Subfertility and reduced progestin synthesis in Pgrmc2 knockout zebrafish.

Progestin receptor membrane component (Pgrmc1 & 2) is a heme-binding protein. Studies on Pgrmc1 have suggested possible roles in heme binding, activation of steroid-synthesizing P450s, along with binding and transferring of membrane proteins. However, the studies of Pgrmc1's paralog, Pgrmc2 are still lacking. In order to determine the physiologic function(s) of Pgrmc2, we generated a zebrafish mutant line (pgrmc2-/-). We found a reduction in both spawning frequency and the number of embryos produced in female pgrmc2-/-. This subfertility is caused by reduced oocyte maturation (germinal vesicle breakdown, GVBD) in pgrmc2-/- in vivo. Nonetheless, oocytes from pgrmc2-/- had similar sensitivity to 17α,20ß-dihydroxy-4-pregnen-3-one (DHP, a maturation induced progestin in zebrafish) compared with wildtype (wt) in vitro. Therefore, we hypothesized that oocyte maturation tardiness found in vivo, could be due to lack of progestin in pgrmc2-/-. Interestingly, we found significant reduced expression of hormones, receptors, and steroid synthesizing enzymes including lhcgr, egfra, ar, and esr2, cyp11a1 and hsd3b1. In addition, DHP levels in pgrmc2-/- ovaries showed a significant decrease compared to those in wt. In summary, we have provided a plausible molecular mechanism for the physiological functions of Pgrmc2 in the regulation of female fertility, likely via regulation of receptors and steroids in the ovary, which in turn regulates oocyte maturation in zebrafish.

Role of sex hormones and the vaginal microbiome in susceptibility and mucosal immunity to HIV-1 in the female genital tract.

While the prevalence of Human immunodeficiency virus-1 (HIV-1) infection has stabilized globally, it continues to be the leading cause of death among women of reproductive age. The majority of new infections are transmitted heterosexually, and women have consistently been found to be more susceptible to HIV-1 infection during heterosexual intercourse compared to men. This emphasizes the need for a deeper understanding of how the microenvironment in the female genital tract (FGT) could influence HIV-1 acquisition. This short review focuses on our current understanding of the interplay between estrogen, progesterone, and the cervicovaginal microbiome and their immunomodulatory effects on the FGT. The role of hormonal contraceptives and bacterial vaginosis on tissue inflammation, T cell immunity and HIV-1 susceptibility is discussed. Taken together, this review provides valuable information for the future development of multi-purpose interventions to prevent HIV-1 infection in women.

In silico predicted structural and functional robustness of piscine steroidogenesis.

Assessments of metabolic robustness or susceptibility are inherently dependent on quantitative descriptions of network structure and associated function. In this paper a stoichiometric model of piscine steroidogenesis was constructed and constrained with productions of selected steroid hormones. Structural and flux metrics of this in silico model were quantified by calculating extreme pathways and optimal flux distributions (using linear programming). Extreme pathway analysis showed progestin and corticosteroid synthesis reactions to be highly participant in extreme pathways. Furthermore, reaction participation in extreme pathways also fitted a power law distribution (degree exponent γ=2.3), which suggested that progestin and corticosteroid reactions act as 'hubs' capable of generating other functionally relevant pathways required to maintain steady-state functionality of the network. Analysis of cofactor usage (O2 and NADPH) showed progestin synthesis reactions to exhibit high robustness, whereas estrogen productions showed highest energetic demands with low associated robustness to maintain such demands. Linear programming calculated optimal flux distributions showed high heterogeneity of flux values with a near-random power law distribution (degree exponent γ≥2.7). Subsequently, network robustness was tested by assessing maintenance of metabolite flux-sum subject to targeted deletions of rank-ordered (low to high metric) extreme pathway participant and optimal flux reactions. Network robustness was susceptible to deletions of extreme pathway participant reactions, whereas minimal impact of high flux reaction deletion was observed. This analysis shows that the steroid network is susceptible to perturbation of structurally relevant (extreme pathway) reactions rather than those carrying high flux.

Piscine follicle-stimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles.

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.

Juvenile offspring of rats exposed to restraint stress in late gestation have impaired cognitive performance and dysregulated progestogen formation.

Gestational stress may have lasting effects on the physical and neurocognitive development of offspring. The mechanisms that may underlie these effects are of interest. Progesterone and its 5α-reduced metabolites, dihydroprogesterone and 5α-pregnan-3α-ol-20-one (3α,5α-THP), maintain pregnancy, have neurotrophic effects, and can enhance cognitive performance. We hypothesized that some of the deleterious effects of gestational stress on the cognitive performance of offspring may be related to progestogen formation. Pregnant rat dams were exposed to restraint under a bright light (thrice daily for 45 min) on gestational days 17-21 or were minimally handled controls. Dams that were exposed to restraint had lower circulating levels of 3α,5α-THP and significantly greater concentrations of corticosterone at the time of birth than did control dams. Male and female offspring, that were gestationally stressed or not, were cross-fostered to non-manipulated dams. Between postnatal days 28-30, offspring were assessed for object recognition, a prefrontal cortex (PFC)-dependent cognitive task. Restraint-exposed offspring performed more poorly in the object recognition task than did control offspring, irrespective of sex. As well, progesterone turnover to its 5α-reduced metabolites in the medial PFC (but not the diencephalon) was significantly reduced among restraint-exposed, compared to control, offspring. Progesterone turnover, and levels of 3α,5α-THP, positively correlated with performance in the object recognition task. Thus, restraint stress in late pregnancy impaired cognitive development and dysregulated progestogen formation in brain.

Development of an animal experimental model to study the effects of levonorgestrel on the human endometrium.

BACKGROUND: This study was designed to develop an animal model to test the response of endometrium to local progestin delivery. METHODS: Proliferative human endometrium was subcutaneously grafted in two groups of SCID mice that received, 2 days before, a subcutaneous estradiol (E(2)) pellet and, for half of them, an additional implant of levonorgestrel (LNG). Mice were sacrificed 1, 2, 3 or 4 weeks after endometrial implantation and grafts were histologically analysed. Proliferation, steroid hormone receptors, blood vessels and stromal decidualization in both groups (E(2) and LNG) were immunohistologically evaluated and compared with proliferative endometrium and endometrium from women with an LNG intrauterine device. RESULTS: Grafts presented normal morphological endometrial characteristics. The expression of progesterone receptors was significantly decreased in glands and stroma of the LNG group as compared with the E(2) group at all times. A significant decrease was also observed in the stromal expression of estrogen receptor-alpha in the LNG group. At 4 weeks, the mean cross-sectional area of vessels was significantly higher after LNG treatment. CONCLUSIONS: These morphological and immunohistochemical characteristics are similar to those observed in women treated with local LNG. This mouse model might facilitate further investigations needed to understand the mechanisms responsible for the breakthrough bleeding frequently observed in progestin users.

Extrapituitary action of gonadotropin-releasing hormone: direct inhibition ovarian steroidogenesis.

Gonadotropin-releasing hormone (GnRH) and its agonistic analogs inhibited the follicle-stimulating hormone (FSH)-induced increase of estrogen and progesterone production in vitro by rat ovarian granulosa cells. Likewise, GnRH analogs inhibited FSH-induced changes in ovarian function in hypophysectomized rats in vivo. These results indicate that GnRH, in addition to its well-known gonadotropin-releasing action in the pituitary, exerts a direct inhibition of ovarian steroidogenesis.

Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function.

A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect and mechanism of cadmium on the progesterone synthesis of ovaries.

The paper presents results of the effect of cadmium on the progesterone synthesis of ovaries. In the current study, we investigated whether Cd also disrupts progesterone synthesis via steroidogenic acute regulatory protein (StAR) and P450 cholesterol side-chain cleavage (P450scc), which play important roles in progesterone synthesis. The Wistar rats were exposed to cadmium in vivo (at 2.5, 5, 7.5mg/kg, as a single s.c. dose). We showed that the serum P(4) and granule cells P(4) of rats were significantly lower than control group. Ovaries granule cells were incubated in Dulbecco-modified Eagle medium +15% fetal bovine serum with 0, 10, 20, or 40 microM CdCl(2) in vitro, progesterone levels were declined in a dose-dependent manner. Our data showed that the expression of StAR and P450scc in vivo or in vitro were inhibited when treated with CdCl(2) (p<0.05). Coculture with 8-bromo-cAMP enhanced progesterone secretion in untreated cultures and reversed the decline in progesterone secretion induced by CdCl(2) treatment; the expression of StAR mRNA and P450scc mRNA in 8-Br-cAMP+40 microM CdCl(2) were significantly higher than 40 microM CdCl(2), and were lower than control group. We concluded that StAR, which delivers cholesterol to the inner mitochondrial membrane, is one site at which Cd interferes with progesterone production in cultured rats ovarian granule cells; P450scc, which conveys cholesterol to pregnenolone, is anther site. The mechanisms were mainly controlled by the cAMP-dependent pathway.

The role of steroids in follicular growth.

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The role of progestins in follicular growth and development is limited, its action confined largely to ovulation, although direct effects on granulosa cell function have been reported. Consistent with these findings, progesterone receptor knockout mice are infertile because they cannot ovulate. Androgens have been shown to promote early follicular growth, but also to impede follicular development by stimulating atresia and apoptosis. The inability of androgens to transduce a signal in mice lacking androgen receptors culminates in reduced fertility. Oestrogens are known to exert effects on granulosa cell growth and differentiation in association with gonadotrophins. Studies with oestrogen receptor knockouts and oestrogen depleted mice have shown us that oestrogen is essential for folliculogenesis beyond the antral stage and is necessary to maintain the female phenotype of ovarian somatic cells. In summary, the action of steroids within the ovary is based on the developmental status of the follicle. In the absence of any single sex steroid, ovarian function and subsequently fertility, are compromised.

Oestrogens and Progestagens: Synthesis and Action in the Brain.

When steroids, such as pregnenolone, progesterone and oestrogen, are synthesised de novo in neural tissues, they are more specifically referred to as neurosteroids. These neurosteroids bind specific receptors to promote essential brain functions. Pregnenolone supports cognition and protects mouse hippocampal cells against glutamate and amyloid peptide-induced cell death. Progesterone promotes myelination, spinogenesis, synaptogenesis, neuronal survival and dendritic growth. Allopregnanolone increases hippocampal neurogenesis, neuronal survival and cognitive functions. Oestrogens, such as oestradiol, regulate synaptic plasticity, reproductive behaviour, aggressive behaviour and learning. In addition, neurosteroids are neuroprotective in animal models of Alzheimer's disease, Parkinson's disease, brain injury and ageing. Using in situ hybridisation and/or immunohistochemistry, steroidogenic enzymes, including cytochrome P450 side-chain cleavage, 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase, cytochrome P450arom, steroid 5α-reductase and 3α-hydroxysteroid dehydrogenase, have been detected in numerous brain regions, including the hippocampus, hypothalamus and cerebral cortex. In the present review, we summarise some of the studies related to the synthesis and function of oestrogens and progestagens in the central nervous system.

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary.

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.

Inhibiting biosynthesis and/or metabolism of progestins in the ventral tegmental area attenuates lordosis of rats in behavioural oestrus.

In the ventral tegmental area (VTA), lordosis of rats is facilitated by 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP). Central 3alpha,5alpha-THP results from metabolism of peripheral progesterone, from the ovaries and/or adrenals, by sequential enzymatic activity of 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase (3alpha-HSOR). In addition, in glial cells, cholesterol is converted into pregnenolone by the P450 side-chain cleavage enzyme (P450scc), which is then metabolized to progesterone by 3beta-hydroxysteroid dehydrogenase, and subsequently reduced to 3alpha,5alpha-THP. We hypothesize that, in the VTA, formation of 3alpha,5alpha-THP by both metabolism and biosynthesis is necessary for facilitation of lordosis of female rats. In Experiment 1, naturally-receptive rats received bilateral VTA infusions of a P450scc inhibitor, digitoxin (1 microg/side); a 5alpha-reductase inhibitor, finasteride (10 microg/side); digitoxin (1 microg/side)+finasteride (10 microg/side); or vehicle and were tested 3 h later for lordosis. In Experiment 2, the effects of VTA infusions of digitoxin, finasteride, digitoxin+finasteride, or vehicle on lordosis and midbrain and plasma 3alpha,5alpha-THP levels were examined. In Experiment 3, we investigated whether infusions of 3alpha,5alpha-THP to the VTA reinstated lordosis and midbrain 3alpha,5alpha-THP levels following administration of inhibitors. VTA infusions of digitoxin, finasteride, or digitoxin+finasteride, significantly and similarly reduced lordosis and midbrain, but not plasma 3alpha,5alpha-THP levels, compared to vehicle. Following receipt of inhibitor infusions, 3alpha,5alpha-THP to the VTA restored lordosis and midbrain 3alpha,5alpha-THP levels. These data suggest that, in the VTA, both central biosynthesis of progesterone and metabolism of progesterone (from central and/or peripheral sources) to 3alpha,5alpha-THP are important for mediating lordosis of rats.

Inhibition of hormone-induced steroidogenesis during cell proliferation in serum-free cultures of rat granulosa cells.

Long term cultures of rat granulosa cells were grown in serum-free medium, consisting of Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient F-12 medium and supplemented with insulin, transferrin, hydrocortisone, and fibronectin (4F medium). In sparse cultures (10(4) cells/cm2), the granulosa cells were steroidogenically responsive to ovine FSH (NIADDK-oFSH-15) during days 1-2 and 10-14 (responsive periods). The major steroids produced were 20 alpha-hydroxyprogesterone (20 alpha-OH-P) and 5 alpha-pregnane, 3 alpha,20 alpha-diol (pregnanediol). However, as of day 3, the cells gradually lost their steroidogenic responsiveness which was inhibited by 88% at day 7 (refractory period). Nevertheless, from day 8 onward, the cells regained their responsiveness which was fully restored at day 12. The transient loss of responsiveness was uniquely associated with progestin biosynthesis, since FSH-induced aromatase activity declined to background levels within 12 days and was never restored again. The loss of progestin responsiveness was not due to lack of cAMP because FSH induced increasing levels of cAMP accumulation, reaching maximal values on day 7 in culture. On the other hand, the onset of the refractory period occurred concomitantly with the entry of the cultured cells into a synchronous proliferation phase, during which the cell population doubled. Thereafter, as DNA synthesis ceased, the cells regained their steroidogenic responsiveness. A deliberate arrest of cell replication, in the presence of excess thymidine or in high density cultures, prevented the temporal loss of activity. The data presented favor the notion that cell proliferation and expression of differentiated functions are inversely related. It is suggested that growth-related processes suppress steroidogenesis by an as yet unknown mechanism.

Mechanisms subserving insulin's differentiating actions on progestin biosynthesis by ovarian cells: studies with cultured swine granulosa cells.

To characterize the nature of insulin action on ovarian cells, an in vitro system of swine granulosa cells was developed in which 3- to 50-fold stimulation of progesterone production was observed in response to insulin under serum-free or sparsely (1%) serum-supplemented conditions. These studies demonstrate that optimal cell density is critical for the full expression of insulin action, and that the dose-dependent responses of granulosa cells to insulin are also significantly influenced by the maturational status of the parent follicle. The striking (greater than 30-fold) responses of medium-sized and healthy preovulatory follicles to insulin were not attributable to the presence or absence of atresia or to a selective inhibition of progesterone's catabolism to 20 alpha-dihydroprogesterone, since 20 alpha-dihydroprogesterone production was also markedly increased in response to insulin. The mechanisms subserving insulin action were explored further by testing the capacity of insulin to 1) increase progesterone accumulation in response to exogenously provided pregnenolone, and 2) stimulate pregnenolone biosynthesis in the presence of trilostane, an inhibitor of pregnenolone metabolism. The effects of insulin were to increase the biosynthesis of progesterone from available pregnenolone and increase the production of pregnenolone from endogenous sterol substrate. The physiological relevance of these differentiative actions of insulin is suggested by insulin's ability to significantly enhance the stimulatory effects of FSH, LH, epinephrine, prostaglandin E2, and cAMP effectors, cholera toxin and 8-bromo-cAMP. In summary, the present studies delineate culture conditions in which swine granulosa cells exhibit a high degree of responsiveness to the stimulatory actions of insulin on progestin biosynthesis. The effects of insulin are critically influenced by cell density, stage of follicle maturation, and the presence or absence of classical ovarian effector hormones with which insulin can interact synergistically. Moreover, our studies indicate that insulin exerts significant actions at several levels of progestin biosynthesis, including the production of pregnenolone, progesterone, and 20 alpha-dihydroprogesterone by granulosa cells. In view of the high concentrations of insulin-like growth factors and somatomedins attained in porcine Graafian follicles, we suggest that trophic actions of insulin or insulin-like peptides and the synergism of insulin with classical ovarian effector hormones are likely to be of physiological importance to the differentiation of granulosa cells in the developing ovarian follicle.

Degradation of rat and human lipoproteins by cultured rat ovary granulosa cells.

We have investigated the degradation of 125I-labeled rat and human lipoproteins by rat ovary granulosa cells cultured in serum-free medium. The granulosa cells degrade rat [125I]iodo high density lipoprotein (HDL) to acid-soluble products, mainly monoiodotyrosine. The degradation of 125I-labeled rat HDL is a specific, saturable, high affinity (Km = 21 micrograms protein/ml) process. In studies of rat [125I]iodo-HDL degradation and progestin (progesterone plus 20 alpha-dihydroprogesterone) production by the same granulosa cell cultures, the cholesterol potentially made available to the cells by degradation can account for the majority of the substrate necessary for the increased progestin production. Granulosa cells degrade human [125I]iodo-HDL by a specific, saturable, high affinity (Km = 20 micrograms protein/ml) process. The degradation of human [125I]iodo-HDL can account for only 20% of the cholesterol substrate necessary for increased progestin production. The degradation of human [125I]iodo-low density lipoprotein (LDL) is saturable and a high affinity (Km = 8 micrograms protein/ml) process, but can be inhibited significantly by a 10-fold excess of unlabeled human HDL. In contrast to both rat [125I]iodo-HDL and human [125I]iodo-HDL, the degradation of human [125I]iodo-LDL can potentially provide twice the cholesterol necessary for increased progestin production. Pronase treatment of the granulosa cells inhibits human [125I]iodo-LDL degradation but stimulates rat [125I]iodo-HDL degradation, indicating that the mechanisms of degradation are separate. The data demonstrate that cultured rat ovary granulosa cells degrade rat HDL, human HDL, and human LDL, and this process has the potential for providing cholesterol for cellular steroid hormone synthesis.

Contribution of granulosa cells and follicular fluid to ovarian estrogen secretion in rhesus monkey in vivo.

In order to ascertain which ovarian cell type within the follicle is the source of preovulatory estrogen secretion in vivo, ovarian venous, as well as peripheral venous, blood was collected prior to, 5 min, 30 min, and 120 min after the removal of follicular fluid and granulosa cells from 17 monkeys. In addition, estrogen, progesterone, and progestins were measured in the peripheral blood, ovarian venous blood, and follicular fluid of the follicle-containing and contralateral ovary in 24 monkeys, in order to prove that the preovulatory follicle is the principal source of estrogen. Estradiol was the principal estrogen and was secreted in larger amounts by the ovary with the large preovulatory follicle (7-10 mm in diameter) compared with the contralateral ovary. In 15 experiments ovarian venous estrogen (3934 +/- 798 pg/ml, mean +/- SE) in the vein draining the large follicle-containing ovary was usually 5-15-fold higher than peripheral plasma estrogen levels which were 307 +/- 31 pg/ml. The contralateral ovary secreted a small amount of estrogen (654 +/- 162 pg/ml). Follicular fluid contained large amounts of estrogen (2754 +/- 695 ng/ml) with levels which did not always correlate well with peripheral plasma or ovarian venous estrogen. Ovaries containing non-preovulatory or recently ovulated follicles secreted less estrogen. The removal of granulosa cells and follicular fluid from the preovulatory follicle led to no significant decrease (P greater than 0.5) in ovarian venous secretion of estrogen after a 5, 30, or 120 min time interval. This would indicate that, within the time constraints of this experiments, the follicular fluid and granulosa cells contribute relatively little to ovarian venous estrogen and that thecal cells of the large preovulatory follicle alone can secrete more of the estrogen into the ovarian vein.

Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells.

In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptor-bound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these cell- and mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with amino-glutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMP-dependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane.

Regulation of 3 beta-hydroxysteroid dehydrogenase delta 5/delta 4-isomerase and cholesterol side-chain cleavage cytochrome P450 by activin in rat granulosa cells.

Activin is a dimeric protein implicated in the local control of follicular steroidogenesis. Using cultured rat granulosa cells, we previously showed that the effect of activin on FSH-induced progesterone synthesis changes with preovulatory follicular development, from positive regulation in nondifferentiated (immature) granulosa cells to negative regulation in preovulatory (mature) granulosa cells. The aim of the present study was to assess development-related effects of activin on the expression of enzymes crucial to progesterone synthesis: cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase (3 beta HSD). Nondifferentiated granulosa cells were isolated from the ovaries of estrogen-pretreated immature female rats that received no other treatment; differentiated granulosa cells were obtained from similar animals treated for 48 h with human FSH to induce preovulatory follicular development. Cells were cultured for 48 h in serum-free medium with and without human FSH and/or recombinant activin-A, and medium was collected for measurement of progestagens (progesterone, pregnenolone, and 20 alpha-dihydroprogesterone). In cultures of nondifferentiated granulosa cells, activin augmented the FSH-induced production of all three steroids. In differentiated granulosa cells, activin suppressed the FSH-stimulated production of progesterone and 20 alpha-dihydroprogesterone, but had no effect on pregnenolone. The presence of exogenous pregnenolone increased the overall production of progesterone, but did not alter qualitative steroidogenic responses to activin. To assess the interaction between FSH and activin on 3 beta HSD and P450scc messenger RNA (mRNA) expression, Northern blot analyses were performed on total RNA isolated from cultured granulosa cells. Treatment in vitro with FSH alone markedly enhanced the abundance of both the 3 beta HSD and P450scc mRNA transcripts in nondifferentiated and differentiated granulosa cells. FSH-stimulated expression of P450scc mRNA was further enhanced by cotreatment of nondifferentiated granulosa cells with activin. However, activin had no consistent effect on FSH-stimulated expression of 3 beta HSD mRNA in nondifferentiated cells. In differentiated granulosa cells, both mRNAs were suppressed more than 50% by the presence of activin. We conclude that the in vitro effects of activin on FSH-induced expression of 3 beta HSD and P450scc mRNAs in rat GC are similar: initially stimulatory (P450scc) or without effect (3 beta HSD), then becoming completely inhibitory. The mechanism of this development-dependent change in the granulosa cell response to activin remains to be elucidated.

Somatomedin-C synergizes with follicle-stimulating hormone in the acquisition of progestin biosynthetic capacity by cultured rat granulosa cells.

We have recently shown that nanomolar concentrations of somatomedin-C (Sm-C), are capable of enhancing the FSH-mediated (but not basal) accumulation of progesterone (Po) by cultured rat granulosa cells. To further characterize this direct cytodifferentiative effect of Sm-C, granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats were cultured under serum-free conditions for up to 96 h. Concurrent treatment with highly purified Sm-C (50 ng/ml) produced 10.2- and 3.6-fold increments in the FSH (20 ng/ml)-stimulated accumulation of Po and 20 alpha-hydroxy-4-pregnen-3-one, respectively. Sm-C-augmented Po biosynthesis was dose- and time dependent, but was independent of the FSH dose employed. Significantly, this effect of Sm-C could not be accounted for by enhancement of cellular viability or plating efficiency, nor by an increase in the number of cells, or their DNA synthesis. Furthermore, specific inhibition of DNA synthesis with cytosine-1-beta-D-arabinofuranoside was without significant effect on the ability of SM-C to enhance FSH-supported Po biosynthesis. Insulin, like Sm-C, also synergized with FSH in the induction of Po biosynthesis. However, insulin [ED50 = 19.2 +/- 1.6 (SE) micrograms/ml] was approximately 4800-fold less potent than Sm-C [ED50 = 4.0 +/- 0.3 (SE) ng/ml] in this regard, and exerted little or no effect at concentrations presumed to saturate the putative high affinity granulosa cell insulin receptor. Although maximal stimulatory doses of Sm-C (75 ng/ml) or insulin (100 micrograms/ml) produced comparable increments in FSH-supported Po biosynthesis, combined treatment with maximal doses of both peptides did not prove additive. Pertinently, the direct cytodifferentiative effect of Sm-C is exerted at (nanomolar) concentrations compatible with its receptor-binding affinity as observed in all other cell types studied. Thus, Sm-C is not likely to be acting through the putative high affinity insulin receptor but rather through its own high affinity recognition sites. Similarly, the cytodifferentiative action of high dose insulin may reflect the consequences of its cross-interaction with the putative Sm-C, rather than the insulin receptor. These findings are in keeping with the suggestion that the granulosa cell may be the site of Sm-C reception and action and that Sm-C of intraovarian or circulatory origin may participate in the differentiation, as well as replication, of the developing granulosa cell.