Mouse Granulosa Cells Research Articles

Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice

To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism. Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured in vitro. The cultured cells were treated with 0, 1, 10, 50, 100, 150, 200 and 500 μmol/L BPA, and the changes in cell proliferation, cell cycle, apoptosis and mitochondrial membrane potential were analyzed with CCK-8 method and flow cytometry. The protein expressions of Bcl-2, Bax, p53 and cyclin D1 in the treated cells were determined with Western blotting. Pregnant ICR mice were treated for a week with BPA at the concentration that produced significant effects on the preantral follicular granulosa cells, and the weight changes of the pregnant mice were recorded. The ovarian tissues of the offspring female mice were weighed at PND 10, 17, 21 and 42 followed by histological observation with HE staining and examination of Bcl-2 mRNA expression level with RT-qPCR. Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (P < 0.05). Exposure to a higher BPA concentration at 200 μmol/L obviously enhanced cell apoptosis by reducing the mitochondrial membrane potential and repressed the cell proliferation (P < 0.05). BPA exposure at 50 μmol/L and 200 μmol/L produced opposite effects on the protein expressions of Bcl-2 (P < 0.01), Bax (P < 0.05) and p53 (P < 0.05) in mouse ovarian preantral follicular granulosa cells. BPA exposure at the doses of 10 and 35 mg/kg caused rapid weight increment of the pregnant mice and changes in ovarian index of the offspring female mice. In the offspring female mice, the changes in Bcl-2 mRNA expression in the ovarian tissue showed a similar pattern to that of ovarian index. Exposure of the pregnant mice to a high BPA concentration at 35 mg/kg resulted in accelerated follicular development into antral follicular stage in PND 21 offspring female mice. BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Global analysis of FSH-regulated gene expression and histone modification in mouse granulosa cells.

Follicle-stimulating hormone (FSH) regulates ovarian follicular development through a specific gene expression program. We analyzed FSH-regulated transcriptome and histone modification in granulosa cells during follicular development. We used super-stimulated immature mice and collected granulosa cells before and 48 h after stimulation with equine chorionic gonadotropin (eCG). We profiled the transcriptome using RNA-sequencing (N = 3/time-point) and genome-wide trimethylation of lysine 4 of histone H3 (H3K4me3; an active transcription marker) using chromatin immunoprecipitation and sequencing (ChIP-Seq; N = 2/time-point). Across the mouse genome, 14,583 genes had an associated H3K4me3 peak and 63-66% of these peaks were observed within ≤1 kb promoter region. There were 72 genes with differential H3K4me3 modification at 48 h eCG (absolute log fold change > 1; false discovery rate [FDR] < 0.05) relative to 0 h eCG. Transcriptome data analysis showed 1463 differentially expressed genes at 48 h eCG (absolute log fold change > 1; FDR < 0.05). Among the 20 genes with differential expression and altered H3K4me3 modification, Lhcgr had higher H3K4me3 abundance and expression, while Nrip2 had lower H3K4me3 abundance and expression. Using ChIP-qPCR, we showed that FSH-regulated expression of Lhcgr, Cyp19a1, Nppc, and Nrip2 through regulation of H3K4me3 at their respective promoters. Transcript isoform analysis using Kallisto-Sleuth tool revealed 875 differentially expressed transcripts at 48 h eCG (b > 1; FDR < 0.05). Pathway analysis of RNA-seq data demonstrated that TGF-βsignaling and steroidogenic pathways were regulated at 48 h eCG. Thus, FSH regulates gene expression in granulosa cells through multiple mechanisms namely altered H3K4me3 modification and inducing specific transcripts. These data form the basis for further studies investigating how these specific mechanisms regulate granulosa cell functions.

Erratum. Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

Study question Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? Summary answer Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. What is known already Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. Study design, size, duration Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. Participants/materials, setting, methods The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. Main results and the role of chance The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. Limitations, reasons for caution The number of PCOS patients in this study was small. Wider implications of the findings This study provides further evidence for metformin as a PCOS treatment. Study funding/competing interest(s) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. Trial registration number N/A.

Follicle Stimulating Hormone Inhibits the Expression of p53 Up-Regulated Modulator of Apoptosis Induced by Reactive Oxygen Species Through PI3K/Akt in Mouse Granulosa Cells.

In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells (GCs). Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced GCs apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. Therefore, we examined whether FSH inhibits the expression of p53 up-regulated modulator of apoptosis (PUMA) induced by reactive oxygen species (ROS) through phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) in mouse GCs. In vivo study: thirty-two-mice were randomly assigned to four groups and given FSH. We found that FSH can inhibit the 3-nitropropionic acid (3-NP) induced apoptosis and PUMA expression in mRNA level. Moreover, In vitro experiment, we found that FSH can inhibit the H(2)O(2)-induced apoptosis and PUMA expression in mRNA level. Additionally, we also found that PI3K/AKT inhibitor LY294002 abolished the downregulation of PUMA mRNA by FSH in vitro, In conclusion, FSH inhibit the expression of PUMA induced by ROS through PI3K/AKT pathway in vivo and vitro.

Boron Attenuates Heat Stress-Induced Apoptosis by Inhibiting Endoplasmic Reticulum Stress in Mouse Granulosa Cells.

Heat stress-induced apoptosis in granulosa cells is mediated by multiple apoptotic signaling pathways, including endoplasmic reticulum (ER) stress. Boron is a naturally occurring trace element with several cytoprotective properties. Nonetheless, the molecular mechanisms involved in the protective functions of boron in granulosa cells undergoing apoptosis caused by heat stress (HS) remain unclear. In this study, we investigated the role of boric acid, a predominant chemical form of boron, in HS-induced apoptotic damage in mouse granulosa cells (mGCs) and explored the underlying mechanisms. We found that HS treatment suppressed cell viability; increased the apoptotic rate of cells; potentiated the activity of caspase-3, a key player in the caspase-mediated apoptotic signaling pathway; and activated ER stress markers, including glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) in mGCs. However, boric acid treatment effectively alleviated the effects of both HS-induced and thapsigargin (an ER stress agonist)-induced apoptosis, such as the enhanced activity of caspase-3 and increase in GRP78 and CHOP expression. Moreover, treatment with 4-phenylbutyrate (4-PBA), an ER stress antagonist, significantly attenuated these HS-induced adverse effects in mGCs. In addition, boric acid supplementation in the culture medium significantly restored the decreased estradiol levels in heat-treated mGCs. The administration of boric acid to female mice previously exposed to hyperthermal conditions effectively restored the levels of serum estradiol in vivo. Collectively, these findings suggest that HS induces apoptosis in mGCs via ER stress pathways and that boron has a protective effect against these adverse effects. This study provides novel insights into the benefits of using boron against heat-induced apoptosis.

OR31-04 Androgen Signaling Regulates Female Fertility Through Modulation of H3K27me3 in Granulosa Cells

Androgens play a major role in female fertility and in women’s health, in general. However, the underlying mechanism and downstream physiological targets of androgen are not fully understood. Androgen actions are mediated by “nuclear” transcriptional signals or “extra-nuclear” kinase actions. Primary androgen receptor (AR) target genes are those at which AR occupies a genomic androgen response element (ARE) and regulates gene transcription. Subsequently, androgens also regulate other genes in an AR-ARE independent fashion, involving membrane-initiated androgen signaling. In the last couple of years, we have reported that androgens may also influence gene expression through histone modifications. We have found that H3K27me3 (tri-methyl lysine 27 histone3) is a downstream target of androgen actions. H3K27me3 is a gene silencing mark, regulated by Enhancer of Zeste Homologue 2 (Ezh2), a histone methyltransferase that promotes tri-methylation of K27 and androgens inhibit the expression and activity of Ezh2. In this study, we report that androgens also regulate the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing the H3K27me3 mark. We find that in granulosa cells (GCs), androgen through the PI3K/Akt pathway, in a transcription-independent fashion, increases hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. ChIP studies reveal increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. To understand the global impact of androgens on ovarian gene expression and the contribution of androgen-induced decrease of H3K27me3, we have performed RNA-seq and ChIP-seq analysis with H3K27me3 antibody in primary mouse GCs treated with DHT or vehicle. Results show 190 significantly differentially expressed genes in DHT treated sample vs vehicle, out of which 129 and 61 genes were significantly up- and downregulated, respectively. Moreover, comparison of the RNA-seq and ChIP-seq data reveals that a number of upregulated genes have significantly lower H3K27me3 enrichment in the enhancer and/or promoter region in the DHT treated samples vs vehicle. This establishes that in the ovary, androgen-induced modulation of H3K27me3 mark through regulation Ezh2 and JMJD3 expression/activity is an important regulatory mechanism for ovarian gene expression. To delineate the physiological importance of JMJD3 in normal ovarian function and female fertility, we have also developed a GC-specific JMJD3 knockout mice. We find that GC-specific JMJD3KO mice are sub-fertile with longer estrous cycles and dysregulated follicular development. This phenotype is very similar to GC-specific ARKO mice. Thus, we propose that one of the critical actions of androgens in regulating follicular function and female fertility is through the regulation of JMJD3 expression.

Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK.

STUDY QUESTIONDoes metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro?SUMMARY ANSWERMetformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs.WHAT IS KNOWN ALREADYPolycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice.STUDY DESIGN, SIZE, DURATIONCumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA.PARTICIPANTS/MATERIALS, SETTING, METHODSThe levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion.MAIN RESULTS AND THE ROLE OF CHANCEThe expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK.LIMITATIONS, REASONS FOR CAUTIONThe number of PCOS patients in this study was small.WIDER IMPLICATIONS OF THE FINDINGSThis study provides further evidence for metformin as a PCOS treatment.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose.TRIAL REGISTRATION NUMBERN/A.

Salt-inducible Kinases Are Critical Determinants of Female Fertility.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3',5'-cyclic adenosine 5'-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Interaction of the mTERT telomerase catalytic subunit with the c-Abl tyrosine kinase in mouse granulosa cells

Context: Oocyte and granulosa cells (GCs) have bidirectional communication and GCs play an important role in folliculogenesis and proliferation of GCs is very important for the development of ovulatory follicle. DNA double-strand breaks activate c-Abl protein tyrosine kinase and c-Abl has a functional role in repairement of DNA and control of telomere.Objective: In this study, we hypothesized that c-Abl has a regulative role on mTERT in mouse ovarian granulosa cells (GCs) and we aimed to detect c-Abl and mTERT interaction in mouse primary culture of GCs.Materials and methods: Mouse ovarian granulosa cell were cultured and siRNA-mediated knockdown approach was used to knockdown c-Abl expression.Results: We showed c-Abl and mTERT immunolocalization in vivo and in vitro mouse GCs. c-Abl and mTERT were constitutively expressed in mouse granulosa cells and c-Abl presented more intense expression in granulosa cells than mTERT expression. The interaction of the c-Abl-mTERT is supported by the exhibition that c-Abl siRNA knockdown cells show decreased mTERT expression. We also present an interaction between c-Abl and mTERT by immunoprecipitation. In addition, our results indicated that the down-regulation of c-Abl was also accompanied by reduced expression of proliferating cell nuclear antigen (PCNA) in GCs.Conclusions: We suggest that mTERT may associate with the c-Abl in mouse GCs and the interactions between c-Abl and mTERT suggest a role for c-Abl in the regulation of telomerase function and proliferation in mouse granulosa cells.

Chlorogenic acid rescues zearalenone induced injury to mouse ovarian granulosa cells.

Zearalenone (ZEA), a toxic substance produced by Fusarium fungi, accumulated in cereals grain and animal feed, causes injury to humans and animals. ZEA can induce obvious reproductive toxicity with the ovarian granulosa cells (GCs) as the main target. However, the study on exploring the protective compounds against ZEA-induced mouse primary ovarian GCs damage remains less. In the current study, the protective effect of 20 compounds derived from traditional Chinese medicines (TCMs) on the injury of mouse GCs caused by ZEA were evaluated using MTT assay and the cell morphology. Our results showed that chlorogenic acid (250, 500, and 1000μg/mL) significantly suppress ZEA-induced GCs death. Western blot analysis suggested chlorogenic acid could rescue the up-regulated apoptosis of GCs induced by ZEA via attenuating the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2 and cleaved-PARP. Our results provide strong evidence that chlorogenic acid warrants further optimization for more potent and safer compounds for against the ZEA lead toxicity to humans and animals.

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.

Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells.

Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6–48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis.

Effect of Vitamin D3 on Mitochondrial Biogenesis in Granulosa Cells Derived from Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is an endocrine disorder diagnosed by anovulation hyperandro- genism. Hyperandrogenism increases apoptosis, which will eventually disturb follicular growth in PCOS patients. Since mitochondria regulate apoptosis, they might be affected by high incidence of follicular atresia. This may cause infertility. Since vitamin D3 has been shown to improve the PCOS symptoms, the aim of study was to investigate the effects vitamin D3 on mtDNA copy number, mitochondrial biogenesis, and membrane integrity of granulosa cells in a PCOS-induced mouse model.Materials and MethodsIn this experimental study, the PCOS mouse model was induced by dehydroepiandrosterone (DHEA). Granulosa cells after identification by follicle-stimulating hormone receptor (FSHR) were cultured in three groups: 1. granulosa cells treated with vitamin D3 (100 nM for 24 hours), 2. granulosa cells without any treatments, 3. Non-PCOS granulosa cells (control group). Mitochondrial biogenesis gene (TFAM) expression was compared between different groups using real-time PCR. mtDNA copy number was also investigated by qPCR. The mitochon- drial structure was evaluated by transmission electron microscopy (TEM). Hormonal levels were measured by an enzymelinked immunosorbent assay (ELISA) kit.ResultsThe numbers of pre-antral and antral follicles increased in PCOS group in comparison with the non-PCOS group. Mitochondrial biogenesis genes were downregulated in granulosa cells of PCOS mice when compared to the non-PCOS granulosa cells. However, treatment with vitamin D3 increased mtDNA expression levels of these genes compared to PCOS granulosa cells with no treatments. Most of the mitochondria in the PCOS group were spherical with almost no cristae. Our results showed that in the PCOS group treated with vitamin D3, the mtDNA copy number increased significantly in comparison to PCOS granulosa cells with no treatments.ConclusionAccording to this study, we can conclude, vitamin D3 improves mitochondrial biogenesis and membrane integrity, mtDNA copy number in granulosa cells of PCOS mice which might improve follicular development and subsequently oocyte quality.

Lipotoxicity Impairs Granulosa Cell Function Through Activated Endoplasmic Reticulum Stress Pathway.

Obesity is closely related to reproductive disorders, which may eventually lead to infertility in both males and females. Ovarian granulosa cells play a critical role during the maintenance of oocyte development through the generation of sex steroids (mainly estradiol and progesterone) and different kinds of growth factors. However, the molecular mechanism of obesity-induced granulosa cell dysfunction remains poorly investigated. In our current study, we observed that high-fat diet feeding significantly increased the level of glucose-regulated protein 78 kDa (GRP78) protein expression in mouse granulosa cells; testosterone-induced estradiol generation was impaired accordingly. To further evaluate the precise mechanism of lipotoxicity-induced granulosa cell dysfunction, mouse primary granulosa cells were treated with palmitate, and the expression levels of ER stress markers were evaluated by real-time PCR and western blot. Lipotoxicity significantly increased ER stress but impaired the mRNA expression of granulosa cell function-related makers, including androgen receptor (Ar), cytochrome P450 family 19 subfamily A member 1 (Cyp19a1), hydroxysteroid 17-beta dehydrogenase 1 (Hsd17b1), and insulin receptor substrate 1 (Irs1). Impaired testosterone-induced estradiol generation was also observed in cultured mouse granulosa cells after palmitate treatment. Insulin augmented testosterone induced estradiol generation through activation of the AKT pathway. However, palmitate treatment abolished insulin-promoted aromatase expression and estradiol generation by the stimulation of ER stress. Overexpression of IRS1 significantly ameliorated palmitate- or tunicamycin-induced impairment of aromatase expression and estradiol generation. Taken together, our current study demonstrated that lipotoxicity impaired insulin-stimulated estradiol generation through activated ER stress and inhibited IRS1 pathway.

The interaction of Wnt signaling members with growth factors in cultured granulosa cells.

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-β) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-β. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-β and EGF groups. FGF-β group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-β was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-β and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.

MC-LR induced overproduction of progesterone via inhibiting miR-3473g: in vitro and in vivo evidence.

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has drawn more and more attention in recent years. In the present study, MC-LR inhibited miR-3473g expression of mouse granulosa cells both in vitro and in vivo. By dual-luciferase reporter assay, we confirmed miR-3473g is able to bind the 3'-UTR of StAR protein (StAR) mRNA and suppress StAR expression. Thus, downregulation of miR-3473g after MC-LR exposure led to StAR overexpression. Excessive StAR probably transported much more cholesterol into the inner membrane of the mitochondria and finally resulted in overproduction of progesterone. Our results revealed that MC-LR exposure was associated with premature luteinization of granulosa cells and may adversely affect women's fertility.

Ovulatory signals alter granulosa cell behavior through YAP1 signaling

BackgroundThe Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation.MethodsTo assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test or Dunnett’s post-hoc test.ResultsOur data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs.ConclusionsOur results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.

Maternal Testosterone Excess Contributes to Reproductive System Dysfunction of Female Offspring Mice.

Hyperandrogenism is considered 1 of the most important characteristics of polycystic ovary syndrome, which affects more than 10% of females of reproductive age and is a common cause of infertility. In addition to the effects on patients themselves, maternal androgen excess has also been reported to impair the growth and development of offspring. In our current study, we found that maternal testosterone (T) treatment during different gestational stages increased the percentage of atretic follicle and decreased corpus luteum formation in female offspring. In addition, decreased serum estradiol and increased T levels were also observed in female offspring of T-treated mice during late gestational stage. Further studies revealed that Forkhead box protein L2 (FOXL2) and Cytochrome P450 family 19 subfamily a member 1 (CYP19A1) expression in granulosa cells of these female offspring mice were decreased. By using mouse primary granulosa cells and the KGN cell line, we demonstrated that decreasing FOXL2 and CYP19A1 levels in ovarian granulosa cells partially may contribute to disturbed sex hormone synthesis in female offspring of T-treated mice during the late gestational stage. Findings from our current study highlight a critical role of excess maternal T exposure, especially during the late gestational stage, which could further lead to aberrant ovary development and sex hormone synthesis in female offspring.

Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.