Primary Rat Granulosa Cells Research Articles

YAP/TAZ-TEAD is a novel transcriptional regulator of genes encoding steroidogenic enzymes in rat granulosa cells and KGN cells

Steroidogenesis in ovarian granulosa cells is regulated by the follicle-stimulating hormone (FSH) via transcriptional regulation of its related genes. We herein showed the involvement of the Hippo pathway in this regulation. In KGN granulosa cell, repression of YAP/TAZ activity induced the expression of CYP11A1, HSD3B2, and CYP19A1 in a TEAD-dependent manner without cAMP stimulation. A selective inhibitor of p38 MAP kinase, suppressed YAP/TAZ knockdown-indued the expression of these genes, suggesting this signal could be involved. The expression of these genes was induced by 8Br-cAMP, whereas that of CYR61 and ADATS1, typical YAP/TAZ-TEAD target genes, was suppressed, suggesting that the cellular signaling of cAMP reduced YAP/TAZ-TEAD activity. The constitutively active mutant YAP canceled the FSH- and 8Br-cAMP-mediated induction of these genes in primary rat granulosa and KGN cells, respectively. Moreover, regulation of steroidogenesis-related genes by YAP/TAZ-TEAD was independent of steroidogenic factor 1, a master gene regulator of steroidogenesis. These results suggest that YAP/TAZ-TEAD is a negative regulator of steroidogenesis and that suppression of YAP/TAZ-TEAD activity by FSH is involved in ovarian steroidogenesis.

Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.

Ameliorative effect of recombinant human lactoferrin on the premature ovarian failure in rats after cyclophosphamide treatments

This study investigated the effect of recombinant human lactoferrin (rhLF) on the premature ovarian failure (POF) of rats. After cyclophosphamide treatments, the POF rats were divided into the following groups: normal control group (NC), low-dose group (LD), medium-dose group (MD) and high-dose group (HD) of rhLF. After drug administrations, the ovarian indexes and hormonal levels were detected. After follicle number count, the proliferation and apoptosis were analyzed with the expressions of genes related with oogenesis, reactive oxygen species (ROS) production and apoptosis detected, followed by the calculation of oxidative stress and protein expressions. After 4-hydroperoxy cyclophosphamide (4-HC) treatments, the effect of rhLF on the proliferation, ROS production and gene expressions of primary rat granulosa cells (GCs) cultured in vitro were detected. After mating, the fertilities of POF rats were recorded. The result showed that the rhLF administrations up-regulated the ovarian index with the number of developing follicles increased and the decreases of hormonal levels conferred. The Ki-67 intensities of the MD and HD groups were up-regulated with the Tunnel intensities decreased. The rhLF treatments significantly promoted the expression of oogenesis, antioxidant and anti-apoptosis related genes. The expression of Bax and Caspase 3 were decreased with the expression of Bcl-2 up-regulated after rhLF administrations. The in vitro treatments of rhLF effectively conferred the toxicity of 4-HC on primary rat GCs. The fertility assessment showed the rhLF treatments up-regulated the offspring’s’ folliculogenesis, which confirmed the ameliorative role of rhLF on the POF damages via the inhibition of ROS production in GCs.

Novel Regulation of PKC-induced Inflammation by Akt and Protein Phosphatase 2A in Ovarian Granulosa Cells

PKC-mediated inflammation is important in ovarian physiology. The roles of Akt and protein phosphatase 2A (PP2A) in PKC-mediated inflammation in ovarian granulosa cells (GCs) remain mostly unclear. PKC activator phorbol 12-myristate 13-acetate induced the Akt phosphorylation in rat primary GCs but reduced the Akt phosphorylation in KGN human GCs. In rat GCs, an inhibitory effect of PI3K inhibitor wortmannin and a stimulatory effect of Akt activator SC79 on PKC-induced cyclooxygenase-2 (COX-2)/PGE2production were noted; wortmannin and SC79 acted oppositely in human GCs. In rat GCs, PP2A inhibitor okadaic acid further enhanced the PKC-mediated promoter activation and elevation of mRNA and protein levels of the COX-2 gene, whereas PP2A activator sodium selenate attenuated the PKC-mediated COX-2 expression and promoter activation. PKC activation did not affect PP2A phosphorylation, but okadaic acid indeed augmented the PKC-induced NF-κB nuclear translocation. Thus, PP2A appears to act as a negative modulator in PKC-mediated cellular inflammation in rat GCs, at least in part due to its attenuating effect on the PKC-induced NF-κB activation.

Role of Klf4 in the Regulation of Apoptosis and Cell Cycle in Rat Granulosa Cells during the Periovulatory Period.

In the ovary, the luteinizing hormone (LH) surge suppresses the proliferation and induces the luteinization of preovulatory granulosa cells (GCs), which is crucial for the survival of terminally-differentiated GCs. Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell cycle and apoptosis in various cell types. The rapid induction of Klf4 expressions by LH was observed in preovulatory GCs. To evaluate whether Klf4 affects GC proliferation and survival, primary rat GCs were isolated from pregnant mare serum gonadotropin-primed Sprague–Dawley rat ovaries and transfected with a Klf4 expression vector or Klf4-specific siRNA, followed by determination of the transcript levels of apoptosis-related and cell cycle-related genes. Cell proliferation, viability, and apoptosis were analyzed by BrdU incorporation, a Cell Counting Kit-8 assay, a bioluminescence caspase 3/7 assay, and flow cytometry. LH treatment increased Klf4 mRNA expression in preovulatory GCs. Transcripts of B-cell lymphoma 2 (Bcl-2) and cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of the cell cycle inhibitor, p21, increased. Altering the expression of Klf4 by overexpression or knockdown consistently affected the expression of Bcl-2 and Cyclin D1. In agreement with this, Klf4 overexpression reduced cell viability, increased the fraction of apoptotic cells, and arrested cell cycle progression in G1 phase. We conclude that Klf4 increases the susceptibility of preovulatory GCs to apoptosis by down-regulating Bcl-2, and promotes LH-induced cell cycle exit. It appears to be a key regulator induced by the LH surge that determines the fate of GCs in preovulatory follicles during the luteal transition.

The effects and possible mechanisms of triclosan on steroidogenesis in primary rat granulosa cells

BackgroundTriclosan (TCS) has been detected in human tissues. It can disrupt steroidogenesis in vivo. The study on the effects of TCS on ovarian granulosa cells was lacking. MethodsPrimary rat granulosa cells (rGCs) were treated with TCS. Concentrations of estradiol (E2), progesterone (P4) in the cell culture supernatants were measured. Microarray was used to measure gene expression profiles. Pathway analysis was performed to identify signaling networks that linked differentially expressed genes (DEGs). Genes related with steroidogenesis were analyzed. ResultsTCS increased E2 and P4 production. A total of 2006 DEGs were identified. Pathway analysis revealed that ovarian steroidogenesis pathway was upregulated. Both PCR and Western-blot demonstrated that the expressions of key genes involved in this pathway were significantly increased. ConclusionsTCS co-administered with follicle-stimulating hormone (FSH) could increase E2 and P4 production in rGCs and up-regulate ovarian steroidogenesis pathway. StAR and aromatase protein were increased by TCS, while P450scc protein wasn’t changed significantly.

1,25-Dihydroxyvitamin D3 modulates the effects of sublethal BPA on mitochondrial function via activating PI3K-Akt pathway and 17β-estradiol secretion in rat granulosa cells

Bisphenol A (BPA), an endocrine-disrupting chemical, is capable of producing reproductive toxicity. BPA results in mitochondrial DNA (mtDNA) deletion and mitochondrial dysfunction; however, the effect of BPA on the mitochondria of ovarian granulosa cells is not clear. Further, 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction, because its receptor, VDR, contributes to the inhibition of oxidative stress and predominantly exists in the nuclei of granulosa cells. Hence, the role of 1,25D3 in BPA-mediated effects on mitochondrial function was examined in this study. Primary rat granulosa cells treated with BPA, 1,25D3, or both were subjected to molecular/biochemical assays to measure cell survival, mtDNA content, mtDNA deletion, superoxide dismutase activity, levels of proteins related to mitochondrial biogenesis, and mitochondrial function. We found that cell viability was dose-dependently reduced and reactive oxygen species (ROS) levels were increased by BPA treatment. BPA administration elevated Mn-superoxide dismutase (MnSOD) expression but negatively regulated total SOD activity. 1,25D3 treatment alone increased 17β-estradiol secretion, ATP production, and cellular oxygen consumption. In cells treated with both agents, 1,25D3 enhanced BPA-induced MnSOD protein upregulation and blocked the BPA-mediated decline in total SOD activity. Furthermore, 1,25D3 attenuated BPA-mediated mtDNA deletion but showed no effect on BPA-induced increases in mtDNA content. Although BPA had no influence on the levels of peroxisome proliferator-activated receptor-γ coactivator-1 α, nuclear respiratory factor-1, mitochondrial transcription factor A, or cytochrome c oxidase subunit IV, 1,25D3 plus BPA markedly increased mitochondrial biogenesis-related protein expression via the PI3K-Akt pathway. Moreover, BPA-mediated negative regulation of cytochrome c oxidase subunit I levels and 17β-estradiol secretion was attenuated by 1,25D3 pre-treatment. Our results suggest that 1,25D3 attenuates BPA-induced decreases in 17β-estradiol and that treatment with 1,25D3 plus BPA regulates granulosa cell mitochondria by elevating mitochondrial biogenesis-related protein levels.

Effect of the interaction of metformin and bone morphogenetic proteins on ovarian steroidogenesis by human granulosa cells

In the present study, we studied the effects of metformin and its interactions with the actions of bone morphogenetic proteins (BMPs) on ovarian steroidogenesis. It was revealed that metformin treatment enhanced progesterone production by human granulosa KGN cells and rat primary granulosa cells induced by forskolin and FSH, respectively. In human granulosa cells, it was found that metformin treatment suppressed phosphorylation of Smad1/5/9 activated by BMP-15 compared with that induced by other BMP ligands. Moreover, metformin treatment increased the expression of inhibitory Smad6, but not of that Smad7, in human granulosa cells, while metformin had no significant impact on the expression levels of BMP type-I and -II receptors. Thus, the mechanism by which metformin suppresses BMP-15-induced Smad1/5/9 phosphorylation is likely, at least in part, to be upregulation of inhibitory Smad6 expression in granulosa cells. The results suggest the existence of functional interaction between metformin and BMP signaling, in which metformin enhances progesterone production by downregulating endogenous BMP-15 activity in granulosa cells.

Interaction between orexin A and bone morphogenetic protein system on progesterone biosynthesis by rat granulosa cells

The involvement of orexins in reproductive function has been gradually uncovered. However, the functional role of orexins in ovarian steroidogenesis remains unclear. In the present study, we investigated the effects of orexin A on ovarian steroidogenesis by using rat primary granulosa cells that express both OX1 and OX2 receptors for orexins. Treatment with orexin A enhanced progesterone, but not estradiol, biosynthesis induced by FSH, whereas it did not affect basal levels of progesterone or estradiol. In accordance with the effects on steroidogenesis, orexin A increased the mRNA levels of progesterogenic enzymes, including StAR, P450scc and 3βHSD, but not P450arom, and cellular cAMP synthesis induced by FSH. Under the condition of blockage of endogenous BMP actions by noggin or BMP-signaling inhibitors, orexin A failed to increase levels of progesterone synthesis induced by FSH treatment, suggesting that endogenous BMP activity in granulosa cells might be involved in the enhancement of progesterone synthesis by orexin A. Treatment with orexin A impaired Smad1/5/9 activation as well as Id-1 mRNA expression stimulated by BMP-6 and BMP-7, the latter of which was reversed by treatment with an OX1 antagonist. It was also found that orexin A suppressed the mRNA expression of both type-I and -II receptors for BMPs and increased that of inhibitory Smad6 and Smad7 in granulosa cells. On the other hand, treatments with BMP-6 and -7 suppressed the expression of OX1 and OX2. Collectively, the results indicated that orexin A enhances FSH-induced progesterone production, at least in part, by downregulating BMP signaling in granulosa cells. Thus, a new role of orexin A in facilitating progesterone synthesis and functional interaction between the orexin and BMP systems in granulosa cells were revealed.

Incretins modulate progesterone biosynthesis by regulating bone morphogenetic protein activity in rat granulosa cells

The effects of incretins on ovarian steroidogenesis have not been clarified. In this study, we investigated the effects of incretins, including GIP and GLP-1, on ovarian steroidogenesis using rat primary granulosa cells. Treatment with incretins significantly suppressed progesterone synthesis in the presence of FSH, and the effect of GIP was more potent than that of GLP-1. In contrast, incretins had no significant effect on estrogen synthesis by rat granulosa cells. In accordance with the effects of incretins on steroidogenesis, GIP and GLP-1 suppressed the expression of progesterogenic factors and enzymes, including StAR, P450scc, 3βHSD, but not P450arom, and cellular cAMP synthesis induced by FSH. In addition, incretins moderately increased FSHR mRNA expression in granulosa cells. Of note, treatment with GIP, but not treatment with GLP-1, augmented Smad1/5/8 phosphorylation and transcription of the BMP target gene Id-1 induced by BMP-6 stimulation, suggesting that GIP upregulates BMP receptor signaling that can inhibit FSH-induced progesterone synthesis in rat granulosa cells. On the other hand, BMP-6 treatment suppressed the expression of GIP receptor but not that of GLP-1 receptor. Expression of the BMP type-I receptor ALK-3 was upregulated by treatment with GIP and GLP-1 and that of ALK-6 was also increased by GIP, while inhibitory Smad6 expression was impaired by GIP and GLP-1 in rat granulosa cells. Collectively, the results indicate that incretins, particularly GIP, impair FSH-induced progesterone production, at least in part, by upregulating BMP signaling in rat granulosa cells. The modulatory effects of incretins on endogenous BMP activity may be applicable to treatment of dysregulated steroidogenesis such as polycystic ovary syndrome.

A regulatory role of androgen in ovarian steroidogenesis by rat granulosa cells

Excess androgen and insulin-like growth factor (IGF)-I in the ovarian follicle has been suggested to be involved in the pathophysiology of polycystic ovary syndrome (PCOS). Here we investigated the impact of androgen and IGF-I on the regulatory mechanism of ovarian steroidogenesis using rat primary granulosa cells. It was revealed that androgen treatment with dihydrotestosterone (DHT) amplified progesterone synthesis in the presence of FSH and IGF-I, whereas it had no significant effect on estrogen synthesis by rat granulosa cells. In accordance with the effects of androgen on steroidogenesis, DHT enhanced the expression of progesterogenic factors and enzymes, including StAR, P450scc and 3βHSD, and cellular cAMP synthesis induced by FSH and IGF-I. Of note, treatment with DHT and IGF-I suppressed Smad1/5/8 phosphorylation and transcription of the BMP target gene Id-1, suggesting that androgen and IGF-I counteract BMP signaling that inhibits FSH-induced progesterone synthesis in rat granulosa cells. DHT was revealed to suppress the expression of BMP-6 receptors, consisting of ALK-2, ALK-6 and ActRII, while it increased the expression of inhibitory Smads in rat granulosa cells. In addition, IGF-I treatment upregulated androgen receptor (AR) expression and DHT treatment suppressed IGF-I receptor expression on rat granulosa cells. Collectively, the results indicate that androgen and IGF-I mutually interact and accelerate progesterone production, at least in part, by regulating endogenous BMP signaling in rat granulosa cells. Cooperative effects of androgen and IGF-I counteract endogenous BMP-6 activity in rat granulosa cells, which is likely to be functionally linked to the steroidogenic property shown in the PCOS ovary.

Interference of Steroidogenesis by Gold Nanorod Core/Silver Shell Nanostructures: Implications for Reproductive Toxicity of Silver Nanomaterials.

As a widely used nanomaterial in daily life, silver nanomaterials may cause great concern to female reproductive system as they are found to penetrate the blood-placental barrier and gain access to the ovary. However, it is largely unknown about how silver nanomaterials influence ovarian physiology and functions such as hormone production. This study performs in vitro toxicology study of silver nanomaterials, focusing especially on cytotoxicity and steroidogenesis and explores their underlying mechanisms. This study exposes primary rat granulosa cells to gold nanorod core/silver shell nanostructures (Au@Ag NRs), and compares outcomes with cells exposed to gold nanorods. The Au@Ag NRs generate more reactive oxygen species and reduce mitochondrial membrane potential and less production of adenosine triphosphate. Au@Ag NRs promote steroidogenesis, including progesterone and estradiol, in a time- and dose-dependent manner. Chemical reactivity and transformation of Au@Ag NRs are then studied by electron spin resonance spectroscopy and X-ray absorption near edge structure, which analyze the generation of free radical and intracellular silver species. Results suggest that both particle-specific activity and intracellular silver ion release of Au@Ag NR contribute to the toxic response of granulosa cells.

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells.

Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria.

Regulatory role of BMP-9 in steroidogenesis by rat ovarian granulosa cells

BMPs expressed in the ovary differentially regulate steroidogenesis by granulosa cells. BMP-9, a circulating BMP, is associated with cell proliferation, apoptosis and differentiation in various tissues. However, the effects of BMP-9 on ovarian function have yet to be elucidated. Here we investigated BMP-9 actions on steroidogenesis using rat primary granulosa cells. BMP-9 potently suppressed FSH-induced progesterone production, whereas it did not affect FSH-induced estradiol production by granulosa cells. The effects of BMP-9 on FSH-induced steroidogenesis were not influenced by the presence of oocytes. FSH-induced cAMP synthesis and FSH-induced mRNA expression of steroidogenic factors, including StAR, P450scc, 3βHSD2 and FSHR, were suppressed by treatment with BMP-9. BMP-9 mRNA expression was detected in granulosa cells but not in oocytes. BMP-9 readily activated Smad1/5/8 phosphorylation and Id-1 transcription in granulosa cells. Analysis using ALK inhibitors indicated that BMP-9 actions were mediated via type-I receptors other than ALK-2, -3 and -6. Furthermore, experiments using extracellular domains (ECDs) for BMP type-I and -II receptor constructs revealed that the effects of BMP-9 were reversed by ECDs for ALK-1 and BMPRII. Thus, the functional receptors for BMP-9 in granulosa cells were most likely to be the complex of ALK-1 and BMPRII. Collectively, the results of the present study showed that BMP-9 can affect luteinization and that there are two possible sources of BMP-9, serum and granulosa cells in the ovary.

The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets.

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

Melatonin counteracts BMP-6 regulation of steroidogenesis by rat granulosa cells

The ovarian bone morphogenetic protein (BMP) system is a physiological inhibitor of luteinization in growing ovarian follicles. BMP-6, which is expressed in oocytes and granulosa cells of healthy follicles, specifically inhibits FSH actions by suppressing adenylate cyclase activity. In the present study, we studied the role of melatonin in ovarian steroidogenesis using rat primary granulosa cells of immature female rat ovaries by focusing on the interaction with BMP-6 activity. Treatment with melatonin had no direct effect on FSH-induced progesterone or estradiol production by granulosa cells, and the results were not affected by the presence of co-cultured oocytes. In addition, synthesis of cAMP by granulosa cells was not significantly altered by melatonin treatment. To elucidate the interaction between activities of melatonin and BMPs, the effect of melatonin treatment on suppression of progesterone synthesis by BMP-6 was investigated. Interestingly, the inhibitory effect of BMP-6 on FSH-induced progesterone production was impaired by co-treatment with melatonin. Granulosa cells express higher levels of MT1 than MT2, and BMP-6 had no significant effect on MT1 expression in granulosa cells. However, BMP-6-induced Smad1/5/8 phosphorylation and Id-1 transcription were suppressed by melatonin, suggesting that melatonin has an inhibitory effect on BMP receptor signaling in granulosa cells. Although the expression levels of ALK-2, -6, ActRII and BMPRII were not affected by melatonin, inhibitory Smad6, but not Smad7, expression was upregulated by melatonin. Thus, melatonin plays a role in the regulation of BMP-6 signal intensity for controlling progesterone production in the ovary. These findings suggest that the effect of melatonin on maintenance of ovarian function is, at least in part, due to the regulation of endogenous BMP activity in granulosa cells.

Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

Canonical WNT Signaling Inhibits Follicle Stimulating Hormone Mediated Steroidogenesis in Primary Cultures of Rat Granulosa Cells

Beta-catenin (CTNNB1), a key component of wingless-type mouse mammary tumor virus integration site family (WNT) signaling, participates in follicle stimulated hormone-mediated regulation of estrogen (E2) production. The purpose of these studies was to determine if CTNNB1’s contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or a canonical member of the WNT signaling pathway, WNT3A, before co-culture and in the presence or absence of FSH for 24 h. Activation of the canonical WNT signaling pathway was determined by dose-dependent induction of Axin2 mRNA expression and stimulation of the CTNNB1/T cell factor promoter-reporter TOPflash. WNT pathway induction was demonstrated at doses of 50 and 500 ng/mL of WNT3A. Granulosa cells treated with WNT3A in combination with FSH had enhanced CTNNB1/T cell factor transcriptional activity above cells treated with WNT3A alone. Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified via quantitative PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased following FSH treatment. Co-incubation of WNT3A and FSH reduced the ability of FSH to stimulate steroidogenic enzymes and subsequent E2 and progesterone (P4) production. Concomitant activation of FSH and WNT pathways results in marked reduction of ovarian differentiation factors, LH receptor (Lhcgr) and inhibin-alpha (Inha). Therefore, WNT inhibits FSH target genes and steroid production associated with maturation and differentiation of the ovarian follicle.

Regulation of Expression and Function of Scavenger Receptor Class B, Type I (SR-BI) by Na+/H+ Exchanger Regulatory Factors (NHERFs)

Scavenger receptor class B, type I (SR-BI) binds HDL and mediates selective delivery of cholesteryl esters (CEs) to the liver, adrenals, and gonads for product formation (bile acids and steroids). Because relatively little is known about SR-BI posttranslational regulation in steroidogenic cells, we examined the roles of Na(+)/H(+) exchanger regulatory factors (NHERFs) in regulating SR-BI expression, SR-BI-mediated selective CE uptake, and steroidogenesis. NHERF1 and NHERF2 mRNA and protein are expressed at varying levels in model steroidogenic cell lines and the adrenal, with only low expression of PDZK1 (NHERF3) and NHERF4. Dibutyryl cyclic AMP decreased NHERF1 and NHERF2 and increased SR-BI mRNA expression in primary rat granulosa cells and MLTC-1 cells, whereas ACTH had no effect on NHERF1 and NHERF2 mRNA levels but decreased their protein levels in rat adrenals. Co-immunoprecipitation, colocalization, bimolecular fluorescence complementation, and mutational analysis indicated that SR-BI associates with NHERF1 and NHERF2. NHERF1 and NHERF2 down-regulated SR-BI protein expression through inhibition of its de novo synthesis. NHERF1 and NHERF2 also inhibited SR-BI-mediated selective CE transport and steroidogenesis, which were markedly attenuated by partial deletions of the PDZ1 or PDZ2 domain of NHERF1, the PDZ2 domain of NHERF2, or the MERM domains of NHERF1/2 or by gene silencing of NHERF1/2. Moreover, an intact COOH-terminal PDZ recognition motif (EAKL) in SR-BI is needed. Transient transfection of hepatic cell lines with NHERF1 or NHERF2 caused a significant reduction in endogenous protein levels of SR-BI. Collectively, these data establish NHERF1 and NHERF2 as SR-BI protein binding partners that play a negative role in the regulation of SR-BI expression, selective CE transport, and steroidogenesis.