Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Steroid regulation of secreted phosphoprotein 1 (SPP1) expression in ovine endometrium.

Secreted phosphoprotein 1 (SPP1) is an extracellular matrix glycoprotein that is highly expressed at the maternal-fetal interface and is a critical mediator of embryo implantation. The objectives of this study were to examine the spatial and temporal cyclical expression patterns and steroid regulation of SPP1 mRNA and protein in ovine endometrium, which may be further indicative of their functionality in embryo implantation. Uterine tissue was obtained following hysterectomy from ovariectomised ewes treated with ovarian steroids. In parallel, invitro culture of endometrial cells was used to investigate the effects of ovarian steroids on SPP1 expression in endometrial and luminal epithelial (LE) cells. A significant sustained mid-luteal phase increase in SPP1 mRNA in intercaruncular regions of the endometrium was observed, indicating that glandular epithelium is likely to be the primary source of SPP1 production. This increase in SPP1 was induced by progesterone treatment and was shown at the protein level by immunohistochemistry analysis. Similarly, treatment of stromal cells with 10ng mL-1 progesterone or in combination with 1ng mL-1 oestradiol significantly increased SPP1 expression (P<0.05). Collectively, expression levels of SPP1 are cycle-dependent and peak in the progesterone-dominant luteal phase. They are dependent on the interaction of uterine LE and stromal cells and may involve paracrine signalling by progesterone receptor-positive stromal cells.

Cytotoxic Effects of the Selective Ligands of Membrane Progesterone Receptors in Human Pancreatic Adenocarcinoma Cells BxPC3.

Progesterone and its synthetic analogues act on cells through different types of receptors, affecting proliferation and apoptosis. These compounds exert their effect through the nuclear receptors and the insufficiently studied membrane progesterone receptors (mPRs) belonging to the progestin and adiponectin Q receptor (PAQR) family. We have identified two selective ligands of mPRs that activate only this type of progesterone receptors - 19-hydroxypregn-4-en-20-one (LS-01) and 19-hydroxy-5ß-pregn-3-en-20-one (LS-02). The goal of this work is to study the effect of these compounds on proliferation and death of human pancreatic adenocarcinoma cells BxPC3 and involvement of the two kinases (p38 MAPK and JNK) in signaling pathways activated by progestins through mPRs. It was shown that progesterone and the compound LS-01 significantly (p < 0.05) inhibited the BxPC3 cell viability, with JNK serving as a mediator. The identified targets of these two steroids are the genes of the proteins Ki67, cyclin D1, PCNA, and p21. Progesterone and the compound LS-01 significantly (p < 0.05) stimulate DNA fragmentation, enhancing the cell death. The p38 mitogen-activated protein kinase (MAPK) is a key mediator of this process. The BCL2A1 protein gene was identified as a target of both steroids. The compound LS-02 significantly (p < 0.05) alters membrane permeability and changes the exposure of phosphatidylserine on the outer membrane leaflet, also enhancing the cell death. This compound acts on these processes by activating both kinases, JNK and p38 MAPK. The compound LS-02 targets the genes encoding the proteins HRK, caspase 9, and DAPK.

Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARß of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABAA receptors.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR ß3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the ß3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Progesterone promotes endothelial nitric oxide synthase expression through enhancing nuclear progesterone receptor-SP-1 formation.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

An agonist for membrane progestin receptor (mPR) induces oocyte maturation and ovulation in zebrafish in vivo.

The maturation and ovulation of fish oocytes are well-characterized biological processes induced by progestins via coordination of nongenomic actions and genomic actions. Previously, we established a procedure that enables the induction of oocyte maturation and ovulation in live zebrafish by simple administration of the natural teleost maturation-inducing hormone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20ß-DHP) into the surrounding water. By this in vivo assay, the potencies of chemicals in inducing or preventing oocyte maturation and ovulation can be evaluated. The potencies of compounds in inducing ovulation of zebrafish oocytes also can be evaluated in vivo with improved in vitro assays. Here, we attempted to evaluate the effect of Org OD 02-0 (Org OD 02), a selective agonist for membrane progestin receptor (mPR), on fish oocyte maturation and ovulation with in vitro and in vivo assays. As reported previously, Org OD 02 triggered oocyte maturation in vitro. The same Org OD 02 triggered oocyte maturation within several hours in vivo. Surprisingly, Org OD 02 even induced ovulation both in in vivo and in vitro. Eggs from Org OD 02-induced ovulation could be fertilized by artificial insemination. The juveniles developed normally. These results indicated that Org OD 02 triggered physiological ovulation in live zebrafish. In summary, we have demonstrated the effect of Org OD 02 on fish oocyte maturation and ovulation in vitro and in vivo. The results suggested that Org OD 02 acted as an agonist not only of mPR but also of nuclear progesterone receptor (nPR).

Maraviroc, tenofovir disoproxil fumarate and dapivirine, activate progesterone receptor B in the absence of progestogens.

Antiretroviral therapy has slowed the HIV/AIDS pandemic and is currently being used as a prophylactic measure for individuals at high risk of infection. However, concerns over adverse effects of long-term use need to be explored. We hypothesize that this may occur, at least in part, through off-target effects via select steroid receptors (SRs) that broadly regulate multiple physiological processes. We investigated the effects of maraviroc (MVC), tenofovir disoproxil fumarate (TDF), and dapivirine (DPV) on progesterone receptor B (PR-B) transcriptional activity. We found that MVC and TDF activate PR-B transcription in the absence of progestogens on a PR-regulated promoter reporter construct and on endogenous PR-regulated genes. MVC and TDF exhibited no direct binding to PR-B; however, increased PR-B phosphorylation was detected with TDF but not MVC. DPV transactivated gilz and ptgs2 in the absence of progestogens and exhibited PR-B binding while showing no effects on phosphorylation, suggesting that it may activate PR-B through a direct mechanism. Our study shows that potential off-target immunomodulatory effects of MVC, TDF and DPV occur in vitro and these are most likely mediated by different mechanisms of PR-B activation.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

The transcriptional repressor GATAD2B mediates progesterone receptor suppression of myometrial contractile gene expression.

The mechanisms whereby progesterone (P4), acting via the progesterone receptor (PR), inhibits proinflammatory/contractile gene expression during pregnancy are incompletely defined. Using immortalized human myometrial (hTERT-HM) cells stably expressing wild-type PR-A or PR-B (PRWT), we found that P4 significantly inhibited IL-1ß induction of the NF-κB target genes, COX-2 and IL-8 P4-PRWT transrepression occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA polymerase II to COX-2 and IL-8 promoters. However, in cells stably expressing a PR-A or PR-B DNA-binding domain mutant (PRmDBD), P4-mediated transrepression was significantly reduced, suggesting a critical role of the PR DBD. ChIP analysis of hTERT-HM cells stably expressing PRWT or PRmDBD revealed that P4 treatment caused equivalent recruitment of PRWT and PRmDBD to COX-2 and IL-8 promoters, suggesting that PR inhibitory effects were not mediated by its direct DNA binding. Using immunoprecipitation, followed by MS, we identified a transcriptional repressor, GATA zinc finger domain-containing 2B (GATAD2B), that interacted strongly with PRWT but poorly with PRmDBD P4 treatment of PRWT hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P4-PRWT transrepression of COX-2 and IL-8 Notably, GATAD2B expression was significantly decreased in pregnant mouse and human myometrium during labor. Our findings suggest that GATAD2B serves as an important mediator of P4-PR suppression of proinflammatory and contractile genes during pregnancy. Decreased GATAD2B expression near term may contribute to the decline in PR function, leading to labor.

The past, present, and future of selective progesterone receptor modulators in the management of uterine fibroids.

Uterine fibroids are common in women of reproductive age and can have a significant impact on quality of life and fertility. Although a number of international obstetrics/gynecology societies have issued evidence-based clinical practice guidelines for the management of symptomatic uterine fibroids, many of these guidelines do not yet reflect the most recent clinical evidence and approved indication for one of the key medical management options: the selective progesterone receptor modulator class. This article aims to share the clinical experience gained with selective progesterone receptor modulators in Europe and Canada by reviewing the historical development of selective progesterone receptor modulators, current best practices for selective progesterone receptor modulator use based on available data, and potential future uses for selective progesterone receptor modulators in uterine fibroids and other gynecologic conditions.

Megestrol Acetate Induces Declarative Memory Changes and Cortisol Suppression in Healthy Volunteers.

BACKGROUND: The effects of the glucocorticoid and progesterone receptor agonist megestrol on declarative memory, and the ability of phenytoin to block these effects, were assessed. METHODS: Healthy volunteers received each medication combination (placebo and megestrol, phenytoin and megestrol, and placebo and placebo) using a randomized, crossover design. The Rey Auditory Verbal Learning Test assessed declarative memory. RESULTS: Megestrol was associated with a significant reduction in declarative memory (p = 0.0008), which was attenuated by phenytoin, and was associated with significant cortisol suppression compared to placebo (p < 0.001). CONCLUSION: Changes in memory and cortisol suppression were found in healthy volunteers given megestrol.

Selective progesterone receptor modulators: current applications and perspectives.

Selective progesterone receptor modulators (SPRMs) are steroid progesterone receptor ligands able to induce agonistic or antagonistic activities. Mifepristone, the class leader, was primarily used for pregnancy termination from the 1980s. Emergency contraception with extended activity was the second major development 30 years later, with mifepristone in some countries and ulipristal acetate world-wide. More recently, ulipristal acetate was released for the treatment of myoma-related uterine bleeding. In addition to a very rapid cessation of bleeding, SPRMs allow a decrease in myoma volume, as do gonadotropin releasing hormone analogs. However, estradiol secretion is not blunted by SPRMs. This offers new alternatives for myoma treatment, especially in women close to menopause. In conclusion, use of SPRMs has allowed significant progress in emergency contraception and treatment of myoma-related symptoms. Numerous future perspectives in women's health care are currently under evaluation.

Agonistic and Antagonistic Effects of Progesterone Derivatives on the Transcriptional Activity of Nuclear Progesterone Receptor B in Yeast Model System.

Identification of progesterone selective agonists and antagonists that act through one of the nuclear progesterone receptor isoforms is of particular importance for the development of tissue-specific drugs in gynecology and anticancer therapy. Fourteen pregna-D'6- and pregna-D'3-pentarane progesterone derivatives with 16α,17α-cycloalkane groups and two progesterone 3-deoxyderivatives were examined for their ability to regulate transcriptional activity of human nuclear progesterone receptor isoform B (nPR-B) expressed in Saccharomyces cerevisiae yeast. Transcriptional activity of nPR-B was measured from the expression of the ß-galactosidase reporter gene with a hormone-responsible element in the promoter. Among the compounds tested, two were full progesterone agonists, four were partial agonists, one compound possessed both agonistic and antagonistic activity, one compound displayed only partial antagonistic activity, and eight compounds did not show any activity. Modifications of the pentarane structure, precisely, introduction of an additional double bound in the A or B rings and/or modification at the 6th position of progesterone, lead to a switch from the complete agonistic activity to partial agonistic or mixed activities. These modifications enable progestins to act as selective modulators of progesterone receptor. Steroids with reduced A-ring and 3-ketogroups lose their ability to regulate PR-B activity. Both 3-deoxycompounds, being selective ligands of progesterone membrane receptors, do not affect PR-B activity.

Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa.

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes during in vitro capacitation and the actions of progesterone (P4) and 17ß-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40-45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERß). ERα was located in the postacrosomal region of all the spermatozoa and ERß on the apical region of 63.7% of the cells. The presence of ERß was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848, P < 0.001). This significantly decreased after in vitro capacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 before in vitro capacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.

Progesterone activates the principal Ca2+ channel of human sperm.

Steroid hormone progesterone released by cumulus cells surrounding the egg is a potent stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them penetrate the egg's protective vestments. Progesterone induces Ca(2+) influx into spermatozoa and triggers multiple Ca(2+)-dependent physiological responses essential for successful fertilization, such as sperm hyperactivation, acrosome reaction and chemotaxis towards the egg. As an ovarian hormone, progesterone acts by regulating gene expression through a well-characterized progesterone nuclear receptor. However, the effect of progesterone upon transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a specialized, non-genomic membrane progesterone receptor. The identity of this non-genomic progesterone receptor and the mechanism by which it causes Ca(2+) entry remain fundamental unresolved questions in human reproduction. Here we elucidate the mechanism of the non-genomic action of progesterone on human spermatozoa by identifying the Ca(2+) channel activated by progesterone. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of progesterone dramatically potentiate CatSper, a pH-dependent Ca(2+) channel of the sperm flagellum. We demonstrate that human CatSper is synergistically activated by elevation of intracellular pH and extracellular progesterone. Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Because our experimental conditions did not support second messenger signalling, CatSper or a directly associated protein serves as the elusive non-genomic progesterone receptor of sperm. Given that the CatSper-associated progesterone receptor is sperm specific and structurally different from the genomic progesterone receptor, it represents a promising target for the development of a new class of non-hormonal contraceptives.

Regulation of uridine diphosphate-glucuronosyltransferase 2B15 expression during ovulation in the rat.

Uridine diphosphate-glucuronosyltransferase 2B15 (UGT2B15) conjugates 5α-androstane-3α, 17ß-diol (3α-diol) to 3α-diol glucuronide (3α-diol G) in steroid target tissues. The present study investigated the regulation of UGT2B15 expression during the ovulatory process in the rat. Real-time PCR analysis revealed that treatment of immature rats with equine chorionic gonadotropin followed by human chorionic gonadotropin transiently stimulated UGT2B15 gene expression in granulosa cells of preovulatory follicles within 6 h. The progesterone receptor antagonist RU486 suppressed the gonadotropin-induced UGT2B15 expression. The expression of UGT2B15 and the levels of 3α-diol G were transiently increased by luteinizing hormone (LH) treatment in cultured preovulatory follicles. The LH-stimulated UGT2B15 mRNA level in cultured preovulatory follicles was inhibited by inhibitors of adenylyl cyclase, phosphoinositide 3-kinase and mitogen-activated protein kinase. Furthermore, a vitamin D receptor agonist (calcitriol) suppressed the LH-stimulated UGT2B15 expression in a dose-dependent manner. Taken together, these results indicate that gonadotropins transiently stimulate UGT2B15 expression and activity in preovulatory follicles, and UGT2B15 mRNA levels are regulated by the progesterone receptor and vitamin D receptor.

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Dual estrogenic regulation of the nuclear progestin receptor and spermatogonial renewal during gilthead seabream (Sparus aurata) spermatogenesis.

Studies in teleosts suggest that progestins have crucial functions during early spermatogenesis. However, the role of the different progestin receptors in these mechanisms is poorly understood. In this work, we investigated the expression pattern and hormonal regulation of the classical nuclear progestin receptor (Pgr) in the gilthead seabream at three different stages of spermatogenesis: the resting (postspawning) phase, onset of spermatogenesis, and spermiation. Immunolocalization experiments using a seabream specific Pgr antibody revealed that the receptor was expressed in Sertoli and Leydig cells, and also in a subset of spermatogonia type A, throughout spermatogenesis. Short-term treatment of testis explants with 17ß-estradiol (E2) increased pgr mRNA expression at all stages, while the progestin 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) had the opposite effect. At the resting stage, Sertoli cell Pgr expression was positively correlated with the occurrence of proliferating spermatogonia type A in the tubules, and both processes were incremented in vitro by E2 likely through the estrogen receptor alpha (Era) expressed in Sertoli and Leydig cells. In contrast, treatment with 17,20ßP downregulated Pgr expression in somatic cells. The androgen 11-ketotestosterone (11-KT) upregulated pgr expression in Leydig cells and promoted the proliferation of mostly spermatogonia type B, but only during spermiation. No relationship between the changes in the cell type-specific expression of the Pgr with the entry into meiosis of germ cells was found. These data suggest a differential steroid regulation of Pgr expression during seabream spermatogenesis and the potential interplay of the E2/Era and 17,20ßP/Pgr pathways for the maintenance of spermatogonial renewal rather than entry into meiosis.

Novel Non-steroidal Progesterone Receptor Ligands Based on m-Carborane Containing a Secondary Alcohol: Effect of Chirality on Ligand Activity.

The progesterone receptor (PR) controls various physiological processes, including the female reproductive system, and nonsteroidal PR ligands are considered to be drug candidates for treatment of various diseases without significant adverse effects. Here, we designed and synthesized m-carborane-based secondary alcohols and investigated their PR-ligand activity. All the synthesized alcohols exhibited PR-antagonistic activity at subnanomolar concentration. Among them, alcohols having a small alkyl side chain and a 4-cyanophenyl group also exhibited PR-agonistic activity in a relatively high concentration range. Optical resolution of secondary alcohols having a methyl side chain was performed, and the PR-ligand activity and PR-binding affinity of the purified enantiomers were examined. The chirality of the secondary alcohol appears to have a more significant influence on PR-agonistic activity than on antagonistic activity.