Isolated Granulosa Cells Research Articles

Dysfunction of Human Estrogen Signaling as a Novel Molecular Signature of Polycystic Ovary Syndrome

Estradiol (E2) is a major hormone-controlling folliculogenesis whose dysfunction may participate in polycystic ovary syndrome (PCOS) infertility. To determine whether both the concentration and action of E2 could be impaired in non-hyperandrogenic overweight PCOS women, we isolated granulosa cells (GCs) and follicular fluid (FF) from follicles of women undergoing ovarian stimulation (27 with PCOS, and 54 without PCOS). An analysis of the transcript abundance of 16 genes in GCs showed that androgen and progesterone receptor expressions were significantly increased in GCs of PCOS (by 2.7-fold and 1.5-fold, respectively), while those of the steroidogenic enzymes CYP11A1 and HSD3B2 were down-regulated (by 56% and 38%, respectively). Remarkably, treatment of GC cultures with E2 revealed its ineffectiveness in regulating the expression of several key endocrine genes (e.g., GREB1 or BCL2) in PCOS. Additionally, a comparison of the steroid concentrations (measured by GC/MS) in GCs with those in FF of matched follicles demonstrated that the significant decline in the E2 concentration (by 23%) in PCOS FF was not the result of the E2 biosynthesis reduction. Overall, our study provides novel hallmarks of PCOS by highlighting the ineffective E2 signaling in GCs as well as the dysregulation in the expression of genes involved in follicular growth, which may contribute to aberrant folliculogenesis in non-hyperandrogenic women with PCOS.

P–713 The effect of aging and polycystic ovarian syndrome on the maturation and apoptosis of granulosa cells from women undergoing controlled ovarian hyperstimulation with recombinant hFSH

Abstract Study question What is the effect of biological aging and polycystic ovarian syndrome (PCOS) on steroidogenic and apoptotic pathways of granulosa cells isolated from women undergoing IVF? Summary answer This analysis revealed that aging as well as PCOS influence the response of women to ovarian stimulation, affecting steroidogenesis and apoptosis on granulosa cells. What is known already Infertility and subfertility affect a significant proportion of humans worldwide. Age-related factors in women lead to a decrease in the ovarian oocyte reserve, and PCOS, a pathological condition characterized by anovulation and hyperandrogenism, are the two most common reasons of subfertility. Despite the progress in recent years, the causes are still ill-defined, and there is a strong need for interventions to ensure good quality oocytes in healthy aging, as well as in PCOS women. Gene expression analyses of granulosa cells isolated during oocyte retrieval are highly informative for understanding the communication between oocytes and follicular cells and optimizing IVF outcomes. Study design, size, duration This report is part of an ongoing study starting from June 2020. 42 women undergoing IVF/ICSI at the University Hospital of Ioannina, Greece, volunteered to participate with informed consent. Participants were divided into three groups: 30- to 35-year-old patients with PCOS, 30- to 35-year-old non-PCOS women, and older women aged 38- to 42-year-old. All participants received 150 – 300IU/d recombinant hFSH. Participants/materials, setting, methods: Granulosa cells (GC) were collected from the follicular fluid at the day of oocyte retrieval using the protocol described by Ferrero et al., 2012 with modifications that allow us to isolate nearly pure populations, also from small or very few follicles. GC purity was determined by FACS and immunocytochemistry. Reverse Transcription-quantitative-PCR was employed to correlate the expression of steroidogenesis- and apoptosis-related genes with the clinical characteristics of each participant, including FSH and progesterone/estradiol serum levels. Main results and the role of chance FACS and immucytochemistry analysis using specific markers of granulosa cells, like FSHR, as well as leucocyte and erythrocyte cell markers (CD45 and Glycophorin) confirmed the purity of the isolated granulosa cell populations. No differences were found in hormone serum levels between 30- to 35-year-old PCOS (Group A) and non-PCOS women of the same age (Group B) whereas progesterone and estradiol serum levels were lower in 38- to 42-year-old women (Group C) compared to group B. RT-qPCR analysis of maturation-related genes (ZP3 and AHR) revealed the presence of more immature follicles in Group A compared to the other groups. The granulosa cells from women of group A were characterized by increased expression of genes involved in steroidogenic pathways (such as FSHR, CYP17A1) compared to group B, as well as of the genes for the estradiol and progesterone receptors (ESR1 and PGRMC1, respectively), and by decreased levels of the apoptosis-related genes BAX and BCL-xL. Importantly, the granulosa cells from the women of Group C were characterized by lower mRNA levels of steroidogenesis-related genes, as well as of ESR1, and by increased apoptosis as revealed by the increased ratio of BAX/BCL2 transcripts, compared to Group A. Limitations, reasons for caution The presented results of gene expression analysis are based on preliminary data as the number of participants is small (6 women per group), but they are part of an ongoing study where the number of participants is increasing in order to improve quantification and reproducibility of our results. Wider implications of the findings: Potential differences in signaling pathways that are crucial for the maturation of granulosa cells and follicles during biological aging or in pathological conditions such as PCOS are expected to provide valuable information concerning the response of individual women to gonadotropin stimulation and might help to design more personalized therapeutic strategies. Trial registration number Not applicable

SOD2 deficiency-induced oxidative stress attenuates steroidogenesis in mouse ovarian granulosa cells

This study investigated the effects of SOD2 (MnSOD)-deficiency-induced excessive oxidative stress on ovarian steroidogenesis in vivo and isolated and cultured granulosa cells using WT and Sod2+/- mice. Basal and 48 h eCG-stimulated plasma progesterone levels were decreased ~50% in female Sod2+/- mice, whereas plasma progesterone levels were decreased ~70% in Sod2+/- mice after sequential stimulation with eCG followed by hCG. Sod2+/- deficiency caused about 50% reduction in SOD2 activity in granulosa cells. SOD2-deficiency also caused a marked reduction in progestins and estradiol in isolated granulosa cells. qRT-PCR measurements indicated that the mRNA expression levels of StAR protein and steroidogenic enzymes are decreased in the ovaries of Sod2+/- mice. Further studies showed a defect in the movement of mobilized cytosolic cholesterol to mitochondria. The ovarian membrane from Sod2+/- mice showed higher susceptibility to lipid peroxidation. These data indicates that SOD2-deficiency induced oxidative stress inhibits ovarian granulosa cell steroidogenesis primarily by interfering with cholesterol transport to mitochondria and attenuating the expression of Star protein gene and key steroidogenic enzyme genes.

The Mechanism of Endoplasmic Reticulum Stress and the Cyclin D2 Expression on Ovarian Granulosa Cell Cycle in Infertile Women with Endometriosis

The endometriosis related to secretory cell namely granulosa cell provides its important organelle of endoplasmic reticulum. This study aimed to analyze the influence of TNF-α against the characteristic of granulosa cell, especially the function in its organelle of endoplasmic reticulum and the cell cycle. This was an observed cross-sectional study. Samples were found by laparoscopy at the time of 17 mm sized follicle was detected. The assessment of peritoneal fluid’s TNF-α level was done by ELISA, the marker of mRNA-GRP78, eIF2α, and CHOP taken from isolated granulosa cell were assessed by qPCR, and flow cytometry was used to observe cell cycle while Western blotting spent to evaluate Cyclin D2. We observed 13 women with endometriosis and 4 women with non-endometriosis. The study showed that peritoneal fluid TNF-α was higher in endometriosis group compared to non-endometriosis (48.66±3.9 vs 37.6±3.8 ng/mL, p0.05) in endometriosis women group. The granulosa cell’s cycle showed the higher result on G0/G1 phase but lowering S/M phase on endometriosis group compared to control. It seemed the influence of peritoneal fluid TNF-α occurred against GRP78. Endometriosis women having higher TNF-α level suggested to lead ER stress were playing the important role in the granulosa cell cycle.

Vitamin D3 affects mitochondrial biogenesis through mitogen‐activated protein kinase in polycystic ovary syndrome mouse model

Polycystic ovarian syndrome (PCOS) is a disorder characterized by oligomenorrhea, anovulation, and hyperandrogenism. Altered mitochondrial biogenesis can result in hyperandrogenism. The goal of this study was to examine the effect of vitamin D3 on mitochondrial biogenesis of the granulosa cells in the PCOS-induced mouse model. Vitamin D3 applies its effect via the mitogen-activated pathway kinase-extracellular signal-regulated kinases (MAPK-ERK1/2) pathway. The PCOS mouse model was induced by the injection of dehydroepiandrosterone (DHEA). Isolated granulosa cells were subsequently treated with vitamin D3, MAPK activator, and MAPK inhibitor. Gene expression levels were measured using real-time polymerase chain reaction. MAPK proteins were investigated by western blot analysis. We also determined reactive oxygen species (ROS) levels with 2', 7'-dichlorofluorescein diacetate. Mitochondrial membrane potential (mtMP) was also measured by TMJC1. Mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-α and nuclear respiratory factor), antioxidant (superoxide dismutase, glutathione peroxidase, and catalase), and antiapoptotic (B-cell lymphoma-2) genes were upregulated in the PCOS mice that treated with vitamin D3 compared with the PCOS mice without any treatment. Vitamin D3 and MAPK activator-treated groups also reduced ROS levels compared with the nontreated PCOS group. In summary, vitamin D3 and MAPK activator increased the levels of mitochondrial biogenesis, MAPK pathway, and mtMP markers, while concomitantly decreased ROS levels in granulosa cells of the PCOS-induced mice. This study suggests that vitamin D3 may improve mitochondrial biogenesis through stimulation of the MAPK pathway in cultured granulosa cells of DHEA-induced PCOS mice which yet to be investigated.

Tyrosine nitrations impaired intracellular trafficking of FSHR to the cell surface and FSH-induced Akt-FoxO3a signaling in human granulosa cells.

Many infertile women suffered from poor ovarian response, and increased reactive oxygen species with age might mediate the poor ovarian response to FSH. In this study, we collected follicular fluids and isolated granulosa cells from female patients. Increased levels of peroxynitrite, tyrosine nitrations of FSH receptor (FSHR) and apoptosis were obviously detectable with decreased FSHR protein expressions in granulosa cells of the poor ovarian responders. In KGN (a human ovarian granulosa cell line) cells, exogenous peroxynitrite could sequester FSHR in the cytoplasm, and these dislocated FSHR might suffer from proteasome-mediated degradations. Here, we identified four peroxynitrite-mediated nitrated tyrosine residues of FSHR. Site-directed mutagenesis of FSHR revealed that Y626 was pivotal for intracellular trafficking of FSHR to the cell surface. Akt-induced inactivation of FoxO3a was required for the repression of FSH on granulosa cell apoptosis. However, peroxynitrite impaired FSH-induced Akt-FoxO3a signaling, while FSHR-Y626A mutant took similar effects. In addition, FoxO3a knockdown indeed impaired FSH-mediated cell survival, while FoxO3a-S253A mutant reversed that significantly.

Activin effects on follicular growth in in vitro preantral follicle culture.

As the follicular environment transits from being activin dominant to inhibin dominant during folliculogenesis, it is assumed that activin plays an important role in the early stage of follicular growth. We examined the effects of activin on morphological, biochemical and molecular changes in isolated preantral follicles. Preantral follicles were mechanically isolated from 14-day old female C57BL/6 mice. Each follicle was cultured and observed for 14 days usingan in vitro follicle culture system containing FSH, FSH + activin A and FSH + inhibin in the culture medium. We subsequently examined FSH receptor (FSH-R) mRNA expression in isolated follicle cultures with or without activin on days 0 and 2. Activin was observed to significantly stimulate follicle enlargement on days 2, 4, 6 and 8, accelerate morphological changes and increase estradiollevels in culture medium on days 4, 12 and 14. In contrast, inhibin did not alter follicular growth. Additionally, activin stimulated the expression of FSH-R mRNA in isolated granulosa cells. It was demonstrated that activin stimulated the growth of preantral follicles, mainly during the early stage of folliculogenesis, by inducing FSH-R expression, in an isolated follicle culture system. J. Med. Invest. 66 : 165-171, February, 2019.

Inhibition of catalase activity with 3-amino-1,2,4-triazole intensifies bisphenol A (BPA)-induced toxicity in granulosa cells of female albino rats.

Exposure to bisphenol A (BPA), an endocrine disruptor and environmental toxicant, is associated with adverse estrogenic effects in both humans and wildlife species. Because the effects of BPA on the ovary at the cellular level are incompletely understood, the present study was designed to investigate the underlying mechanism of granulosa cell injury following BPA exposure. Eight-week-old female Wistar rats were treated with BPA (25 mg/kg BW/day for 9 days, intraperitonially) with or without pretreatment of the catalase-specific blocker 3-amino-1,2,4-triazole (ATZ; 1 g/kg BW/day for 5 days, intraperitonially). Different oxidative and antioxidant stress parameters, pro-inflammatory cytokines, and hormonal levels were measured. Catalase expression in isolated granulosa cells was analyzed by Western blot. There were noticeable increases in both nitric oxide and lipid peroxidation levels in the granulosa cells of the BPA-treated group with or without pretreatment with ATZ. Compared with the controls, BPA exposure resulted in a significant increase in pro-inflammatory cytokine levels that was further increased following pretreatment with ATZ. Results of the hormonal assays clearly showed a significant decrease in both estrogen and progesterone levels. In contrast, there was a significant increase in both serum follicle-stimulating hormone and luteinizing hormone levels following BPA exposure, with or without ATZ pretreatment. Results of Western blot analysis demonstrated decreased expression of catalase in the BPA-treated group and a further decrease in expression in the group treated with both BPA and ATZ. Our data suggest that catalase plays a role in mediating reproductive damage to granulosa cells exposed to BPA.

Granulosa cells from human primordial and primary follicles show differential global gene expression profiles.

Can novel genetic candidates involved in follicle dormancy, activation and integrity be identified from transcriptomic profiles of isolated granulosa cells from human primordial and primary follicles? The granulosa cell compartment of the human primordial and primary follicle was extensively enriched in signal transducer and activator of transcription 3 (STAT3) and cAMP-response element binding protein (CREB) signalling, and several other putative signalling pathways that may also be mediators of follicle growth and development were identified. Mechanistic target of rapamycin kinase (mTOR) signalling and the factors Forkhead Box L2 (FOXL2) and KIT proto-oncogene receptor tyrosine kinase (KITL) may be involved in defining the early steps of mammalian follicular recruitment through complex bidirectional signalling between the oocyte and granulosa cells. cAMP/protein kinase K (PKA)/CREB signalling is a feature of FSH-induced regulation of granulosa cell steroidogenesis that is essential to normal human fertility. A class comparison study was carried out on primordial follicles (n = 539 follicles) and primary follicles (n = 261) follicles) donated by three women having ovarian tissue cryopreserved before chemotherapy. RNA samples from isolates of laser capture micro-dissected oocytes and follicles from the primordial and primary stage, respectively, were sequenced on the HiSeq Illumina platform. Data mapping, quality control, filtering, FPKM (fragments per kilobase of exon per million) normalization and comparisons were performed. The granulosa cell contribution in whole follicle isolates was extracted in silico. Modelling of complex biological systems was performed using Ingenuity Pathway Analysis (IPA). For validation of transcriptomic findings, we performed quantitative RT-PCR of selected candidate genes. Furthermore, we interrogated the in situ localization of selected corresponding proteins using immunofluorescence. Our differentially expressed gene analysis revealed a number of transcripts in the granulosa cells to be significantly down- (736 genes) or up- (294 genes) regulated during the human primordial-to-primary follicle transition. The IPA analysis revealed enriched canonical signalling pathways not previously associated with granulosa cells from human primordial and primary follicles. Immunofluorescent staining of human ovarian tissue explored the intra-ovarian localization of FOG2, and FOXL2, which revealed the presence of forkhead box L2 (FOXL2) in both oocytes and granulosa cells in primary follicles, with a more enriched staining in the granulosa cells in primary follicles. Friend of GATA 2 (FOG2) stained strongly in oocytes in primordial follicles, with a shift towards granulosa cell as follicle stage advanced. http://users-birc.au.dk/biopv/published_data/ernst_et_al_GC_2017/. This is a descriptive study, and no functional assays were employed. The study was based on a limited number of patients, and it is acknowledged that natural biological variance exists in human samples. Strict filters were applied to accommodate the in silico extraction of the granulosa cell contribution. In support of this, quantitative RT-PCR was used to confirm selected candidate genes, and immunofluorescent staining was employed to interrogate the intra-ovarian distribution of selected corresponding proteins. Moreover, it is unknown whether the primordial follicles analysed represent those still in the resting pool, or those from the cohort that have entered the growing pool. We present, for the first time, a detailed description of global gene activity in the human granulosa cell compartment of primordial and primary follicles. These results may be utilized in the development of novel clinical treatment strategies aimed at improving granulosa cell function. E.H.E. was supported by the Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.L.H. was supported by a grant from Fondens til Lægevidenskabens Fremme and Kong Christian Den Tiendes Fond. No authors have competing interests to declare.

Effect of Threonine on the Proliferation and Viability of Porcine Granulosa Cells

The proliferation and viability of granulosa cells directly affects the development and function of follicles. The current study examined the effect of threonine on cell morphology and cell cycle of porcine granulosa cells. It would help to understand hormone secretion, reproductive health, and disease prevention and treatment in females. Granulosa cells were isolated from pig ovaries, and different passages of cells were stained and identified by FSHR antibody. Threonine was added to the culture medium, and cells were stained with HE to examine the changes in granulosa cell morphology. Cell viability was measured using the CCK-8 assay, and cell cycle phase was determined by flow cytometry. Immunofluorescence staining showed >98% FSHR-positive cells demonstrating that the isolated granulosa cells were >95% pure. HE-staining of different passages of granulosa cells showed that cells from the second to fourth passage have stable morphology, whereas those from the fifth and subsequent passages began to degenerate and degrade. CCK-8 results showed that =0.5 mM threonine can significantly enhance granulosa cell viability after 0.5, 1 and 2 h of treatment, and cells treated with 1 mM threonine for 4 h had the highest cell viability. In contrast, 5 and 10 mM threonine significantly reduced cell viability, indicating that only a certain amount of threonine can enhance granulosa cell viability. Flow cytometry results demonstrated that the percentage of G1 phase cells was higher in the 0.1, 0.5 and 1 mM groups than in the control group, but lower in the 10 mM group than in control group. While the 5 mM group had the highest percentage of S phase cells, the 10 mM group had no S phase cells. In contrast, the percentage of G2 phase cells was much higher in the 10 mM group than in the control group and other dose groups, indicating that an appropriate amount of threonine can promote granulosa cell proliferation, but excessive threonine can inhibit cell proliferation.

Effect of Vitamin D3 on Biosynthesis of Estrogen in Porcine Granulosa Cells via Modulation of Steroidogenic Enzymes

Vitamin D3 is a fat-soluble secosteroid responsible for enhancing intestinal absorption of calcium, iron, and other materials. Vitamin D3 deficiency, therefore, can cause health problems such as metabolic diseases, and bone disorder. Female sex hormones including estrogen and progesterone are biosynthesized mainly in the granulosa cells of ovary. In this study, we isolated granulosa cells from porcine ovary and cultured for the experiments. In order to examine the effect of vitamin D3 on the ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by real-time PCR and Western blot assay. The production of estrogen from the granulosa cells was also measured by the ELISA assay. Genes associated with follicle growth were not significantly altered by vitamin D3. However, it increases expression of genes involved in the estrogen-biosynthesis. Further, estrogen concentrations in porcine granulosa cell-cultured media increased in response to vitamin D3. These results showed that vitamin D3 is a powerful regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Metabolic state defines the response of rabbit ovarian cells to leptin

Leptin is a hormone that mediates the effect of the metabolic state on several biological functions, including reproduction. Leptin affects reproductive functions via alterations in the release of hormonal regulators. However, the extent to which caloric restriction (CR) can affect the complex processes of reproduction by other mechanisms, such as altering ovarian functions via direct binding/response to leptin, is unknown. Therefore, the aim of the present study was to show basic ovarian cell functions and CR on the response of ovarian cells to leptin. Female rabbits were subjected to 50% CR restriction for 10days before ovulation. On the day of ovulation, both control and CR animals were sacrificed. Isolated granulosa cells were cultured for 2days with and without leptin (100ng/ml), and the accumulation of various markers was evaluated using immunocytochemistry; i.e., cell proliferation (PCNA and cyclin B1), apoptosis (bax), MAP/ERK1,2 kinase (MAPK), protein kinase A (PKA), and IGF-I. In addition, the release of IGF-I and estradiol (E2) by cells cultured with and without leptin (1, 10, 100, 1000, or 10,000ng/ml) was assessed by radioimmunoassay (RIA). In the granulosa cells of control animals, leptin promoted cyclin B1, MAPK, and PKA accumulation, but not that of PCNA, and reduced bax and IGF-I accumulation. These cells responded to leptin by increased IGF-I, but not E2 release. In cells of CR animals, leptin increased cyclin B1 accumulation, but decreased PCNA, MAPK, and IGF-I expression. Bax and PKA were not affected. Leptin resulted in a decrease in IGF-I release. CR modulated the influence of leptin on E2 release dose dependently, i.e., E2 increased at 10 and decreased at 10,000ng/ml. Therefore, CR modified the influence of leptin on PCNA, E2, bax, PKA, MAPK, and IGF-I release, but it did not change the effect of leptin on cyclin B1 and IGF-I accumulation within the cells. Our data showed that leptin directly affected proliferation, apoptosis, and hormone release by ovarian cells, probably via PKA- and MAPK-dependent pathways. Furthermore, it was demonstrated that nutrition could influence reproduction by affecting the response of ovarian cells to leptin.

Androgen receptor-mediated non-genomic effects of vinclozolin on porcine ovarian follicles and isolated granulosa cells: Vinclozolin and non-genomic effects in porcine ovarian follicles

The present study investigated the influence of the androgen receptor (AR) agonists testosterone (T) and dihydrotestosterone (DHT), and vinclozolin (Vnz), a fungicide with antiandrogenic activity, on non-genomic signal transduction within ovarian follicles. Porcine granulosa cells (GCs) isolated from mature follicles were cultured for 48h. For the last 24h of culture, they were exposed to T (10−7M), DHT (10−7M), Vnz (1.4×10−5M), T and Vnz (T+Vnz), or DHT and Vnz (DHT+Vnz) at the same concentrations. To better imitate in vivo conditions, whole follicles (4–6mm in diameter) were incubated (24h) in an organ culture system with the same factors. Expression of AR mRNA and protein was determined by real-time PCR and western blot analyses. To demonstrate AR localization in cultured GCs and whole follicles, immunocytochemistry and immunohistochemistry were performed, respectively. To elucidate the possible non-genomic action of Vnz in GCs, protein expression and the activity of ERK1/2 and Akt kinases were determined by western blot and ELISA analyses. The immunocytochemistry and immunohistochemistry results showed that exposure of GCs and follicles to Vnz resulted in cytoplasmic and perinuclear AR localization. Real-time PCR and western blot analysis showed that AR mRNA and protein expression increased (P≤0.001) in GC cultures after combined treatment with an androgen and Vnz. In whole follicles, such treatment also increased AR mRNA with a decrease in the respective protein expression (P≤0.001). Moreover, addition of T or DHT with Vnz increased the activity of ERK1/2 and Akt kinases in cultured GCs (P≤0.001). The results suggest a novel mechanism for Vnz action in porcine ovarian follicles on both AR mRNA and protein levels. Thus, this environmental antiandrogen activates non-genomic signaling pathways, as indicated by the increased activity of both investigated kinases observed within minutes of Vnz addition. Given the widespread presence of Vnz in the environment, elucidation of its non-genomic action should be the subject of studies on female fertility.

Effect of vitamin D3 on production of progesterone in porcine granulosa cells by regulation of steroidogenic enzymes.

1,25-dihydroxyvitamin D3 (VD3), an active form of Vitamin D, is photosynthesized in the skin of vertebrates in response to solar ultraviolet B radiation (UV-B). VD3 deficiency can cause health problems such as immune disease, metabolic disease, and bone disorders. It has also been demonstrated that VD3 is involved in reproductive functions. Female sex hormones such as estrogen and progesterone are biosynthesized mainly in ovarian granulosa cells as the ovarian follicle develops. The functions of sex hormones include regulation of the estrus cycle and puberty as well as maintenance of pregnancy in females. In this study, we isolated granulosa cells from porcine ovaries and cultured them for experiments. To examine the effects of VD3 on ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by Real-time PCR and Western blotting assay. Production of progesterone from granulosa cells was also measured by ELISA assay. As a result, transcriptional and translational regulation of progesterone biosynthesis-related genes in granulosa cells was significantly altered by VD3. Furthermore, progesterone concentrations in porcine granulosa cell-cultured media decreased in response to VD3. These results show that VD3 was a strong regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Pro-nerve growth factor in the ovary and human granulosa cells.

Pro-nerve growth factor must be cleaved to generate mature NGF, which was suggested to be a factor involved in ovarian physiology and pathology. Extracellular proNGF can induce cell death in many tissues. Whether extracellular proNGF exists in the ovary and may play a role in the death of follicular cells or atresia was unknown. Immunohistochemistry of human and rhesus monkey ovarian sections was performed. IVF-derived follicular fluid and human granulosa cells were studied by RT-PCR, qPCR, Western blotting, ATP- and caspase-assays. Immunohistochemistry of ovarian sections identified proNGF in granulosa cells and Western blotting of human isolated granulosa cells confirmed the presence of proNGF. Ovarian granulosa cells thus produce proNGF. Recombinant human proNGF even at high concentrations did not affect the levels of ATP or the activity of caspase 3/7, indicating that in granulosa cells proNGF does not induce death. In contrast, mature NGF, which was detected previously in follicular fluid, may be a trophic molecule for granulosa cells with unexpected functions. We found that in contrast to proNGF, NGF increased the levels of the transcription factor early growth response 1 and of the enzyme choline acetyl-transferase. A mechanism for the generation of mature NGF from proNGF in the follicular fluid may be extracellular enzymatic cleavage. The enzyme MMP7 is known to cleave proNGF and was identified in follicular fluid and as a product of granulosa cells. Thus the generation of NGF in the ovarian follicle may depend on MMP7.

Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period

Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare's serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus-oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15:Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

The Absence of ER-β Results in Altered Gene Expression in Ovarian Granulosa Cells Isolated From In Vivo Preovulatory Follicles

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-β is highly expressed in ovarian granulosa cells, and mice lacking ER-β are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERβ-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERβ-null ovaries. ERβ-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERβ in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERβ-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERβ-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

Palmitic acid in chicken granulosa cell death-lipotoxic mechanisms mediate reproductive inefficacy of broiler breeder hens

In vivo and in vitro approaches were used to elucidate mechanisms of palmitate-induced cytotoxicity of follicle granulosa cells in fuel-overloaded broiler hens. In contrast to their energy-restricted counterparts, broiler breeder hens fed ad libitum for 2 wk had dyslipidemia, atresia within hierarchical ovarian follicles, and a 34% reduction in egg production (P < 0.05). Based on vital staining of freshly isolated granulosa cells with annexin V/propidium iodide, there were increases in apoptosis consistent with suppressed Akt activation (P < 0.05). Supplementing primary granulosa cell cultures with 0.5 mM palmitate for 48 or 96 h increased apoptosis (P < 0.05). Palmitate-induced cell death was accompanied by increased acyl-CoA oxidase, carnitine palmitoyl transferase-1, serine palmitoyl transferase, and sphingomyelinase transcripts and increased concentrations of proinflammatory interleukin-1β (P < 0.05). Triacsin-C inhibition of fatty acyl-CoA synthesis blunted interleukin-1β production and rescued granulosa cultures from palmitate-induced cell death. That there was partial to complete prevention of cell death with addition of the free radical scavenger pyrrolidine dithiocarbamate, the sphingomyelinase inhibitor imipramine, or the de novo ceramide synthesis inhibitor fumonisin B1, supported the notion that palmitate-induced granulosa cell cytotoxicity operated through a palmitate-derived metabolite. Palmitoyl-CoA may be channeled into β-oxidation and/or into bioactive metabolites that increase free radical generation, an inflammatory response, and ceramide production. In conclusion, palmitate-derived metabolites activated apoptotic machinery in avian granulosa cells, which caused ovarian follicular atresia and reduced egg production in fuel-overloaded broiler breeder hens.

The Effects of Platelet-Derived Growth Factor BB on Ovine Granulosa Cell Function.

Platelet derived growth factors (PDGF) have been shown to modulate ovarian follicular development in some species. However, relatively little is known regarding the potential role of PDGF in regulating granulosa cell function in ruminants. Oocytes and granulosa cells in growing ovine follicles express mRNA encoding both PDGFA and PDGFB and follicular atresia appeared to cause decreased expression in the granulosa cells. However, with in situ hybridization, mRNAs for PDGF receptors (PDGFRA and PDGFRB) were not detectible in the granulosa cells. Thus, it is not clear whether PDGF can regulate granulosa cell function in vitro. Therefore, our aim was to determine the effects of PDGF on thymidine incorporation and progesterone production in sheep granulosa cells. Granulosa cells were collected from ovine follicles 1-2 mm in diameter. The incorporation of 3H-thymidine was determined during a 24 hour culture (20,000 viable cells per well) with M199 media. Secretion of progesterone was measured following culture of granulosa cells (100,000 viable cells per well) in an enriched McCoys 5a media with 30 ng/ml androstenedione, 3 ng/ml ovine FSH and 1 ng/ml IGF-1. Cells were cultured for 6 days with media being replaced every 48 hrs. The concentration of progesterone was measured by RIA in the media collected at the end of the culture period. The amount of DNA at the end of the culture period was also measured using binding of Hoechst 33258 dye. Triplicate wells were included in each assay and a minimum of 3 independent granulosa cell cultures were examined. The effects of human PDGFBB (10 ng/ml) was compared to vehicle treated control cells using paired t-tests. Addition of PDGFBB increased (P<0.05) 3H-thymidine incorporation 4.33 ± 1.10 fold compared to control cultures. Progesterone concentrations in PDGFBB treated granulosa cells decreased (P<0.01) to 0.33 ± 0.03 of those observed in control cultures with no effect on DNA content (1.06 ± 0.09 fold relative to control cultures). Given that PDGFBB regulated ovine granulosa cell function even though mRNA encoding PDGF receptors could not be localized to granulosa cells by in situ hybridization, RT-PCR was used to determine if culture of the granulosa cells might induce expression of PDGF receptors. Total cellular mRNA was extracted from freshly isolated granulosa cells or those cultured for 2 days (2 pools each). One pool of freshly isolated granulosa cells and both pools of cultured granulosa cells contained both PDGFRA and PDGFRB mRNA. Therefore, PDGFs are likely regulators of granulosa cell function in sheep although differences in receptor expression requires further exploration.

Differential expression of CTGF in pre- and post-ovulatory granulosa cells in the hen ovary is regulated by TGFβ1 and gonadotrophins

Connective tissue growth factor (CTGF) is a cysteine-rich, matrix-associated heparin-binding protein that is important in many cell types as a regulator of cell proliferation, angiogenesis, cell remodelling and other cellular processes. CTGF is necessary for normal follicle growth and luteinisation in mammals. The avian follicular hierarchy provides an excellent experimental model to study developmental events, particularly the role of cellular remodelling factors in the process of folliculogenesis. In this study, we examined CTGF expression and regulation in the hen ovary. CTGF expression was increased considerably as follicular development proceeds in pre-ovulatory follicles, peaking in expression at the time of ovulation. Immunohistochemistry revealed that CTGF protein was concentrated in the cytoplasm of follicular granulosa cells throughout the ovulation cycle. We isolated granulosa cells from the follicles at two key stages of the ovulation cycle (in terms of cellular alteration): during pre-ovulatory growth and during post-ovulatory regression. Follicle-stimulating hormone (FSH) and luteinising hormone (LH) inhibited CTGF expression in pre-ovulatory granulosa cells but stimulated CTGF expression in post-ovulatory granulosa cells. Moreover, TGFβ1 stimulated CTGF expression in both pre- and post-ovulatory granulosa cells. Nevertheless, TGFβ1 could rescue the inhibition of gonadotrophins on pre-ovulatory granulosa CTGF expression but could not further stimulate CTGF expression in gonadotrophin-treated post-ovulatory granulosa cells. The results of this study indicate that CTGF expression in avian granulosa cells is modulated by a combination of gonadotrophins and TGFβ1 according to the different stages of follicle maturation and degradation. The results also suggest that the gonadotrophic action on post-ovulatory follicles in the avian ovary differs from the gonadotrophin-induced luteinisation in mammals.