increased cAMP accumulation from granulosa cells, cotreatment with YC-1 markedly inhibited FSH-stimulated cAMP levels., Conclusion(s): These findings demonstrate the expression of sGC subunits in rat granulosa cells and indicate that activation of sGC increases cGMP levels, which are associated with inhibition of FSH-stimulated E2 production and cAMP accumulation.

The present study examined the gonadotropin surge-inducing actions of estradiol (E(2)), both alone and with progesterone (P(4)), in middle-aged, early persistent-estrous (PE) female rats that had become PE within 35 days. In addition, we also assessed the effect of P(4) on the mating-induced gonadotropin surges in these acyclic animals. Early PE rats were ovariectomized and received E(2) implants (Day 0). On Day 4, an s.c. injection of P(4) (0.5 mg/ 100 g body weight) at 1200 h markedly increased plasma P(4) and elicited both LH and FSH surges, whereas vehicle-treated controls displayed no rise in P(4) or gonadotropins. This observation confirms that at middle age, female rats no longer respond to the positive-feedback stimulation of E(2) on gonadotropin surges whenever the estrous cyclicity ceases. As PE continued, such a surge-inducing action of E(2) plus P(4) became diminished after 75 days of PE and disappeared thereafter. When caged with males, vehicle-treated early PE rats display a mating-induced increase in P(4) from the adrenal along with small gonadotropin surges. The amplitude of these mating-induced gonadotropin surges was enhanced by supplementation with exogenous P(4) in early PE rats. Our findings indicate that during the early phase of PE, the surge-inducing action of E(2) and P(4) remains intact but deteriorates as PE continues. Thus, a deficiency in P(4) secretion during aging may contribute to the diminished gonadotropin surge response in the hypothalamic-pituitary axis and the subsequent cessation of estrous cyclicity.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.

Objective: To determine the effects of nitric oxide (NO) and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells., Design: Basic research study., Setting: University research laboratory., Animal(s): Primary cell culture of granulosa cells obtained from estrogen-treated, immature Sprague-Dawley female rats., Intervention(s): Functionally immature rat granulosa cells were incubated for 48 hours with media alone; FSH; forskolin; the NO generator DETA/NO; an inhibitor of soluble guanylyl cyclase (ODQ); and/or a cell-permeable cGMP analog., Main Outcome Measure(s): Media concentrations of inhibin A were measured by solid-phase immunosorbent assay. Inhibin alpha and betaA subunit mRNA levels were determined by Northern and slot blot analyses., Result(s): Whereas FSH caused a 20-fold increase in inhibin A levels compared with untreated granulosa cells, the NO generator DETA/NO significantly inhibited FSH-stimulated inhibin A concentrations. Similarly, cotreatment with FSH plus dibutyryl cGMP significantly attenuated inhibin A concentrations, compared with those in cells treated with FSH alone. Incubation with forskolin (FSK) stimulated inhibin A levels sevenfold, whereas cotreatment with FSK plus DETA/NO or FSK plus dibutyryl cGMP effectively decreased inhibin A concentrations. The effects of NO on inhibin A levels were not prevented by cotreatment with an inhibitor of soluble guanylyl cyclase. In addition, there was no influence of DETA/NO or dibutyryl cGMP on inhibin subunit mRNA levels., Conclusion(s): These findings indicate that NO and cGMP can attenuate inhibin A concentrations through actions at one or more post-FSH receptor sites. These influences may reflect inhibition of inhibin A secretion, rather than gene expression and protein synthesis. In addition, NO decreases inhibin A concentrations through both cGMP-dependent and -independent pathways. These results suggest local roles for NO and cGMP in the regulation of granulosa cell function.

The production of a viable oocyte is dependent upon the critical influences of gonadotrophins on follicular development, granulosa cell maturation, ovulation, and luteinization. While the effects of LH and FSH are due in large part to cyclic AMP-dependent signalling mechanisms, it is clear that a number of other factors modulate the actions of gonadotrophins on the ovary via activation of alternative signalling pathways. In this regard, recent studies indicate that the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) mediates a wide range of influences on the ovary. Nitric oxide (NO) is a major regulator of cGMP production via its action on soluble guanylyl cyclase, while natriuretic peptides activate receptors with intrinsic guanylyl cyclase activities. In addition, other factors known to influence ovarian functions are now recognized to act via NO/cGMP pathways. This report will review these previous findings and present new data demonstrating the inhibitory influence of cGMP on cAMP-stimulated LH receptor expression in cultured granulosa cells.

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.

This study examined the effects of aging on LH surge magnitude, ovulation, and ovarian expression of tissue-type plasminogen activator (tPA), a protease implicated in follicular rupture. While mean LH levels and ovulation rates were similar in middle-aged cyclic and young groups, there was a significant correlation between peak LH levels and ovulation rates in individual rats, such that females with lower LH surges ovulated fewer ova. In a separate experiment, proestrous LH levels were characterized in young and middle-aged rats, followed by in situ hybridization analysis of ovarian tPA mRNA. In young proestrous rats, tPA expression was observed in thecal-interstitial cells and oocytes, but not granulosa cells, prior to the LH surge. After the LH surge, there was a marked increase in tPA mRNA levels in granulosa cells of preovulatory, but not smaller follicles, peaking at 0200 hr estrus. By 0500 hr estrus, ovarian tPA expression declined, and ovulation had occurred. In contrast, LH-induced follicular tPA mRNA levels were dramatically lower in middle-aged rats with attenuated LH surges, and persisting preovulatory follicles were common in ovaries of these females on estrus morning. These findings suggest that age-related declines in ovulatory function result in part from altered induction of ovarian tPA expression, likely due to decreased proestrous LH secretion.

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.

WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.

The present study examined the effects of progesterone (P4) treatments on estrous cyclicity and the loss of ovarian follicles during aging. Young rats received repeated treatments with P4 or empty implants between 3.5 and 8 mo of age. At 8 mo, ovaries were obtained from some animals to determine the numbers of resting follicles, and estrous cycle patterns and hormone levels were determined from other groups of treated females. In contrast to the cyclic increases in P4, estradiol (E2), LH, and FSH in control animals, P4-implanted rats exhibited elevated serum P4 but low E2, LH, and FSH levels. After implant treatments, the follicular reserve was significantly (p < 0.05) larger in P4-treated females (2012 +/- 297 resting follicles per ovary, n = 5 rats per group) than in regularly cyclic control rats (713 +/- 226 follicles per ovary, n = 7). The effects of P4 implants on the follicular reserve were associated with a subsequently higher incidence of regular estrous cycles after P4 treatment. These results demonstrate that P4 prevents cyclic increases in E2 secretion and is associated with a conservation of the ovarian follicular reserve and the maintenance of regular estrous cycle patterns, indicating a protective effect of P4 on the age-related loss of ovarian follicles.

Reproductive aging in the female rat is associated with the gradual loss of regular ovulatory function, decreased fertility, and smaller litter sizes. In the present study, we assessed ovarian ovulatory responsiveness to exogenous gonadotropin stimulation in young and middle-aged cyclic females and in middle-aged acyclic persistent-estrous (PE) rats. The ovulatory response to human chorionic gonadotropin (hCG) was dose-dependent in both young and middle-aged cyclic rats, with the percentages of rats ovulating and the numbers of ova shed per rat increasing with the dose of hCG administered. At the highest dose tested (10 mIU hCG/g bw), the range in ovulation rates among middle-aged cyclic rats (0-18 ova shed/rat) was greater than that in young animals (12-18 ova/rat). However, there were no statistically significant differences in either the percentages of females ovulating or in the mean ovulation rates between young and middle-aged cyclic groups. In contrast to the normal ovulatory responses observed in most middle-aged cyclic animals, response to hCG was markedly impaired in PE females of the same age. Middle-aged PE rats consistently failed to ovulate in response to a dose of hCG (10 mIU/g bw), which elicited high ovulation rates in young rats. At an even higher dose (20 mIU/g bw), only minimal ovulatory responses were observed (1.8 +/- 0.8 ova/rat; 80% of rats ovulating). Thus, most middle-aged regularly cyclic females maintain a similar ovulatory responsiveness to hCG as young rats, suggesting that decreased ovulation rates during aging may be related to attenuated preovulatory LH surges. However, impaired ovulatory responses were observed in middle-aged PE females, indicating altered ovarian function in acyclic animals, which may contribute to their anovulatory state.

The stimulatory effects of gonadotropins on antral and preovulatory follicles are well known, but conflicting results have been reported regarding the gonadotropin responsiveness and dependency of preantral follicles. Taking advantage of the relatively uniform development of the first wave of follicles in the postnatal rat ovary, we evaluated the role of endogenous and exogenous gonadotropins on preantral follicle development. Reduction of the high levels of gonadotropins present in juvenile rats by either hypophysectomy (at Day 15) or GnRH antagonist treatment (starting from Day 11 of age) resulted in decreased ovarian weight at Day 19 of age that was associated with a reduced number of developing follicles and increased atresia of remaining follicles. In contrast, treatment with FSHctp (a long-acting FSH agonist) in intact (Days 5-19 of age), hypophysectomized (Days 15-19), or GnRH antagonist-treated (Days 11-19) animals resulted in increased ovarian weight and follicle development as determined histologically and by inhibin-alpha expression. A dose-dependent stimulatory effect of hCG on ovarian weight was seen when animals were cotreated with FSHctp and the GnRH antagonist. At low doses of hCG, augmentation of antral follicle formation occurred, whereas higher doses of hCG led to morphological signs of luteinization. These findings demonstrate the important role of endogenous gonadotropins in preantral follicle development and indicate that preantral follicles are highly responsive to exogenous gonadotropins.

Superoxide dismutases (SOD) modulate oxygen free radical metabolism and influence second messenger signaling in a variety of cell types. We have investigated the influence and possible mechanisms of action of SOD on aromatase activity in cultured rat granulosa cells. Although treatment of granulosa cells with FSH (0.3-30 ng/ml) resulted in a dose-dependent stimulation of estrogen levels, cotreatment of cells with SOD (10(-6) M) significantly attenuated estrogen production at the highest doses of FSH. The effects of SOD were dose dependent between 10(-7)-10(-5) M, with increasing amounts of SOD causing decreasing concentrations of estrogen. Cotreatment of cells with catalase (1500 U/ml) failed to prevent the inhibitory influence of SOD on estrogen production, indicating that the effects of SOD were not due to accumulation of hydrogen peroxide. Although incubation with either forskolin or (Bu)2cAMP alone stimulated estrogen production from granulosa cells, cotreatment with SOD significantly attenuated estrogen levels, indicating that SOD can inhibit aromatase activity at one or more post-FSH receptor sites. Treatment of cells with SOD, FSH, or forskolin resulted in small, but significant, increase in cGMP concentrations. In contrast, cotreatment of cells with FSH plus SOD as well as forskolin plus SOD had a marked synergistic effect on cGMP content, increasing cGMP levels over 100-fold. Incubation of granulosa cells with (Bu)2cGMP (2 mM) significantly decreased FSH-induced estrogen levels in a dose-dependent manner (0.25-2 mM). In addition, (Bu)2cGMP attenuated both forskolin- and (Bu)2cAMP-induced estrogen production. In contrast to the effects of (Bu)2cGMP and SOD on estradiol levels, these agents had no significant effect on progesterone production by cultured granulosa cells. These results demonstrate attenuated induction of aromatase activity by FSH in cultured rat granulosa cells cotreated with SOD, suggesting a potential modulatory role of this antioxidant on granulosa cell functions. The findings that SOD and activators of the cAMP-dependent signaling pathway synergistically increase the levels of the second messenger cGMP and that (Bu)2cGMP treatment attenuates FSH-, forskolin-, and cAMP-induced aromatase activity suggest a potential mechanism of SOD action and demonstrate the antagonist action of cGMP on cAMP-mediated estrogen production.

Chronic GnRH agonist therapy lowers androgens and decreases androgen-dependent hair shaft diameter, but the resulting induction of hypoestrogenemia has limited its usefulness as a single agent. Estrogen- and progestin-containing oral contraceptives also reduce circulating androgen levels and are commonly used empirically for the treatment of hirsutism, but have not been evaluated in a blinded randomized controlled fashion. The present study is the first double masked trial to evaluate the combination use of a GnRH agonist and an estrogen-containing oral contraceptive and tests our hypothesis that these could synergistically reduce androgen levels and suppress hormone-dependent hair growth while avoiding the symptoms and risks of agonist-induced hypoestrogenemia. We enrolled 64 women in a 24-week blinded randomized controlled trial to compare placebo, nafarelin (NAF; 400 micrograms, intranasal spray, twice daily), norethindrone (1 mg), and ethinyl estradiol (NOR 1/35; 0.035 mg, daily, for 3 of 4 weeks), or combined use of NAF and NOR 1/35 for 24 weeks. At baseline and every 8 weeks, we measured gonadotropins, estrogens, androgens, and hair growth. Bone density was assessed by dual energy x-ray adsorptiometry, and hot flashes were measured objectively. Baseline total testosterone (T), free T, percent free T, and sex hormone-binding globulin-binding capacity were similar among groups. With treatment, significant reductions (P = 0.01) in total T were seen with combination and NAF only therapy. Significant increases (P < 0.001) in the sex hormone-binding globulin-binding capacity were seen in women given NOR 1/35 alone or in combination with NAF. Free T levels decreased to approximately half of baseline levels with combination treatment (17.9 to 6.4 nmol/L; P < 0.001) and NOR 1/35 alone (20.8 to 10.2 nmol/L; P < 0.001). There was a significant decrease in hair shaft diameter after combination therapy (P < 0.05) that was not seen with either agent alone. Combination therapy also prevented the hot flashes and bone loss that occurred with agonist alone. In summary, our results demonstrate that combination GnRH agonist and low dose oral contraceptive therapy is more effective than either agent alone in the treatment of hirsutism and avoids the hypoestrogenic complications that occur with agonist only therapy.

Objective: To evaluate follicular FSH and LH requirements during suppression of endogenous gonadotropins with the GnRH antagonist Nal-Glu and whether LH-like activity could be supplied by administering subcutaneous hCG., Design: Randomized clinical trial., Participants: Thirty-two normally cycling females in the late follicular phase (dominant follicle mean diameter > or = 14 mm)., Intervention: Twelve normal women were randomized to receive 150 IU IM FSH with or without 75 IU SC hCG; 11 subjects were randomized to receive 225 IU FSH with or without 50 IU SC hCG; 9 women received 150 or 225 IU IM hMG. Subjects returned the next day for repeat blood sample and ultrasound., Results: Continued follicular maturation, as evidenced by rising E2 levels, correlated with serum immunoactive and bioactive FSH levels and was unrelated to bioactive LH-hCG. Two hundred twenty-five international units of exogenous FSH consistently supported follicular maturation. There was a similar increase in mean follicular diameter in women with an E2 rise versus those with a plateau or fall. In subjects receiving SC mini-dose hCG, serum bioactive LH-hCG levels were increased significantly and were similar to levels before Nal-Glu., Conclusions: During administration of a GnRH-a, the maturing follicle appears to require only FSH support. In markedly hypogonadotropic women, mini-dose hCG may be a more practical alternative to recombinant LH to promote normal follicle maturation.

Ovulation and subsequent luteal tissue formation are preceded by midcycle surges of both LH and FSH. Although LH has been widely known as the luteinizing hormone, a potential role for FSH in the luteinization process is possible. Our earlier studies using recombinant FSH (rcFSH) without LH contamination have shown that treatment with a surge dose of rcFSH induces ovulation of mature follicles in hypophysectomized rats. The present studies examined further whether FSH alone is sufficient to induce normal corpus luteum formation. Immature hypophysectomized rats were implanted with an estrogen pellet (10 mg diethylstilbestrol). Two days later, a minipump releasing 4 IU rcFSH/day was placed to induce follicular growth. Forty-eight hours after FSH treatment, both DES pellet and FSH minipump were removed, and rats were injected with a single sc dose of 40 IU rcFSH, 5 micrograms hCG, or saline. For some animals, oviducts were excised the following day to determine the number of ovulated oocytes. The remaining animals received, 2 days later, sc injections of 125 micrograms ovine PRL twice daily for 3 days to maintain luteal function. All rats that received a surge dose of rcFSH or hCG ovulated similar numbers of oocytes, whereas none of the control animals did. Ovaries and blood samples were obtained 5 days after the gonadotropin surge. rcFSH and hCG significantly increased ovarian weight to 73.9 +/- 4.8 and 94.7 +/- 5.6 mg, respectively, compared to 10.0 +/- 0.5 mg in controls. Serum progesterone levels were increased by 192- and 102-fold in rcFSH- and hCG-treated animals, respectively, compared with those in the saline-treated rats. rcFSH and hCG also induced a marked elevation of ovarian [125I]hCG binding (4.2 +/- 0.2 and 3.7 +/- 0.1 ng/mg ovary, respectively), whereas ovaries from control animals exhibited low binding (0.6 +/- 0.1 ng/mg ovary). These gonadotropin-induced increases in [125I]hCG binding were associated with similar elevations in the levels of three LH receptor transcripts of 2.5, 4.2, and 7.0 kilobases. Also, levels of the ovarian cholesterol side-chain cleavage enzyme (CYP 11A) mRNA (2 kilobases) were low in control animals, but increased 20.5- and 14.3-fold after surge doses of rcFSH and hCG, respectively. Accompanied by biochemical signs of luteinization, morphological features typical of luteinized ovaries were found in both rcFSH and hCG groups, showing the formation of large polyhedral lutein cells and small spindle-shaped lutein cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The induction of granulosa cell differentiation and follicle maturation is dependent upon the stimulatory actions of FSH. Our recent studies used recombinant DNA technology to fuse the carboxyl-terminal peptide (CTP) of hCG beta-subunit to the carboxyl-terminus of the FSH beta-subunit. The resulting FSH analog has identical in vitro receptor-binding and biological activities as wild-type FSH (WT-FSH), but an increased circulating half-life. The present studies examined further the ability of FSH with one (FSH-CTP1) or two (FSH-CTP2) appended CTPs to promote granulosa cell differentiation and follicle ovulatory potential. WT-FSH, FSH-CTP1, and FSH-CTP2 were produced from Chinese hamster ovary cells transfected with the common alpha-subunit and respective beta-subunit. Hormone concentrations were quantitated by RIA, and relative levels confirmed by radioligand receptor assay. Both FSH-CTP1 and FSH-CTP2 retained full FSH receptor-binding activity, but did not bind LH receptors. To compare in vivo bioactivity, immature estrogen-primed female rats received ip injections of FSH or the agonists at 0 and 24 h. At 48 h, substantial stimulation (up to 2.5-fold) of ovarian weight was induced by 1.0 and 3.0 IU/day FSH-CTP1 or FSH-CTP2, whereas a higher dose (10 IU/day) of WT-FSH was required for an 1.8-fold stimulation. Although the in vivo potencies of FSH-CTP1 and FSH-CTP2 were similar, FSH-CTPs were about 10-fold more potent than WT-FSH in inducing granulosa cell aromatase activity and LH receptors. We further reduced the frequency of hormone administration. Increasing doses (1-10 IU) of a single ip injection of FSH-CTP1 resulted in dose-dependent increases in granulosa cell aromatase activity and LH receptor content 48 h later. Although a single injection (10 IU) of WT-FSH had no effect, the same total dose of WT-FSH administered as four 2.5-IU injections 12 h apart was effective. To test the ovulatory potential of ovarian follicles, rats received a single injection of FSH-CTP1, followed 52 h later by 5 IU hCG to induce ovulation. Although hCG did not induce ovulation in females receiving a single dose (10 IU) of WT-FSH, 20 +/- 2 and 43 +/- 5 ovulated ova/rat were found in animals primed with 3 and 10 IU FSH-CTP1, respectively. Because twice daily injections of WT-FSH (2.5 IU/injection) also increased the ovulatory potential of the ovary, the enhanced effectiveness of FSH-CTP1 appears to be related to its increased circulating half-life.(ABSTRACT TRUNCATED AT 400 WORDS)

The anterior pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), act upon the ovary and testis via occupancy of specific cell membrane receptors, resulting in increased cAMP production, steroidogenesis, and expression of differentiation-related genes. Recent cloning of the cDNAs for LH and FSH receptors allows the analysis of mRNA levels for these receptors in gonadal tissues. This review summarizes progress in elucidating the molecular basis of LH and FSH receptor gene regulation in the ovary and testis during different physiologic states.

On the basis of earlier observations of changing testicular LH receptor levels during postnatal development of the rat, we analyzed the levels of LH receptor mRNA transcripts in testes of rats at ages 5-70 days. Extracted testis RNA, prepared for Northern blotting, was hybridized with a specific LH receptor cRNA probe derived from subcloned cDNA corresponding to the extracellular domain of the receptor. Six LH receptor mRNA transcripts with molecular sizes of 7.8, 7.0, 4.2, 2.5, 1.8, and 1.2 kb were identified. Of these, the 1.2- and the 1.8-kb mRNA transcripts presumably code for truncated forms of LH receptor. At 5 days, only the 1.8- and the 4.2-kb mRNA transcripts were observed. Additional 7.0- and 1.2-kb transcripts appeared at 10 and 15 days, respectively. From the age of 25 days through adulthood, all six mRNA transcripts were observed. Densitometric analyses revealed that the amounts of the 7.0- and 1.8-kb mRNA transcripts correlated well with LH receptor levels, while the 4.2-kb transcript showed high levels earlier in life with poor correlation to LH receptor number. Because the 1.8 kb receptor transcript lacked transmembrane domains, the present results suggest the 7.0-kb LH receptor transcript as the likely candidate to encode the functional receptor. These data provide the basis for future analyses of the molecular regulation of LH receptor expression.

Follitropin (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. FSH is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of anovulatory women. One issue regarding the clinical use of FSH is its short half-life in the circulation. To address this point, we constructed chimeric genes containing the sequence encoding the C-terminal peptide of the chorionic gonadotropin beta subunit (CG beta) fused to the translated sequence of the human FSH beta subunit (FSH beta). This region of CG beta is important for maintaining the prolonged plasma half-life of human CG dimer. The presence of the C-terminal peptide sequence did not significantly affect assembly of FSH beta with the alpha subunit or secretion of the dimer. In vitro receptor binding and steroidogenic activity of dimer bearing the FSH beta-C-terminal peptide chimera were the same as wild-type FSH. However, both the in vivo potency and half-life in circulation of the dimer bearing either one or two C-terminal peptide units were enhanced. Dimers containing FSH beta-CG beta chimeras could serve as potent FSH agonists for clinical use, and the present strategy may have wide applications for enhancing the in vivo half-life of diverse proteins.

The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2-day culture period. However, inclusion of FSH (30 ng/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (1-100 ng/ml) increased FSH receptor mRNA levels in a dose-dependent manner (ED50, 4.5 ng/ml), with a maximal 5.9 +/- 0.7-fold increase observed in response to 30 ng/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 microM), suggesting the involvement of cAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with epidermal growth factor (EGF; 0.3-10 ng/ml), basic fibroblast growth factor (bFGF; 1-30 ng/ml), or insulin-like growth factor-I (IGF-I; 1-30 ng/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 ng/ml) on its own receptor mRNA levels. Similarly, GnRH (10-1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (ID50, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-D-Phe1,D-pCl-Phe2,D-Trp3,6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitory actions of EGF, bFGF, and GnRH. Thus, the use of cultured rat granulosa cells provides a model system to analyze the hormonal regulation of FSH receptor gene expression in the ovary.

Objective: To examine the possible impact of abnormal adrenal steroidogenesis on the ovarian dysfunction seen in polycystic ovarian disease (PCOD)., Design: Prospective analysis of blood sampling monthly for 6 months, then three times weekly for 90 days., Setting: Tertiary institutional outpatient care., Participants: Six anovulatory women with a diagnosis of PCOD., Intervention: Six-month suppression with gonadotropin-releasing hormone agonist (GnRH-a) followed by suppression with dexamethasone (DEX) for 90 days., Main Outcome Measures: Serum levels of testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), cortisol, estradiol (E2), progesterone (P), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and bioactive LH., Results: Gonadotropin-releasing hormone agonist administration suppressed greater than 60% of the circulating levels of T and A, suggesting an ovarian origin. Minimal changes of DHEA, DHEAS, and cortisol were seen. With the addition of DEX, there was greater than 90% suppression of the total circulating A, T, DHEA, DHEAS, and cortisol, supporting the adrenal origin of the non-GnRH-a suppressible androgens. Excessive ovarian T and A secretion returned during the 90-day recovery study period in spite of rises of FSH concentrations that changed the ratio of FSH to LH in all subjects. Four of the six women failed to ovulate. In comparison of the women who did and did not ovulate during recovery, no differences in absolute levels or changes in concentrations of steroids or gonadotropins could be detected., Conclusions: Using sequential and simultaneous administration of GnRH-a and DEX, we were able to delineate the contributions of the ovaries and adrenals to the abnormal steroidogenesis seen in PCOD. Despite prolonged suppression of ovarian and then adrenal steroidogenesis, ovarian dysfunction, evidenced by abnormal androgen production, returned with cessation of agonist administration.

The actions of gonadotropins on ovarian differentiation are associated with dynamic changes in gonadotropin receptor content, presumably due to modulation of receptor gene expression. The present studies used a reverse transcription-polymerase chain reaction to obtain a rat FSH receptor cDNA fragment, followed by synthesis of a labeled cRNA probe to examine the regulation of FSH receptor mRNA levels during follicular maturation, ovulation, and luteinization. Northern blot analysis of ovarian RNA with the FSH receptor probe revealed two predominant hybridization signals of 7.0 and 2.5 kilobases (kb) as well as minor signals of 4.2 and 1.8 kb. Treatment of immature rats with PMSG (10 IU) to induce follicular development resulted in increased FSH receptor mRNA levels 24 h after treatment, with a further increase at 52 h, coincident with increased [125I]FSH binding. Subsequent treatment with an ovulatory dose of hCG decreased FSH binding and receptor mRNA levels by 6 h, with a maximal inhibition at 24 h after hCG. In luteinized ovaries obtained 3 and 5 days after hCG treatment, the 7.0-kb FSH receptor mRNA increased again, but no concomitant elevation of [125I]FSH binding was detected. We recently demonstrated that FSH treatment alone is capable of inducing follicular growth and ovulation, thus providing a unique model to evaluate the effects of FSH on regulation of its receptor gene. Immature hypophysectomized estrogen-treated rats were implanted with an osmotic minipump delivering recombinant human FSH (rcFSH; 4 IU/day) to stimulate follicle growth, followed 52 h later with a single injection (20 IU) of rcFSH to induce ovulation. Stimulation of follicular growth with rcFSH increased both FSH receptor binding and mRNA levels. In contrast, the ovulatory dose of rcFSH decreased FSH binding and receptor message levels within 12 h. Thus, gonadotropin regulation of ovarian FSH receptor content during follicular growth, ovulation, and luteinization is associated with similar changes in FSH receptor message levels. Also, studies using rcFSH demonstrate that both up- and down-regulation of FSH receptor gene expression can be induced by the homologous hormone at different stages of follicle development.

The action of LH is mediated through specific plasma membrane receptors that are both up- and down-regulated in the ovary during the reproductive cycle. Using immature rats treated with PMSG and hCG as a model system, we have studied the regulation and distribution of LH receptor mRNA in different cell types during follicle development, ovulation, and luteinization by Northern blot and in situ hybridization. In untreated rats, LH receptor mRNA was below the detection level in granulosa cells, cumulus cells, and oocytes, while low levels of LH receptor mRNA were found in the thecal cells. After stimulation with PMSG, expression of LH receptor mRNA was enhanced in the thecal-interstitial cells, while a more dramatic increase in receptor mRNA abundance took place in granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor mRNA varied among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than granulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking detectable hybridization signal. The uneven expression of LH receptor mRNA endows different ovarian cells with varying hormonal responsiveness. After an ovulatory dose of hCG, LH receptor mRNA levels were dramatically decreased, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor message was again increased. Our results reveal that the previously documented regulation of the LH receptor-binding activity during ovarian development correlates with expression of the LH receptor transcripts, suggesting that the LH receptor gene is regulated in a complex manner during the periovulatory period to achieve cell-specific expression together with gonadotropin induction and suppression of receptor gene activity.

Although earlier reports suggest a stimulatory effect of FSH on Leydig cell function, controversy exists due to unavailability of FSH preparations free of contaminating LH. Recent availability of recombinant human FSH preparations made it possible to reinvestigate this question. Immature male rats were hypophysectomized (21-22 days old at surgery) and implanted with osmotic minipumps releasing 8 IU recombinant FSH or 18 IU purified human pituitary FSH (hpFSH)/day, whereas control animals received vehicle alone. After 7 days of treatment, testicular weight increased in the recombinant FSH and hpFSH-treated animals to values 2.3- and 2.5-fold those of controls, respectively. Analyses of the steroidogenic capacity of Leydig cells in testes of rats treated with recombinant FSH or hpFSH also revealed 2.9- and 3.8-fold androgen production in vitro compared to controls. In these rats recombinant FSH and hpFSH increased the LH receptor number in testicular homogenate by 50% and 70%, respectively. The increase in LH receptor number was associated with increases in the LH receptor mRNA levels. In hypophysectomized control rats, small seminiferous tubules contained spermatogonia and zygotene/early pachytene spermatocytes. In contrast, treatment with either FSH preparation enhanced the progression of meiosis, as evidenced by large number of pachytene spermatocytes and appearance of round spermatids. The present results show that LH-free recombinant FSH, like purified pituitary FSH, is capable of increasing the LH receptor content and steroidogenic responsiveness of Leydig cells through paracrine mechanisms together with a stimulatory effect on spermatogenesis. These observations suggest that prepubertal elevation of FSH secretion may be important for increasing Leydig cell steroidogenic capacity and spermatogenic progression.

The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.

Regularly cyclic, middle-aged female rats exhibit a decreased incidence of fertility, and those females that are fertile produce smaller litters. This decreased litter size is directly related to a reduced number of normal blastocysts available for implantation. Recent evidence indicates that embryonic abnormalities in middle-aged rats become apparent as early as Day 2 of pregnancy. Inasmuch as the semicircadian secretion of prolactin (PRL) is essential for the rescue of corpora lutea during early gestation and luteal production of progesterone (P) and estradiol (E2) in sufficient quantities is obligatory for embryonic development and implantation, the present study examined the profiles of plasma PRL, P, and E2 during the first 3 days of pregnancy in both young and middle-aged rats and assessed the embryonic development in these same animals. Regularly cyclic, middle-aged (9-11 mo) and young (4-5 mo) rats were cannulated via the right jugular vein on Diestrus Day 2 and mated with fertile males on proestrus. The next morning, sperm in the vaginal lavage confirmed mating, and that day was designated Day 1 of pregnancy. Beginning at 1400 h on Day 1 and continuing to 2400 h on Day 2, serial blood samples were taken at 2-h intervals for PRL assay. In the first experiment, samples were also collected at 8-h intervals during Days 1-3 for measurement of plasma P.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of plasma membrane receptors by homologous hormones has been found in diverse cell types. In testicular Leydig and ovarian luteal cells, treatment with LH/hCG decreases LH receptor content. Although suppression of LH-binding sites may result from ligand-induced receptor internalization, sequestration, and/or phosphorylation, the gonadotropins may also regulate receptor mRNA levels. We examined the regulation of testis LH receptor mRNAs in adult rats that received 10 or 200 IU hCG, using cRNA probes derived from the 5' extracellular domain (EC) or the 3' transmembrane domain (TM) of the rat receptor cDNA. Probe EC hybridized to predominant signals of 7 and 1.8 kilobases (kb) and weaker signals of 4.2 and 2.5 kb. However, probe TM hybridized to the three larger forms of the LH receptor mRNA, but not to the 1.8-kb species, suggesting that the latter form lacks the transmembrane domain. After 6 and 12 h of treatment with 200 or 10 IU hCG, respectively, hybridization to the larger mRNA species decreased by more than 60%, preceding decreases in testicular [125I]hCG binding. These transcripts were further inhibited (greater than 93%) between 24-72 h after hCG treatment and returned to 40% and 100% of control levels by days 6 and 9, respectively. In contrast, the truncated 1.8-kb LH receptor transcript was not affected by hCG treatment, indicating a differential suppressive effect of the ligand on its receptor mRNA levels. In the ovary, hybridization to probe EC revealed four transcripts with similar sizes as those found in the testes, with a predominant 7-kb species.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.

Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.

Regularly cyclic, middle-aged female rats exhibit a decreased incidence of fertility, and those females that are fertile produce small litters. These decreases in fertility and litter size are associated with reduced numbers of normal blastocysts formed and implanted, suggesting that pre- and/or peri-implantation failures may be the causes for these aging-related reproductive declines. The present study examined the relationships and influence of circulating estradiol (E2) and progesterone (P) levels on early embryonic development and implantation in middle-aged rats. Serial blood samples obtained from cannulated, middle-aged pregnant rats revealed minor decreases in plasma P and increases in E2 levels during Days 2-4 of pregnancy, compared to young pregnant rats, resulting in significantly (p less than 0.001) decreased plasma P/E2 ratios. These alterations in endogenous hormone secretion in middle-aged pregnant rats were associated with fewer normal blastocysts on Day 5 of pregnancy and reduced numbers of normally implanting embryos. Correlation analysis further revealed a significant (p less than 0.05) inverse relationship between mean circulating E2 levels and numbers of normal conceptuses on Day 12 of gestation. Moreover, s.c. administration of P implants (in Silastic) to middle-aged pregnant rats increased serum P levels by about 34-40 ng/ml, and significantly (p less than 0.05) reduced the incidence of abnormal embryos before implantation. In contrast, treatment with E2 minipumps produced a sustained rise in serum E2 (by about 7-15 pg/ml) and resulted in the complete absence of embryos in the reproductive tracts by Day 5 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.

Induction of follicular growth by PMSG is associated with increased ovarian LH receptor content, whereas the preovulatory surge of LH decreases LH binding sites, followed by a secondary increase in receptor numbers coincident with corpora lutea formation. Based on the recently reported LH receptor cDNA sequence, we have performed a reverse transcription-polymerase chain reaction to obtain LH receptor cDNA clones and generated a 32P-labeled cRNA probe to examine the dynamic changes in ovarian LH receptor mRNA levels during gonadotropin induction of follicular growth, ovulation and luteinization. Northern blot analysis of ovary RNA revealed hybridization signals of about 7.0, 4.2, 2.5 and 1.8 kb, with no hybridization to nongonadal tissues. PMSG increased the intensity of all four LH receptor messages at 24 h, preceding an increase in LH receptor number, with peak LH receptor mRNA and receptor content observed at 52 h. Treatment with hCG resulted in decreased LH receptor binding and mRNA levels by 6 h after injection, with maximal inhibition (greater than 85%) of message at 12 to 24 h after hCG treatment. Subsequently, LH receptor message levels increased again at 3 days after hCG, concomitant with increased [125I]hCG binding. A further increase in LH receptor content, but not message levels, was observed 5 days after hCG. These results demonstrate that the induction of LH receptors by PMSG is preceded by increased LH receptor mRNA levels. Furthermore, ligand-induced down-regulation of the LH receptor following an ovulatory dose of hCG is associated with decreased LH receptor message content, followed by increases in LH receptor message levels and binding sites during subsequent luteinization. Thus, the up- and down-regulation of ovarian LH receptors during follicle growth, ovulation and luteinization is probably due, at least in part, to changes in receptor message modulation.

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether discernible alterations in neuroendocrine and/or ovarian function precede the loss of regular oestrous cycles in ageing female rats, the present study examined the pattern of gonadotrophin secretion near the time of ovulation and the pattern of ovarian steroid secretion in the early morning of pro-oestrus in middle-aged (10-12 months old) females displaying regular oestrous cycles and compared these with young (4 months old) animals. In addition, the subsequent reproductive patterns in these animals were observed and correlations between the changes in hormonal profiles and the decline in regular reproductive cyclicity were established. In middle-aged females which subsequently ceased to display regular oestrous cycles (middle-aged non-regular; M-NR) within 1-2 months, the pro-oestrous surge of LH was significantly reduced in magnitude. There was no difference in the LH surge between young females and middle-aged animals which maintained regular oestrous cycles (middle-aged regular; M-R) for at least 2 months. There also was no difference in the magnitude of the pro-oestrous FSH surge or in the secondary rise in FSH in the early morning of oestrus among young, M-R and M-NR females. In a separate group of middle-aged females which subsequently became M-NR, serum concentrations of both oestradiol and testosterone in the early morning of pro-oestrus were markedly raised over those in the young and M-R groups.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently reported that successive treatments of young virgin rats with progesterone (P) implants produce elevated circulating P and consistently low estradiol (E2) concentrations, and subsequently delay the aging-associated reproductive decline. Inasmuch as E2 has been implicated in causing the loss of regular estrous cyclicity in aging rats, the present study examined if the concomitant presence of moderately increased circulating E2 levels could counteract the effects of P implants on reproductive aging. Starting at 3 1/2 mo and continuing to 8 mo of age, regularly cyclic, virgin rats received either s.c. Silastic implants of P (P-implanted), blank Silastic implants (virgin controls), or P + E2 implants (P + E2-implanted) for 3 wk, followed by implant removal for 1 wk. Each of these implant treatments was repeated in the same female rats 5 times. Blood samples were obtained on different days of the estrous cycle from the control group and on Day 11 of successive treatments with P or P + E2 implants for measurements of serum P and E2 values. At 8 1/2 and 10 mo of age, estrous cyclicity of these same virgin rats was again monitored, and 10-mo-old regularly cyclic females from each treatment group were mated with young fertile males to complete term pregnancies. While virgin controls showed cyclic increases in E2 and P secretion during the estrous cycle, P-implanted virgins exhibited consistently low serum E2 and moderately increased P levels during 5 successive treatments. The latter indicates a potent inhibition of ovarian E2 secretion by P implants.(ABSTRACT TRUNCATED AT 250 WORDS)

The progressive cessation of regular ovulatory function in aging female rats is preceded by a significant decrease in the magnitude of the proestrous LH surge during regular estrous cycles. However, our recent study has demonstrated that normal LH secretion and regular estrous cycles can be maintained for an extended period of time in aging females housed with fertile males and allowed to undergo repeated pregnancies. Since progesterone (P) secretion is persistently increased in pregnant rats, the present study examined whether repeated increases in circulating progesterone accounted for these results. Starting at 8 months of age and continuing to 13 months, multiparous rats were grouped and treated as follows: controls: females were housed five per cage; mated: five females were housed with one fertile male and allowed to undergo repeated pregnancies; and P-implanted: females were housed five per cage and implanted sc with Silastic capsules containing P for 3 of every 4 weeks. During the 4.5 months of study, serum concentrations of estradiol (E2) in the P-implanted rats remained between 13 and 27 pg/ml, similar to levels in pregnant females (8-26 pg/ml) of the mated group. These E2 values were less than the preovulatory increase in serum E2 on proestrus (mean +/- SE, 56 +/- 10 pg/ml) in cyclic control females. In contrast, serum P values were persistently elevated in both the pregnant and the P-implanted rats, although the values in the latter (27-55 ng/ml) were about one third to one half of those in the former group (117-125 ng/ ml). All treatments were stopped at 13 months of age, and estrous cycle patterns were determined thereafter. Between 13 and 17 months of age, the percentages of regularly cycling rats were significantly (P less than 0.01) greater in the mated group (50%, 36%, and 15% at 13, 15, and 17 months, respectively) than in the control group (23%, 20%, and 9%, respectively). During this same period, 50% of the females from the P-implanted group continued to display regular cycles. By the age of 11 months, 8 of 21 untreated retired breeder females exhibited attenuated LH surges on proestrus and subsequently ceased to display regular estrous cycles within 2 months, whereas the other 13 rats showed normal LH and FSH surges and continued to maintain regular cycles. In contrast to these, aged female rats from the previously mated (15-month-old) and the P-implanted (19-month-old) groups exhibited normal profiles of proestrous LH and FSH surges during regular estrous cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Inoculation of cyclic female rats with the prolactin (Prl)/growth hormone-secreting pituitary tumor, MtT.W15, resulted in a cessation of estrous cyclicity within 5--10 days. Associated with this acyclicity was a persistently low serum concentration of estradiol and marked increases in both circulating Prl and progesterone. At Day 26 of acyclicity, basal serum luteinizing hormone (LH) values measured in samples taken every 20 min from 0900--1100 h were significantly reduced when compared to cyclic, nontumor animals on diestrus Day 2. There was no difference in basal follicle-stimulating hormone (FSH) concentrations. In a separate group of acyclic, tumor-bearing females 42--56 days after transplantation, a single s.c. injection of 20 micrograms estradiol benzoate (EB) at 1030 h elicited significant increases in both serum LH and FSH values between 1700 and 1830 h on the next day. The magnitude of the LH surge was reduced and that of FSH was increased in tumor-bearing animals when compared to cyclic, nontumor females given a similar EB injection on diestrus Day 1. These results demonstrate that chronic hyperprolactinemia is associated with inhibition of basal LH secretion and ovarian estrogen production and an increase in circulating progesterone concentrations. Nevertheless, the stimulatory feedback effects of estrogen on LH and FSH release are still present and functioning in acyclic female rats under chronically hyperprolactinemic conditions. These data suggest that the cessation of regular ovulatory cycles associated with hyperprolactinemia may be due to a deficiency of LH and/or estrogen secretion, but not to a lack of central nervous system response to the stimulatory feedback action of estrogen.

In multiparous rats, the incidence of regular estrous cyclicity and fertility decreases markedly at middle age. However, recent studies have shown that repeated pregnancies or progesterone (P) implants can subsequently cause retired breeder females to maintain regular cyclicity for an extended period of time; these results suggest a P-mediated deceleration of reproductive aging. In the present study, we examined the relation of ovarian steroid levels in young virgin females to their subsequent estrous cyclicity and reproductive function during aging as compared to multiparous females. Beginning at 4 mo of age and continuing to 6 mo of age, regularly cyclic virgin rats received either consecutive P implants (n = 41) or no implants (controls, n = 45) for 3 wk, followed by implant removal for 1 wk. Additional females (n = 72) were mated and allowed to undergo repeated pregnancies at 4, 6 1/2, and 8 mo of age. Blood samples were obtained throughout the estrous cycle (virgin females), during pregnancy (multiparous rats), and on Day 11 of successive treatments with P implants (virgins with P implants) for P, estradiol (E2), and testosterone (T) measurements. Subsequently, regularly cyclic females from all three groups were mated with fertile males to undergo term pregnancies at 10 and 12 mo of age. While the virgin controls showed cyclic increases in P, T, and E2 secretion during their estrous cycles, the P-implanted females had persistently low E2 and high P and T levels during treatment, which indicates an inhibition of ovarian E2 synthesis by P.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.

In female rats ovariectomy (OVX) on the morning of diestrus day 2 resulted in a prompt (within 8 h) and gradual increase in LH release, whereas a similar operation on estrus did not raise serum LH until 24 h later. In proestrous females, OVX in the morning neither prevented the anticipated LH surge on that evening, nor increased LH release on the next morning. Since circulating concentrations of estradiol (E2) are progressively increased during diestrus day 2 and proestrus, this pattern of E2 secretion may affect the acute increase in LH secretion following OVX. To test such a hypothesis, we examined the effects of large, sustained or large, transient increases in circulating estrogen on the subsequent increase in gonadotropin secretion after OVX. On the morning of diestrus day 1 a subcutaneous injection of 20 micrograms estradiol benzoate (EB) produced prompt and large increases in both serum E2 and estrone (E1) for about 2 days, despite OVX of females at 24 h after the EB injection. An injection of 20 micrograms 17 beta-estradiol (E2) also promptly increased both serum E2 and E1. micrograms E2 injection were twice that observed after 20 micrograms EB, and both E2 and E1 returned to low baseline values within 24 h after the E2 injection. In contrast, administration of 1 microgram EB only produced small and transient rises in serum E2 and E1.(ABSTRACT TRUNCATED AT 250 WORDS)