Philip S. LaPolt

The Luteinizing Hormone Surge Is Preceded by an Estrogen-Induced Increase of Hypothalamic Progesterone in Ovariectomized and Adrenalectomized Rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17β-estradiol (50 µg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.

Estrogen Induces de novo Progesterone Synthesis in Astrocytes

The brain is an established target for peripheral steroids, but also expresses steroidogenic enzymes and is capable of de novo ‘sex’ steroid synthesis (neurosteroidogenesis) independent of peripheral steroidogenic organs. In adrenalectomized and ovariectomized rats that do not have peripheral sources of steroids, estrogen treatment increased progesterone levels specifically in the hypothalamus, indicating that estrogen stimulates progesterone neurosteroidogenesis. Recent studies have demonstrated that specific cell types preferentially secrete specific steroids, and that astrocytes are the primary progesterone synthesizing cells in the nervous system. We hypothesized that estrogen could directly induce de novo synthesis of progesterone in astrocytes. To determine whether estrogen stimulates progesterone synthesis in astrocytes, astrocyte-enriched cultures were grown to confluence, then grown for an additional 48 h in an estrogen- and phenol-free Dulbecco’s Modified Eagle Medium (DMEM) and then treated with either 17β-estradiol or steroid-free media. After culturing for 48 h in steroid-free, phenol red-free DMEM, low levels of progesterone were detected in the media, whereas progesterone levels were significantly increased in the media of astrocytes cultured in DMEM with 17β-estradiol (10–7–10–4M). To determine whether estrogen regulated the mRNA expression of progesterone synthetic enzymes, P-450 side-chain cleavage and 3β-hydroxysteroid dehydrogenase, control and 17β-estradiol-treated astrocytes were harvested and prepared for Northern and slot blot analysis. Expression levels of enzyme mRNAs were very low and 17β-estradiol did not significantly increase mRNA levels of either steroidogenic enzyme. These results suggest that estrogen directly stimulated the de novo synthesis of neuroprogesterone in astrocytes, and demonstrate the potential for estrogen to regulate reproductive physiology and behavior through the paracrine actions of astrocyte-derived progesterone.

Roles of cyclic GMP in modulating ovarian functions

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.

Influences of Age and Ovarian Follicular Reserve on Estrous Cycle Patterns, Ovulation, and Hormone Secretion in the Long-Evans Rat

Abstract This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E2) 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 E2 secretion, as preovulatory E2 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.

Long-term administration of gonadotropin-releasing hormone agonist and dexamethasone: assessment of the adrenal role in ovarian dysfunction *

OBJECTIVEnTo examine the possible impact of abnormal adrenal steroidogenesis on the ovarian dysfunction seen in polycystic ovarian disease (PCOD).nnnDESIGNnProspective analysis of blood sampling monthly for 6 months, then three times weekly for 90 days.nnnSETTINGnTertiary institutional outpatient care.nnnPARTICIPANTSnSix anovulatory women with a diagnosis of PCOD.nnnINTERVENTIONnSix-month suppression with gonadotropin-releasing hormone agonist (GnRH-a) followed by suppression with dexamethasone (DEX) for 90 days.nnnMAIN OUTCOME MEASURESnSerum 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.nnnRESULTSnGonadotropin-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.nnnCONCLUSIONSnUsing 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.

Cell-Specific Expression and Regulation of Soluble Guanylyl Cyclase α1 and β1 Subunits in the Rat Ovary

Abstract 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 α1 and β1 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 α1 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 α1 immunoreactivity. In gonadotropin- treated immature rats, intense sGC α1 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 α1 immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC β1, indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC α1 and β1 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.

Temporal Changes Occur in the Neuroendocrine Control of Gonadotropin Secretion in Aging Female Rats: Role of Progesterone

Abstract The present study examined the gonadotropin surge-inducing actions of estradiol (E2), both alone and with progesterone (P4), in middle-aged, early persistent-estrous (PE) female rats that had become PE within 35 days. In addition, we also assessed the effect of P4 on the mating-induced gonadotropin surges in these acyclic animals. Early PE rats were ovariectomized and received E2 implants (Day 0). On Day 4, an s.c. injection of P4 (0.5 mg/ 100 g body weight) at 1200 h markedly increased plasma P4 and elicited both LH and FSH surges, whereas vehicle-treated controls displayed no rise in P4 or gonadotropins. This observation confirms that at middle age, female rats no longer respond to the positive-feedback stimulation of E2 on gonadotropin surges whenever the estrous cyclicity ceases. As PE continued, such a surge-inducing action of E2 plus P4 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 P4 from the adrenal along with small gonadotropin surges. The amplitude of these mating-induced gonadotropin surges was enhanced by supplementation with exogenous P4 in early PE rats. Our findings indicate that during the early phase of PE, the surge-inducing action of E2 and P4 remains intact but deteriorates as PE continues. Thus, a deficiency in P4 secretion during aging may contribute to the diminished gonadotropin surge response in the hypothalamic-pituitary axis and the subsequent cessation of estrous cyclicity.

Influences of Age and Reproductive Status on Ovarian Ovulatory Responsiveness to Gonadotropin Stimulation

Abstract 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.

Effects of aging on luteinizing hormone secretion, ovulation, and ovarian tissue-type plasminogen activator expression.

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.

Effects of nitric oxide and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells

OBJECTIVEnTo determine the effects of nitric oxide (NO) and cGMP on inhibin A and inhibin subunit mRNA levels from cultured rat granulosa cells.nnnDESIGNnBasic research study.nnnSETTINGnUniversity research laboratory.nnnANIMAL(S)nPrimary cell culture of granulosa cells obtained from estrogen-treated, immature Sprague-Dawley female rats.nnnINTERVENTION(S)nFunctionally 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.nnnMAIN OUTCOME MEASURE(S)nMedia 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.nnnRESULT(S)nWhereas 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.nnnCONCLUSION(S)nThese 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.