Cultured Granulosa Cells Research Articles

Expression and regulation of stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) in equine and bovine preovulatory follicles

The interaction between stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) has been implicated in leukocyte attraction, tissue remodeling and angiogenesis. The objective of the present study was to characterize the expression and regulation of SDF1 and CXCR4 in equine follicles during the ovulatory process. Equine preovulatory follicles were isolated during estrus 0–39h after hCG treatment. Follicle wall preparations (theca interna with attached granulosa cells) and isolated preparations of granulosa cells and theca interna were obtained, and total RNA extracts were analyzed by RT-PCR/Southern blot. Results showed that levels of CXCR4 transcripts were induced by hCG in follicles at 36h post-hCG (P<0.05 vs 0h), with the induction observed in both granulosa and theca cells. Immunoblotting and immunohistochemical analyses confirmed an increase in CXCR4 protein in follicles after hCG treatment. In contrast, levels of SDF1 transcripts were very low in granulosa cells but high in theca interna cells throughout most of the ovulatory period. Studies in vivo performed with bovine preovulatory follicles collected 0–24h post-hCG revealed a marked and significant up-regulation of CXCR4 transcripts after hCG (P<0.05), as observed in equine follicles. A similar pattern of CXCR4 mRNA up-regulation was observed in cultures of bovine granulosa cells treated with forskolin (P<0.05). This forskolin-dependent induction of CXCR4 mRNA was suppressed by co-treatment with inhibitors of PKA, ERK1/2 and EGFR, and by the progesterone receptor antagonist RU486 (P<0.05), underscoring the contribution of multiple signaling pathways. In complementary studies, treatment of bovine granulosa cells with EGF or the hypoxia mimetic cobalt chloride significantly increased CXCR4 transcript levels, whereas co-treatment with forskolin and a CXCR4 antagonist repressed the expression of several ovulation-related genes. Collectively, this study describes for the first time the gonadotropin-dependent up-regulation of CXCR4 transcript in ovarian follicles of large monoovulatory species, provides some insights into the regulation of CXCR4 gene expression in granulosa cells, and identifies a potential link between follicular SDF1/CXCR4 activation and the regulation of ovulation-related genes.

MicroRNA miR-513a-3p acts as a co-regulator of luteinizing hormone/chorionic gonadotropin receptor gene expression in human granulosa cells

The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is essential for normal male and female reproductive processes. The spatial and temporal LHCGR gene expression is controlled by a complex system of regulatory mechanisms which are crucial for normal physiological function, especially during the female cycle. In this study, we aimed to elucidate whether microRNAs are involved in this network and play a role in regulating LHCGR expression. Computational analysis predicted that miR-513a-3p interacts with the LHCGR mRNA via three binding sites located in the 3′UTR region, enabling a synergistic action. Moreover, using a luciferase-based reporter assay we found that miR-513a-3p targets the LHCGR, resulting in a significant down-regulation of its expression. In human primary granulosa cell cultures we detected a dynamic, inversely associated expression pattern of miR-513a-3p and the LHCGR. In addition, transfection with miR-513a-3p or its specific inhibitor led to a down- or up-regulation at the LHCGR mRNA level, respectively. An increased amount of miR-513a-3p resulted in the down-regulation of the LHCGR mRNA, reflected by the attenuation of cAMP synthesis after hormonal stimulation. In conclusion, these data demonstrate that miR-513a-3p is involved in the control of the LHCGR expression by an inversely regulated mechanism at the post-transcriptional level and show for the first time that this kind of post-transcriptional process contributes to the multifaceted system of the human LHCGR regulation.

Constitutive and follicle-stimulating hormone-induced action of somatostatin receptor-2 on regulation of apoptosis and steroidogenesis in bovine granulosa cells

In the present study, we employed primary bovine culture of granulosa cells (GCs) as a cellular model to study the potential involvement of somatostatin receptor 2 (SSTR2) in ovarian function. The results showed that bovine GCs expressed SST2 receptor and further found that SSTR2 was possibly regulated by follicle-stimulating hormone (FSH), as a significant increase in protein level of SSTR2 was observed in FSH-treated GCs. For further analysis, endogenous SSTR2 expression was disrupted using small inhibitory RNA (siRNA) and the efficacy of differential silencing of endogenous SSTR2 expression was measured both at transcriptional and translational levels. Transient blockage of SSTR2 evidenced its constitutive action on GCs, as it significantly increased level of cAMP (2.4-folds) and basal progesterone production (∼2-fold, P<0.05) with significant increase (P<0.05) in mRNA levels of StAR and P450ssc without altering estradiol concentration and aromatase mRNA expression. Furthermore, silencing of SSTR2 reduced GCs apoptosis (52.5%, P<0.05) and increased cell proliferation, which was further corroborated by up-regulation in protein expressions of B-cell leukemia/lymphoma 2 (Bcl-2), inhibition of caspase3 and mRNA level of bcl2-associated-X protein (Bax). These results provide evidence that SSTR2 subtype controls GCs apoptosis, proliferation and hormonal secretions through selective constitutive action, independently of somatostatin (SST). Given the local inhibitory actions of SSTR2 on the gonads, we further found that apoptosis in ssRNAi-2 transfected cells decreased (6.8% vs 1.9%, P<0.05) more strongly on FSH treatment. Apoptotic protein expressions and steroid hormone mRNA levels were correlated with a relative decrease in apoptosis and increase in progesterone production. Our results suggest that SSTR2 may play a crucial role as a local inhibitor of FSH action on GCs apoptosis and steroidogenesis.

Non-canonical progesterone signaling in granulosa cell function

It has been known for over 3 decades that progesterone (P4) suppresses follicle growth. It has been assumed that P4 acts directly on granulosa cells of developing follicles to slow their development, as P4 inhibits both mitosis and apoptosis of cultured granulosa cells. However, granulosa cells of developing follicles of mice, rats, monkeys, and humans do not express the A or B isoform of the classic nuclear receptor for P4 (PGR). By contrast, these granulosa cells express other P4 binding proteins, one of which is referred to as PGR membrane component 1 (PGRMC1). PGRMC1 specifically binds P4 with high affinity and mediates P4's anti-mitotic and anti-apoptotic action as evidenced by the lack of these P4-dependent effects in PGRMC1-depleted cells. In addition, mice in which PGRMC1 is conditionally depleted in granulosa cells show diminished follicle development. While the mechanism through which P4 activation of PGRMC1 affects granulosa cell function is not well defined, it appears that PGRMC1 controls granulosa cell function in part by regulating gene expression in T-cell-specific transcription factor/lymphoid enhancer factor-dependent manner. Clinically, altered PGRMC1 expression has been correlated with premature ovarian failure/insufficiency, polycystic ovarian syndrome, and infertility. These collective studies provide strong evidence that PGRMC1 functions as a receptor for P4 in granulosa cells and that altered expression results in compromised reproductive capacity. Ongoing studies seek to define the components of the signal transduction cascade through which P4 activation of PGRMC1 results in the regulation of granulosa cell function.

Establishment and validation of a model for non-luteinized human mural granulosa cell culture

Cell culture techniques of human mural granulosa cells (MGCs) serve as a major in vitro tool. However, the use of luteinized MGCs has major limitations due to their luteinized state. Our aim was to establish a standardized protocol for the culture of MGCs as a model for different stages of folliculogenesis. We showed that early-non-luteinized, preovulatory-non-luteinized and luteal-MGCs have distinct gene expression pattern. After 4days of incubation of luteinized-MGCs, ovulatory genes mRNA’s achieve expression levels similar to the early non-luteinized follicles. FSH stimulation for 48h of these 4days cultured MGCs showed ovulatory genes mRNA’s expression similar to the pre-ovulatory non-luteinized follicles. These FSH-stimulated cells responded to hCG stimulation in a pattern similar to the response of pre-ovulatory follicles.This novel model may provide a standardized research tool for delineation of the molecular processes occurring during the latter stages of follicular development in the human ovary.

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.

Comparison of Medium Supplementation on Proliferation and Hormone Production of Bovine Granulosa Cells in a Defined Culture System

Aims: The objective of this study was to develop a serum -free bovine granulosa cell (GC) culture system in which estradiol (E2) production could be maintained in a defined media, with polyvinyl alcohol (PVA), insulin and insulin

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for Original Research Article British Microbiology Research Journal, 4(4): 418-427, 2014 419 their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers

BackgroundHeifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.ResultsGranulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.ConclusionsThese results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

CANDLES, an assay for monitoring GPCR induced cAMP generation in cell cultures

BackgroundG protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to GαS stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells.ResultsCANDLES (C yclic A MP iN direct D etection by L ight E mission from S ensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement.ConclusionsHere we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells.

Sphingosine-1-Phosphate may Decrease the Cytotoxic Effect of Doxorubicin on Human Granulosa Cells

Context: The increase in malignancy of young women in the recent decades, combined with a significant improvement in long term survival after gonadotoxic chemotherapy, have brought about a ubiquitous interest in preservation of fertility in these young patients. The present study examines the effects of Sphingosine-1-Phosphate (S1P) on primary human granulosa cell cultures in-vitro as a possible protecting factor against Doxorubicin (DOX) and Cyclophosphamide associated toxicity. Understanding cytotoxic effects and gonadotoxicity in human luteinized Granulosa Cells (GC) may contribute to our understanding and preventing follicle loss. Study objective: To examine the possible protective effect of S1P on chemotherapy induced gonadotoxicity, in human luteinized Granulosa Cells (GCs). Design: Human GC’s were donated by women undergoing follicular aspiration for in vitro fertilization (IVF), after informed consent and institutional approval by ethics committee (IRB, Helsinki). The GCs were separated from RBC’s by centrifugation on ficoll and plated on multiwell plates for Lactate Dehydrogenase (LDH) assay, and on 6 well plates for flow cytometry. Each experiment was conducted in triplicates and repeated at least three times. Results: S1P significantly protected GCs against Doxorubicin (DOX) toxicity, but inconsistently against Cyclophosphamide. Conclusion: S1P may minimize the gonadotoxic effect of chemotherapy on human luteinized granulosa cells.

Hypoxia Promotes Progesterone Synthesis During Luteinization in Bovine Granulosa Cells

To determine whether hypoxia has an effect on luteinization, we examined the influence of hypoxia on a model of bovine luteinizing and non-luteinizing granulosa cell culture. The granulosa cells were obtained from small antral follicles (≤ 6 mm in diameter). To induce luteinization, the cells were treated for 24 h with insulin (2 µg/ml), forskolin (10 µM) or insulin in combination with forskolin at 20% O2. After 24 h, progesterone (P4) production was higher in the treated cells, which we defined as luteinizing granulosa cells, than in non-treated cells, which we defined as non-luteinizing granulosa cells. P4 production by non-luteinizing granulosa cells was not affected by hypoxia (24 h at 10% and 5% O2), while P4 production by granulosa cells treated with insulin in combination with forskolin was significantly increased under hypoxia (24 h at 10% and 5% O2). Because hypoxia affected P4 production by the luteinizing granulosa cells but not by the non-luteinizing granulosa cells, hypoxia seems to promote P4 production during, rather than before, luteinization. In the cells treated with insulin in combination with forskolin, mRNA and protein expression of steroidogenic acute regulatory protein (StAR) and protein expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) increased under 10% O2, while mRNA and protein expressions of key protein and enzymes in P4 biosynthesis did not increase under 5% O2. The overall results suggest that hypoxia plays a role in progressing and completing the luteinization by enhancing P4 production through StAR as well as 3β-HSD expressions in the early time of establishing the corpus luteum.

Luteinizing hormone, insulin like growth factor-1, and epidermal growth factor stimulate vascular endothelial growth factor production in cultured bubaline granulosa cells

The objective of this study was to characterize in vitro expression and secretion of vascular endothelial growth factor (VEGF) in bubaline granulosa cells (GC), grown in serum containing media supplemented with luteinizing hormone (LH), insulin like growth factor-1 (IGF-1), and epidermal growth factor (EGF) at three different doses and time durations. GCs were collected from ovarian follicles of varying diameters [Gp-I (small), 4–6mm; Gp-II (medium), 7–9mm; Gp-III (large), 10–13mm; Gp-IV (pre-ovulatory), >13mm]. In general, each of the three treatments resulted in a dose as well as time dependent increase in the mRNA expression and secretion of VEGF in the cultured GCs of Gp-IV follicles. These results were well supported by our observations on immunocytochemistry in Gp IV granulosa cell culture (GCC). We also looked into the expression dynamics of an anti-apoptotic factor – proliferating cellular antigen (PCNA) and a pro-apoptotic factor – Bcl-2-associated X protein (BAX) in GCs of Gp IV follicles on treatments with LH, IGF-1, and EGF to evaluate their cytoprotective/anti-apoptotic property. Relative expressions of PCNA and BAX showed a mutually opposite trend with the PCNA expression increasing and BAX expression decreasing with increase in dose and time to reach the zenith (P<0.05) and nadir (P<0.05) at the highest dose(s) at the maximum time duration (72h) for PCNA and BAX respectively on treatment with all the three factors. Thus, it can be concluded that LH, IGF-1, and EGF treatments have a cytoprotective/anti-apoptotic effect and stimulate VEGF production in granulosa cells of bubaline pre-ovulatory follicles.

Role of microRNA-136-3p on the Expression of Luteinizing Hormone-Human Chorionic Gonadotropin Receptor mRNA in Rat Ovaries1

MicroRNAs (miRNAs) are small noncoding RNAs that interact with mRNAs and trigger either translation repression or RNA cleavage of target genes. In this study, we investigated whether miRNA was involved in down-regulation of the luteinizing hormone receptor (LHR) in rat ovaries. An miRNA microarray was used to analyze the overall miRNA expression profile of rat ovaries in association with the down-regulation of LHR mRNA. We found that 23 miRNAs were highly expressed during this period. Combining these results with data from a bioinformatics database, clustering analysis led us to focus on miR-136-3p for further analysis. In both in vivo and in vitro studies, miR-136-3p expression levels were increased at 6 h after human chorionic gonadotropin (hCG) administration, concurrent with down-regulation of LHR mRNA. Moreover, transfection of cultured granulosa cells with miR-136-3p resulted in a significant decrease in LHR mRNA levels in comparison with those of cells transfected with negative control. In contrast, transfection with a miR-136-3p inhibitor increased LHR mRNA levels. Finally, cotransfection of granulosa cells with a miR-136-3p inhibitor and a reporter vector containing the 3'-untranslated region (UTR) of LHR mRNA and Renilla luciferase coding sequence revealed that miR-136-3p bound directly to the 3'-UTR of LHR mRNA. These data demonstrated that miR-136-3p participated in the down-regulation of LHR mRNA by binding directly to LHR mRNA.

Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.

Ovarian, metabolic and endocrine indexes in dairy cows with different body condition scores

1. In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes S.K. Tripathi, M. Farman, S. Nandi, S Mondal, PSP. Gupta, V.Girish Kumar Animal Reproduction Science CrossRef

Prostate Androgen-Regulated Mucin-Like protein 1: A Novel Regulator of Progesterone Metabolism

The LH surge reprograms preovulatory follicular cells to become terminally differentiated luteal cells which produce high levels of progesterone and become resistant to apoptosis. PARM1 (prostate androgen regulated mucin-like protein 1) has been implicated in cell differentiation and cell survival in nonovarian cells, but little is known about PARM1 in the ovary. This study demonstrated that the LH surge induced a dramatic increase in Parm1 expression in periovulatory follicles and newly forming CL in both cycling and immature rat models. We further demonstrated that hCG increases Parm1 expression in granulosa cell cultures. The in vitro up-regulation of Parm1 expression was mediated by hCG-activated multiple signaling pathways and transcriptional activation of this gene. Parm1 knockdown increased the viability of cultured granulosa cells but resulted in a decrease in progesterone levels. The inhibitory effect of Parm1 silencing on progesterone was reversed by adenoviral mediated add-back expression of Parm1. Parm1 silencing had little effect on the expression of genes involved in progesterone biosynthesis and metabolism such as Scarb1, Ldlr, Vldlr, Scp2, Star, Cyp11a1, Hsd3b, and Srd5a1, while decreasing the expression of Akr1c3. Analyses of culture media steroid levels revealed that Parm1 knockdown had no effect on pregnenolone levels, while resulting in time-dependent decreases in progesterone and 20α-dihydroprogesterone and accelerated accumulation of 5α-pregnanediol. This study revealed that the up-regulation of Parm1 expression promotes progesterone and 20α-dihydroprogesterone accumulation in luteinizing granulosa cells by inhibiting progesterone catabolism to 5α-pregnanediol. PARM1 contributes to ovulation and/or luteal function by acting as a novel regulator of progesterone metabolism.

Expression, Regulation, and Promoter Activation of Vanin-2 (VNN2) in Bovine Follicles Prior to Ovulation1

Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum‐free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre‐luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from 10 women undergoing treatment for assisted reproduction. The GC were cultured in α‐MEM containing IGF‐I, STI, androstenedione, PVP‐40 (chemically defined medium) or TCM‐199 containing FSH/serum. After 48, 96 and 144h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (Electron Microscopy). Data were analyzed by Anova and linear mixed‐effects regression (SAS version 9.0). Cells cultured in α‐MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

Characterization of the Gonadotropin Releasing Hormone Receptor (GnRHR) Expression and Activity in the Female Mouse Ovary

GnRH agonists (GnRHa) are increasingly used for fertility preservation in women undergoing gonadotoxic chemotherapy. However, the protective mechanisms of action for these compounds have not yet been elucidated. In this study, we aimed to determine whether GnRHa have a direct effect on ovarian granulosa cells. GnRH receptor (GnRHR) expression was determined in mouse somatic and gonadal tissues including granulosa/cumulus cells and oocytes using quantitative RT-PCR and immunohistochemistry. Granulosa cells were isolated from mouse ovaries primed with pregnant mare serum gonadotropin. Response to GnRHa in cultured granulosa cells was assessed by determining the increase of intracellular cAMP and by assessing phosphorylation of downstream mediators of GnRH signaling: ERK and p38. To measure intracellular cAMP in our system, the cells were transfected with a cAMP-responsive luciferase reporter plasmid and stimulated with GnRHa. For all experiments, pituitary tissue and/or the αT3-1 mouse pituitary cell line were used as controls. GnRHR mRNA and protein were detected in mouse ovaries, granulosa/cumulus cells, and oocytes. After GnRHa stimulation at various time intervals, we were unable to detect a cAMP increase or activation of the ERK or p38 signaling pathway in cultured primary mouse granulosa cells, whereas activation was detected in the control αT3-1 mouse pituitary cells. In this study, we have not detected activation of the canonical GnRH signaling pathways in mouse ovarian somatic cells. Our findings suggest that the mechanism of action of GnRHa in the ovary is either below the detection level of our experimental design or is different from that in the pituitary.