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.

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.

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.

Comparison of progestin transcriptional profiles in rat mammary gland using Laser Capture Microdissection and whole tissue-sampling.

INTRODUCTION: Investigation of molecular mechanisms by gene expression profiling gains increasingly importance in preclinical safety evaluation. However, assigning expressed genes to specific cell populations is nearly impossible if the investigated RNA originates from whole tissue extracts. In this regard, Laser Capture Microdissection (LCM) can be used to detect changes specific to individual cell types. The objective of this study was to investigate the use of LCM for characterisation of progestin-related gene expression changes in the mammary gland. Thus, transcriptional profiles of the mammary gland of rats treated with a non-steroidal progesterone-receptor ligand, promegestone, medroxyprogesterone acetate, progesterone or vehicle were compared using whole tissue homogenates or LCM-captured epithelial cells. METHODS: Total RNA from 30 mammary glands was isolated from snap-frozen specimen of the whole tissue and from approximately 25.000-30.000 cells of cresyl violet stained frozen sections employing LCM. After amplification of averaged 0.2µg total RNA of LCM-captured samples, RNA was labelled, hybridised to Affymetrix GeneChips and analysed. RESULTS: LCM-captured samples showed up to 3-fold more differentially expressed probe sets (progesterone) and up to 10-fold more downregulated (promegestone) probe sets than whole tissue samples implying high cell specificity. Moreover, mammary gland specific differentiation markers like whey acidic protein, alpha lactalbumin, casein alpha s1 and casein kappa showed up to 3.4-fold (alpha lactalbumin, vehicle) higher expression values. Multivariate data analyses revealed a clear separation of gene expression profiles according to the method used, suggesting an amplification dependent bias. DISCUSSION: LCM transcriptional profiling provides highly cell-specific information. An amplification dependent bias was observed. The technical variability was shown to be smaller than the biological variability. For progestin-related transcriptional profiling of the mammary gland, whole tissue-sampling proved to yield more informative results. Therefore LCM should only be considered when cell-type specific gene expression profiles are necessary for an in depth evaluation.

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.

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.

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.

Ontogeny of uteroplacental progestagen production in pregnant mares during the second half of gestation.

In pregnant mares during late gestation, little, if any, progesterone (P4) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P4 known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5 alpha-pregnane-3,20-dione (5 alphaDHP) and 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha 5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20 alpha 5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5 alphaDHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.

Biosynthesis of steroids in ovarian follicles of red seabream, Pagrus major (Sparidae, Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro.

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.

Regulation of human endometrial transforming growth factor beta1 and beta3 isoforms through menstrual cycle and medroxyprogesterone acetate treatment.

The progesterone-induced differentiation of endometrial tissue from proliferative into secretory and decidua seems to be modulated by locally produced hormones and cytokines. Transforming growth factor beta (TGFbeta). a cytokine produced by endometrial cells, has been shown to modulate endometrial cell proliferation in vitro. Our aim was to evaluate the effects of medroxyprogesterone acetate (MPA) and the influence of menstrual cycle on the expression of TGFbeta1 and TGFbeta3 in human endometrium in vivo. In a double-blind, placebo-controlled trial, 46 healthy women with regular menstrual cycles received either MPA (10 mg/day) or placebo during 10 days. Endometrial and blood samples were collected 8-12 hours after the last MPA or placebo administration. Patients were classified into three groups according to biopsy dating and treatment: proliferative [tissue]/placebo, secretory [tissue]/placebo and secretory [tissue]/MPA. The immunohistochemical distribution of TGFbeta1 and TGFbeta1 mRNA was similar in all groups. Immunoreactive TGFbeta3 was present in the epithelium in 9.1% of proliferative samples, in 41.2% of secretory/placebo samples and in 87.5% of secretory/MPA samples (p=0.001). In the stroma, the frequency of TGFbeta3 staining was markedly increased after treatment with MPA (62.5%) compared to placebo (proliferative: 9.1%; secretory: 5.9%; p=0.005). The levels of TGFbeta3 mRNA increased during the secretory phase and were higher in the MPA-treated group, being directly correlated with morphological endometrial differentiation. It is concluded that MPA administration to healthy women increased TGFbeta3 but did not change TGFbeta1 gene and protein expression in the endometrium. This finding suggests that TGFbeta3 may be a local factor mediating progesterone- and progestogen-induced endometrial differentiation.

Adrenal progestogen and androgen production in 21-hydroxylase-deficient nonclassic adrenal hyperplasia is partially independent of adrenocorticotropic hormone stimulation.

OBJECTIVE: To test the hypothesis that adrenal steroidogenesis in nonclassic adrenal hyperplasia (NCAH) patients is, at least in part, independent of adrenocorticotropic hormone (ACTH) control. DESIGN: Prospective controlled clinical study. SETTING: Patients and healthy volunteers in an academic research environment. PATIENT(S): Four patients with 21-hydroxylase (21-OH) deficient NCAH and four healthy control women. INTERVENTION(S): Patients received the long-acting gonadotropin-releasing hormone analog (GnRH-a) leuprolide acetate (3.75 mg/month IM) on weeks 0 and 4; and dexamethasone (DEX) in weekly incremented doses (0.25 mg/day, 0.50 mg/day, 1.0 mg/day, and 1.5 mg/day), beginning on weeks 4, 5, 6, and 7, respectively. MAIN OUTCOME MEASURE(S): The levels of 17-hydroxyprogesterone (17-HP), progesterone (P4), androstenedione (A4), dehydroepiandrosterone sulfate (DHS), and cortisol (F) were measured at the beginning of weeks 0, 4, 5, 6, 7, and at the end of the study (beginning of week 8). RESULT(S): Patients and controls had a similar median age and body mass index (BMI). There were no significant decreases in the median levels of the studied hormones following 4 weeks of treatment with GnRH-a only, in either NCAH patients or controls. Analysis of individual hormonal values demonstrated that by the end of the study (after DEX of 1.5 mg/day during a week) only 2 of 4, 0 of 4, 3 of 4 and 3 of 4 NCAH patients had 17-HP, P4, A4, and DHS levels within the range of control values, respectively. CONCLUSION(S): Ovarian and incremental adrenal suppression did not fully suppress progestogen and androgen production in all of the study patients with 21-OH-deficient NCAH, suggesting that their production was partially independent of ACTH stimulation. Potentially in these patients subtle degrees of adrenocortical hyperplasia and/or abnormal enzymatic kinetics are responsible for the nonsupressibility.

Tokishakuyakusan directly attenuates PACAP's luteolytic action on luteal function in the rat ovary.

We investigated the potential direct effects of Tokishakuyakusan (TS) on progestin [progesterone and 20alpha-hydroxyprogesterone (20alpha-OH-P)] and cyclic adenosine-3',5'-monophosphate (cAMP) production in cultured rat luteal cells. In addition, we examined whether TS regulates the inhibitory effects of pituitary adenylate cyclase-activating polypeptide (PACAP), a newly found peptide, on luteinizing hormone (LH)-stimulated progesterone production. TS significantly stimulated progesterone, but not 20alpha-OH-P, production and cAMP accumulation through 24 hours of culture. PACAP-38 significantly elevated progesterone, 20alpha-OH-P and cAMP levels at all concentrations studied. On the other hand, PACAP-38 inhibited the production of progesterone and the accumulation of cAMP enhanced by LH, while the ratio of progesterone to 20alpha-OH-P was significantly decreased by PACAP-38 + LH. Concomitant treatment with TS and PACAP-38 + LH increased the ratio of progesterone to 20alpha-OH-P more than with PACAP-38 + LH. The present data have demonstrated that TS stimulates progesterone production in rat luteal cells, reconfirming our previous evidence that TS stimulates luteal steroidogenesis. The data further suggest that TS tends to attenuate PACAP's inhibition of LH-stimulated progesterone production, suggesting a luteotrophic effect within the corpus luteum.

Dependence on prolactin of the luteolytic effect of prostaglandin F2alpha in rat luteal cell cultures.

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10, 100 and 1000 ng/ml). Following 48 hours of incubation, the levels of progesterone, 20 alpha-hydroxy-4-pregnene-3-one (20 alpha-OH-P) and adenosine 3',5'-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20 alpha-OH-P in a dose-dependent manner (p < 0.01 and 0.05, respectively, ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each, ANOVA). Conversely, LH-stimulated 20 alpha-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20 alpha-OH-P production in LH-stimulated cells, PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.