Nuclei Of Granulosa Cells Research Articles

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.

Di-(2-ethylhexyl) phthalate exposure induces ferroptosis by regulating the Nrf2-mediated signaling pathway in mouse ovaries

Di-(2-ethylhexyl) phthalate (DEHP), an endocrine-disrupting chemical present in plasticized products, exerts strong modulation on the anatomy and function of the female reproductive system. However, the potential mechanisms underlying DEHP-induced regulation of prepubertal female reproductive toxicity have not yet been elucidated. Therefore, this study was designed to elucidate the molecular mechanism of ovarian injury induced by DEHP exposure in mice. Elevated serum mono-2-ethylhexyl phthalate (MEHP) concentrations, decreased levels of ovarian hormones (E2 and P4), and observed ovarian injury were found after DEHP exposure. Ovarian transcriptome analysis revealed significant alterations in ferroptosis-associated gene expression, with potential regulation by Nrf2. Subsequent analysis of ferrous iron, malondialdehyde (MDA), Western blotting, and immunofluorescence of the ovaries confirmed the RNA-seq findings. Transcriptome analysis of granulosa cells revealed a direct or indirect regulatory relationship between Nrf2 and downstream ferroptosis-related proteins following MEHP exposure. Further experiments demonstrated that ferrostatin-1 attenuated MEHP-induced ferroptosis in granulosa cells. Additionally, Nrf2 stabilization and accumulation in the nucleus of granulosa cells were observed following MEHP treatment. RNAi-mediated knockdown of Nrf2 exacerbated MEHP-induced ferroptosis in granulosa cells. This evidence indicates that DEHP exposure induces ferroptosis through regulation of the Nrf2-mediated signaling pathway in mouse ovaries, laying the groundwork for future studies aiming to develop therapeutic strategies against DEHP toxicity.

Effect of the gonadotropin surge on steroid receptor regulation in preovulatory follicles and newly formed corpora lutea in the cow

The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA—estrogen related receptor alpha and ESRRB—estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2–3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular–luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.

Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways

BackgroundPseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro.MethodsPrimary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting.ResultsVirus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection.ConclusionsThe data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

SRSF1 is essential for primary follicle development by regulating granulosa cell survival via mRNA alternative splicing.

Granulosa cell abnormalities are characteristics of premature ovarian insufficiency (POI). Abnormal expression of serine/arginine-rich splicing factor 1 (SRSF1) can cause various diseases, but the role of SRSF1 in mouse granulosa cells remains largely unclear. In this study, we found that SRSF1 was expressed in the nuclei of both mouse oocytes and granulosa cells. The specific knockout of Srsf1 in granulosa cells led to follicular development inhibition, decreased granulosa cell proliferation, and increased apoptosis. Gene Ontology (GO) analysis of RNA-seq results revealed abnormal expression of genes involved in DNA repair, cell killing and other signalling pathways. Alternative splicing (AS) analysis showed that SRSF1 affected DNA damage in granulosa cells by regulating genes related to DNA repair. In summary, SRSF1 in granulosa cells controls follicular development by regulating AS of genes associated with DNA repair, thereby affecting female reproduction.

Nuclear lncRNA NORSF reduces E2 release in granulosa cells by sponging the endogenous small activating RNA miR-339

BackgroundFunctioning as a competing endogenous RNA (ceRNA) is the main action mechanism of most cytoplasmic lncRNAs. However, it is not known whether this mechanism of action also exists in the nucleus.ResultsWe identified four nuclear lncRNAs that are presented in granulosa cells (GCs) and were differentially expressed during sow follicular atresia. Notably, similar to cytoplasmic lncRNAs, these nuclear lncRNAs also sponge miRNAs in the nucleus of GCs through direct interactions. Furthermore, NORSF (non-coding RNA involved in sow fertility), one of the nuclear lncRNA acts as a ceRNA of miR-339. Thereby, it relieves the regulatory effect of miR-339 on CYP19A1 encoding P450arom, a rate-limiting enzyme for E2 synthesis in GCs. Interestingly, miR-339 acts as a saRNA that activates CYP19A1 transcription and enhances E2 release by GCs through altering histone modifications in the promoter by directly binding to the CYP19A1 promoter. Functionally, NORSF inhibited E2 release by GCs via the miR-339 and CYP19A1 axis.ConclusionsOur findings highlight an unappreciated mechanism of nuclear lncRNAs and show it acts as a ceRNA, which may be a common lncRNA function in the cytoplasm and nucleus. We also identified a potential endogenous saRNA for improving female fertility and treating female infertility.

1,25-Dihydroxyvitamin D3 alleviates hyperandrogen-induced ferroptosis in KGN cells

PurposeHyperandrogenism, one of the most frequent causes of anovulation in women, increases the risk of metabolic disorders in patients with polycystic ovary syndrome (PCOS). Ferroptosis, characterized by iron-dependent lipid peroxidation, has provided new insight into the progression of PCOS. 1,25-dihydroxyvitamin D3 (1,25D3) may play a role in reproduction because its receptor, VDR, which contributes to the inhibition of oxidative stress, is primarily located in the nuclei of granulosa cells. This study has therefore investigated whether 1,25D3 and hyperandrogenism affect granulosa-like tumor cells (KGN cells) through ferroptosis.MethodsKGN cells were treated with dehydroepiandrosterone (DHEA) or pretreated with 1,25D3. Cell viability was evaluated with the cell counting kit-8 (CCK-8) assay. The mRNA and protein expression levels of ferroptosis-related molecules, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member (SLC7A11), and long-chain acyl-CoA synthetase 4 (ACSL4), were assessed via qRT–PCR and western blot. The concentration of malondialdehyde (MDA) was measured by ELISA. The rates of reactive oxygen species (ROS) production and lipid peroxidation were assessed via photometric methods.ResultsDecreased cell viability, suppression of GPX4 and SLC7A11 expression, increased expression of ACSL4, elevated levels of MDA, accumulation of ROS, and increased lipid peroxidation, which are changes representative of ferroptosis, were observed in KGN cells after treatment with DHEA. Pretreatment with 1,25D3 in KGN cells significantly prevented these changes.ConclusionsOur findings demonstrate that 1,25D3 attenuates hyperandrogen-induced ferroptosis of KGN cells. This finding might lead to new insights into the pathophysiology and therapy of PCOS and provides new evidence for the treatment of PCOS with 1,25D3.

MicroRNA 195-5p Targets Foxo3 Promoter Region to Regulate Its Expression in Granulosa Cells.

Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.

Hypermethylation-mediated downregulation of lncRNA PVT1 promotes granulosa cell apoptosis in premature ovarian insufficiency via interacting with Foxo3a.

Long noncoding RNA PVT1 is involved in the progression of female gynecological cancers. However, the role of PVT1 in ovarian granulosa cell apoptosis-mediated premature ovarian insufficiency (POI) remains unclear. This study aims to elucidate the role of PVT1 in ovarian granulosa cell apoptosis-mediated POI. The expression of PVT1 was compared between ovarian tissues from POI patients and normal controls. The methylation level in the PVT1 promoter region was detected by methylation-specific polymerase chain reaction. The interaction between PVT1 and forkhead box class O3A (Foxo3a) was confirmed by RNA pull-down and RNA immunoprecipitation assays. Granulosa cell apoptosis was detected using flow cytometry. The effect of PVT1 on transcription activity of Foxo3a was detected by luciferase reporter assay. The expression of PVT1 was low in the POI ovarian tissues compared with the controls, and such a low expression was related to the hypermethylation of the PVT1 promoter. PVT1 was localized in both the cytoplasm and the nucleus of granulosa cells. We determined that PVT1 could bind with Foxo3a and that downregulating PVT1 by small interfering RNAs inhibited Foxo3a phosphorylation by promoting SCP4-mediated Foxo3a dephosphorylation, resulting in an increase in Foxo3a transcription activity. Moreover, downregulating PVT1 promoted granulosa cell apoptosis by increasing the Foxo3a protein levels. An in vivo experiment showed that the injection of PVT1 overexpressing vectors restored the ovarian function in POI mice. Hypermethylation-induced downregulation of PVT1 promotes granulosa cell apoptosis in POI by inhibiting Foxo3a phosphorylation and increases the Foxo3a transcription activity.

FSH promotes the proliferation of sheep granulosa cells by inhibiting the expression of TSP1

Thrombospondin (TSP1) plays an important role as an antiangiogenic factor in the reproductive system of female mammals. However, its expression and function in sheep are still unclear. In the present research, the Altay sheep (a native Chinese breed) was used to analyze the expression of TSP1 in the ovary and its potential function in granulosa cells. TSP1 was widely expressed in most tissues, as shown by qPCR. In the ovary, TSP1 mRNA expression decreased during follicular to luteal growth. The TSP1 protein was expressed in a wide variety of follicles of different diameters and localized to the cytoplasm and nucleus of granulosa cells. In in vitro studies, follicle-stimulating hormone (FSH) significantly inhibited the expression of TSP1 in sheep granulosa cells. Functionally, FSH- and TSP1-specific siRNAs can promote the proliferation of sheep granulosa cells. In contrast, TSP1 mimetic peptide, ABT510, offsets the proliferation of sheep granulosa cells. Different signaling pathway inhibitors all promoted FSH-inhibited TSP1 expression, but each inhibitor had different effects on TSP1. Among them, the PI3K and ERK pathway inhibitors significantly promoted TSP1 expression and inhibited the proliferation of sheep granulosa cells.

Expression, location and biological effects of four and a half LIM domain protein2 (FHL2) on granulosa cells in ovine.

Previous studies have shown that four and a half LIM domain protein 2(FHL2) plays an essential role in the regulation of follicular development in mammals. Although the FHL2 genes of human and mouse have been well characterized, the expression and location of FHL2 in ovary and the biological functions of FHL2 on granulosa cells (GCs) of ovine are still not clear. In this study, full-length complementary DNA (cDNA) of FHL2 from ovine follicular GCs was amplified by real-time PCR (RT-PCR). The expression and location of FHL2 in ovary and GCs of ovine were studied by immunohistochemistry and immunofluorescence, and the biological effects of FHL2 on the cell proliferation, cell apoptosis, cell cycles and expression level of related genes of ovine GCs were also explored by overexpression or knockdown of FHL2. The results indicated that FHL2 was expressed in ovine follicular GCs and the sequence of the FHL2 cDNA was consistent with that predicted in GenBank, which did not cause an amino acid change. According to the results, FHL2 was expressed in ovine ovary and mainly located in the cytoplasm and nucleus of GCs. In addition, overexpression of FHL2 significantly reduced the cell viability, promoted the cell apoptosis and decreased the percentage of G0/G1 and S phase cells. RT-PCR showed that overexpression of FHL2 significantly increased the mRNA expression level of Bax and decreased the expression of Bcl-2 and the Bcl-2/Bax mRNA ratio compared with the control group. Besides, the knockdown of FHL2 gene in ovine GCs significantly improved the cell viability, suppressed the cell apoptosis, decreased the mRNA expression level of Caspase-3 gene, increased the Bcl-2/Bax mRNA ratio and increased the percentage of S and G2/M phase cells. Our results suggest that FHL2 may play an important role in the biological functions of GCs in ovine.

Long noncoding RNA HCP5 participates in premature ovarian insufficiency by transcriptionally regulating MSH5 and DNA damage repair via YB1.

The genetic etiology of premature ovarian insufficiency (POI) has been well established to date, however, the role of long noncoding RNAs (lncRNAs) in POI is largely unknown. In this study, we identified a down-expressed lncRNA HCP5 in granulosa cells (GCs) from biochemical POI (bPOI) patients, which impaired DNA damage repair and promoted apoptosis of GCs. Mechanistically, we discovered that HCP5 stabilized the interaction between YB1 and its partner ILF2, which could mediate YB1 transferring into the nucleus of GCs. HCP5 silencing affected the localization of YB1 into nucleus and reduced the binding of YB1 to the promoter of MSH5 gene, thereby diminishing MSH5 expression. Taken together, we identified that the decreased expression of HCP5 in bPOI contributed to dysfunctional GCs by regulating MSH5 transcription and DNA damage repair via the interaction with YB1, providing a novel epigenetic mechanism for POI pathogenesis.

The effects of magnesium sulfate on cyclophosphamide-induced ovarian damage: Folliculogenesis

Cyclophosphamide (CYP) is one of the alkylating chemotherapeutic agents and its adverse effects on folliculogenesis in the ovary are well-known due to the previous scientific research on this topic. Magnesium has various effects in organisms, including catalytic functions on the activation and inhibition of many enzymes, and regulatory functions on cell proliferation, cell cycle, and differentiation. In this study, the effects of magnesium sulfate (MgSO4) on CYP induced ovarian damage were investigated. Immature Wistar-Albino female rats of 28-days were treated with pregnant mare serum gonadotrophin (PMSG) to develop the first generation of preovulatory follicles. Rats of the experimental groups were then treated with either CYP (100 mg/kg, i.p) and MgSO4 (270 mg/kg loading dose; 27 mg/kg maintenance doseX12, i.p) solely or in combination. Following in-vivo 5-bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. In the ovaries, added to the evaluation of general morphology and follicle count; BrdU and TUNEL-labeling, cleaved caspase-3 and p27 (cyclin-dependent kinase inhibitor) staining was also performed immunohistochemically and an ultrastructural evaluation was performed by transmission electron microscopy (TEM). The number of primordial follicles were decreased and multilaminar primary and atretic follicles were increased in CYP group. After MgSO4 treatment, while primordial follicle pool were elevated, the number of atretic follicles were decreased. Additionally, decreased BrdU-labeling, increased cleaved caspase 3 immunoreactivity and increased TUNEL labeling were observed in CYP group. In CYP treated animals, observations showed that while MgSO4 administration caused no alterations in BrdU proliferation index and caspase-3 immunoreactivity, it significantly reduced the TUNEL labeling. It was also observed that, while p27 immunoreactivity significantly increased in the nuclei of granulosa and theca cells in the CYP group; MgSO4 treatment significantly reduced these immunoreactivities. The ultrastructural observations showed frequent apoptotic profiles in granulosa and theca cells in both early and advanced stages of follicles in the CYP group and the MgSO4 treatment before the CYP application led to ultrastructural alleviation of the apoptotic process. In conclusion, our data suggest that MgSO4 may provide an option of pharmacologic treatment for fertility preservation owing to the beneficial effects of on chemotherapy-induced accelerated follicular apoptotic process, and the protection of the primordial follicle pool.

SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFβ-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFβ1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFβ signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.

Vitamin D3 Regulates Follicular Development and Intrafollicular Vitamin D Biosynthesis and Signaling in the Primate Ovary.

There is an increasing recognition that vitamin D plays important roles in female reproduction. Recent studies demonstrated that 1α,25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, improved ovarian follicle survival and growth in vitro. Therefore, we investigated the direct effects of VD3 at the specific preantral and antral stages of follicular development, and tested the hypothesis that vitamin D receptor (VDR) and enzymes critical for vitamin D biosynthesis are expressed in the primate ovary. Fourteen adult rhesus macaques provided ovarian tissue. Secondary and antral follicles were isolated for PCR analysis on VDR, vitamin D3 25-hydroxylase, and 25-hydroxyvitamin D3-1α-hydroxylase. VDR protein localization was determined by immunohistochemistry on ovarian sections. Isolated secondary follicles were cultured under conditions of control and VD3 supplementation during the preantral or antral stage. Follicle survival, growth, steroid and anti-Müllerian hormone (AMH) production, as well as oocyte maturation were evaluated. In vivo- and in vitro-developed follicles were also assessed for genes that are critical for vitamin D biosynthesis and signaling, gonadotropin signaling, steroid and paracrine factor production, and oocyte quality. The mRNA encoding VDR, 25-hydroxylase, and 1α-hydroxylase was detectable in in vivo- and in vitro-developed preantral and antral follicles. The 25-hydroxylase was elevated in cultured follicles relative to in vivo-developed follicles, which further increased following VD3 exposure. VD3 treatment increased 1α-hydroxylase in in vitro-developed antral follicles. The absence of VD3 during culture decreased VDR expression in in vitro-developed antral follicles, which was restored to levels comparable to those of in vivo-developed antral follicles by VD3 supplementation. Positive immunostaining for VDR was detected in the nucleus and cytoplasm of granulosa cells and oocytes. While only survival was improved in preantral follicles treated with VD3, VD3 supplementation promoted both survival and growth of antral follicles with increased estradiol and AMH production, as well as oocyte maturation. Thus, Vitamin D biosynthesis and signaling systems are expressed in primate ovarian follicles. Our findings support a role for VD3 in regulating follicular development in a stage-dependent manner, as well as the intrafollicular vitamin D biosynthesis and signaling, directly in the ovary.

Down-regulation of long non-coding RNA MALAT1 inhibits granulosa cell proliferation in endometriosis by up-regulating P21 via activation of the ERK/MAPK pathway.

Is there a specific mechanism underlying the association between lung adenocarcinoma transcript 1 (MALAT1) and endometriosis-related infertility? The down-regulation of MALAT1 in endometriosis granulosa cells (GCs) may have an adverse effect on the growth and development of oocytes by inhibiting GC proliferation, due to cell cycle-dependent mechanisms that enhance P21 expression through activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. The association between endometriosis and infertility is well supported throughout the literature, and endometriosis per se and its surgical treatment have an adverse effect on the ovarian reserve and on oocyte development. MALAT1, one of the most extensively expressed and evolutionarily conserved transcripts, has been implicated to play a role in human development and many diseases. However, little is known about the role of MALAT1 long non-coding RNA (lncRNA) in endometriosis and its associated infertility. We measured MALAT1 lncRNA expression levels in GCs from 52 endometriosis patients and 52 controls. Also, MALAT1 was knocked down in a human GC tumor-derived cell line, KGN, to investigate the role of MALAT1 and its molecular mechanism in cell proliferation. GCs were collected from women with or without endometriosis undergoing IVF or ICSI treatment. All endometriosis patients were diagnosed by laparoscopy or laparotomy, and control patients were limited to male factor or tubal disease and had a normal ovarian reserve. Quantitative real-time PCR (qRT-PCR) was used to measure the differential expression levels of MALAT1 lncRNA between endometriosis patients and controls. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic values of MALAT1 in endometriosis. In the KGN cell line, MALAT1 was knocked down with locked nucleic acid GapmeRs. Cell counting kit-8 assays, ethynyl-2-deoxyuridine assays and flow cytometry were used to study the role of MALAT1 in cell proliferation and cell-cycle progression, and western blotting was performed to detect the potential underlying mechanism. We first found that MALAT1 lncRNA was significantly down-regulated in endometriosis GCs and was associated with the antral follicle count (R = 0.376, P < 0.001 versus control). In addition, MALAT1 lncRNA levels were significantly lower in the GCs of infertile women with advanced stages of endometriosis (P = 0.01 versus control). The ROC curves illustrated strong separation between all the endometriosis patients and the control group (AUC: 0.705; 95% CI: 0.606-0.804; P < 0.001), Stage I-II and control group (AUC: 0.651; 95% CI: 0.536-0.767; P = 0.016), and Stage III-IV and control group (AUC: 0.827; 95% CI: 0.718-0.936; P < 0.001). MALAT1 lncRNA was primarily localized in the nuclei of GCs. We found a negative correlation between MALAT1 lncRNA and P21 mRNA in the GCs from patients (R = -0.628; P < 0.001). MALAT1 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression. In addition, MALAT1 knockdown induced an increase in both the mRNA and protein levels of P21, and of P53, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal protein kinase (p-JNK) protein levels, as well as causing a decrease in cyclin dependent kinase 2 (CDK2), cyclin D1 and p-P38 MAPK protein levels. Furthermore, inhibition of the ERK/MAPK pathway with U0126, the up-regulation of p-ERK1/2, P21 and P53, and the down-regulation of CDK2 and cyclin D1 by the knockdown of MALAT1 were all attenuated by MALAT1 knockdown. Therefore, MALAT1 may regulate GC proliferation through P21/P53-dependent control of the cell cycle, and the ERK/MAPK pathway participates in this process. None. The hormonal treatment used in IVF and surgical removal of endometriotic lesions may have altered MALAT1 expression in GCs. The ovarian granulosa-like tumor cell line, KGN, was used for further functional and mechanistic studies due to the difficulties in obtaining human GCs in sizable amounts and maintaining primary cultures. Our finding represents the first example of an lncRNA-based mechanism in endometriosis GCs. Women with endometriosis show altered MALAT1 expression levels in GCs that may impair fertility by regulating the function of GCs. Therefore, analysis of MALAT1 and its molecular mechanisms of action provide new insights into the pathogenesis of endometriosis and its associated infertility. This work was supported by the National Natural Science Foundation of China (grant number: 81671524) and the National key research and development program of China (grant number: 2017YFC1001100). The authors declare there is no conflict of interest.

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

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

Nuclear exclusion of SMAD2/3 in granulosa cells is associated with primordial follicle activation in the mouse ovary.

Maintenance and activation of the limited supply of primordial follicles in the ovary are important determinants of reproductive lifespan. Currently, the molecular programme that maintains the primordial phenotype and the early events associated with follicle activation are not well defined. Here, we have systematically analysed these events using microscopy and detailed image analysis. Using the immature mouse ovary as a model, we demonstrate that the onset of granulosa cell (GC) proliferation results in increased packing density on the oocyte surface and consequent GC cuboidalization. These events precede oocyte growth and nuclear translocation of FOXO3a, a transcription factor important in follicle activation. Immunolabelling of the TGFβ signalling mediators and transcription factors SMAD2/3 revealed a striking expression pattern specific to GCs of small follicles. SMAD2/3 were expressed in the nuclei of primordial GCs but were mostly excluded in early growing follicles. In activated follicles, GC nuclei lacking SMAD2/3 generally expressed Ki67. These findings suggest that the first phenotypic changes during follicle activation are observed in GCs, and that TGFβ signalling is fundamental for regulating GC arrest and the onset of proliferation.

Morphological study of apoptosis in granulosa cells and ovulation in a model of atresia in rat preovulatory follicles.

SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus-oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.

The role of fructose‑1,6‑bisphosphatase 1 in abnormal development of ovarian follicles caused by high testosterone concentration.

The present study aimed to identify the molecular mechanisms underlying the effects of the fructose-1,6-bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism-induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca-interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1×10−5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist-flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription-quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14-day-cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator-activated receptor γ coactivator-1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose-6-phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism-induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.