Human Granulosa Cells Research Articles

A Longitudinal Study of the Relationship of Adiponectin with Reproduction in Infertile Women Undergoing IVF/ICSI Treatment, and an Experimental Study in Human Granulosa Cells.

This study investigated the roles of adiponectin in IVF treatment during Phase I (the basal stage before gonadotropin administration), Phase II (approximately 8 days after gonadotropin administration), and Phase III (on the ovum pick-up day), as well as the effects of adiponectin on CYP19A1 and the FSH receptor (FSHR) mRNA expression in a human granulosa-like tumor cell line (KGN). In human subjects (a longitudinal study, n = 30), blood samples were collected in all phases, while follicular fluid (FF) was only collected in Phase III. The participants were classified into successful and unsuccessful groups based on the determination of fetal heartbeats. KGN cells were treated with adiponectin/FSH/IGF-1 (an experimental study, n = 3). There was no difference in the adiponectin levels between successful and unsuccessful pregnancies in the FF (Phase III) and in serum (all phases), as well as among the three phases in both groups. Serum FSH (Phase I) was positively associated with serum adiponectin in the unsuccessful group, but it had a negative association in the successful group (all phases). Serum adiponectin and serum FSH (Phase I) were positively correlated in the unsuccessful group, whereas they were negatively correlated (all phases) in the successful group. The serum adiponectin levels (Phase III) were significantly higher than in the FF in unsuccessful pregnancies, but there was no difference in successful pregnancies. FF adiponectin concentrations were negatively correlated with serum LH in successful subjects. In KGN cells, adiponectin had no influence on CYP19A1 and FSHR mRNA expression. High adiponectin levels in serum compared to FF (Phase III) in unsuccessful subjects might negatively impact IVF treatment.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

ABCC4 is a PGE2 efflux transporter in the ovarian follicle: A mediator of ovulation and a potential non-hormonal contraceptive target.

The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non-hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility.

CLPP inhibition triggers apoptosis in human ovarian granulosa cells via COX5A abnormality-Mediated mitochondrial dysfunction.

Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function before the age of 40years. It is confirmed to have a strong and indispensable genetic component. Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins, which is necessary to maintain mitochondrial function. Previous findings have shown that the variation in CLPP is closely related to the occurrence of POI, which is consistent with our findings. This study identified a novel CLPP missense variant (c.628G > A) in a woman with POI who presented with secondary amenorrhea, ovarian dysfunction, and primary infertility. The variant was located in exon 5 and resulted in a change from alanine to threonine (p.Ala210Thr). Importantly, Clpp was mainly localized in the cytoplasm of mouse ovarian granulosa cells and oocytes, and was relatively highly expressed in granulosa cells. Moreover, the overexpression of c.628G > A variant in human ovarian granulosa cells decreased the proliferative capacity. Functional experiments revealed that the inhibition of CLPP decreased the content and activity of oxidative respiratory chain complex IV by affecting the degradation of aggregated or misfolded COX5A, leading to the accumulation of reactive oxygen species and reduction of mitochondrial membrane potential, ultimately activating the intrinsic apoptotic pathways. The present study demonstrated that CLPP affected the apoptosis of granulosa cells, which might be one of the mechanisms by which CLPP aberrations led to the development of POI.

A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells.

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar three-dimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S- and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6β1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Laminin-mimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6β1.

Pterostilbene ameliorates oxidative damage and ferroptosis in human ovarian granulosa cells by regulating the Nrf2/HO-1 pathway

The survival of ovarian granulosa cells is of great significance to the physiological maintenance of the ovary. Oxidative damage to the ovarian granulosa cells can lead to various diseases related to ovarian dysfunction. Pterostilbene exerts many pharmacological effects, such as anti-inflammatory and cardiovascular protective effects. Moreover, pterostilbene was shown to have antioxidant properties. This study aimed to investigate the effect and underlying mechanism of pterostilbene on oxidative damage in ovarian granulosa cells. Ovarian granulosa cell (OGC) lines COV434 and KGN were exposed to H2O2 to establish an oxidative damage model. After treatment with different concentrations of H2O2 or pterostilbene, the cell viability, mitochondrial membrane potential, oxidative stress, and iron levels were detected, and the expression of ferroptosis-related and Nrf2/HO-1 signaling pathway-related proteins were evaluated. Pterostilbene treatment could effectively improve cell viability, reduce oxidative stress, and inhibit ferroptosis stimulated by H2O2. More importantly, pterostilbene could up-regulate Nrf2 transcription by stimulating histone acetylation, and inhibition of Nrf2 signaling could reverse the therapeutic effect of pterostilbene. In conclusion, this research shows that pterostilbene protects human OGCs from oxidative stress and ferroptosis through the Nrf2/HO-1 pathway.

Assessment of Epiregulin Effect and its Combination with Gonadotropins on Proliferation, Apoptosis, and Secretory Activity by Human Ovarian Cells.

The release of epidermal growth factor ligand epiregulin (EREG) by human ovarian granulosa cells, its direct action on basic ovarian cell functions, and interrelationships with gonadotropins were investigated. We examined (1) the ovarian production of EREG (the time-dependent accumulation of EREG in the medium incubated with human ovarian granulosa cells, and (2) the effect of the addition of EREG (0, 1, 10, and 100ng.ml-1) given alone or in combination with FSH or LH (100ng.ml-1) on basic granulosa cells functions. Viability, proliferation (accumulation of PCNA and cyclin B1) and apoptosis (accumulation of bax and caspase 3), the release of steroid hormones (progesterone, testosterone, and estradiol), and prostaglandin E2 (PGE2) were analyzed by using the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. A significant time-dependent accumulation of EREG in a medium cultured with human granulosa cells with a peak at 3 and 4days was observed. The addition of EREG alone increased cell viability, proliferation, progesterone, testosterone, and estradiol release, decreased apoptosis, bud did not affect PGE2 release. The addition of either FSH or LH alone increased cell viability, proliferation, progesterone, testosterone, estradiol, and PGE2 release and decreased apoptosis. Furthermore, both FSH and LH mostly promoted the stimulatory action of EREG on granulosa cell functions. These results demonstrated, that EREG produced by ovarian cells can be an autocrine/paracrine stimulator of human ovarian cell functions. Furthermore, they demonstrate the functional interrelationship between EREG and gonadotropins in the control of ovarian functions.

Divergent roles of Sirtuin 1 in human granulosa-lutein cells: similarities to hCG.

Sirtuin 1 (SIRT1) is an NAD+ dependent deacetylase that modifies gene expression through histone deacetylation. It also deacetylates non-histone substrates, e.g. tumor suppressor p53, NOS3, HIF1A, NFKB, FOXO3a, PGC-1α, and PPARγ. Consequently, it regulates a wide range of physiological functions including cell cycle control, energy expenditure, oxidative stress response, apoptosis, and aging. SIRT1 is expressed in ovarian granulosa cells (GCs) of various species including humans at different stages of the reproductive cycle. The importance of SIRT1 in female reproduction is supported by the findings that SIRT1-knockout mice exhibit defects in reproductive tissue development. These mice were found to have a thin-walled uterus, small ovaries, with follicles present but no corpora lutea. This review aims to provide state-of-the-art information on SIRT1's mode of action and its roles in human granulosa-lutein cells and GCs from other species where data is available. It also discusses the overlapping actions of SIRT1 and hCG on the production of critical GC-borne factors.

PPM1K-regulated impaired catabolism of branched-chain amino acids orchestrates polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common diseases with the coexistence of reproductive malfunction and metabolic disorders. Previous studies have found increased branched chain amino acid (BCAA) levels in women with PCOS. However, it remains unclear whether BCAA metabolism is causally associated with the risk of PCOS. The changes of BCAA levels in the plasma and follicular fluids of PCOS women were detected. Mendelian randomization (MR) approaches were used to explore the potential causal association between BCAA levels and the risk of PCOS. The function of the gene coding the protein phosphatase Mg2+/Mn2+-dependent 1K (PPM1K) was further explored by using Ppm1k-deficient mouse model and PPM1K down-regulated human ovarian granulosa cells. BCAA levels were significantly elevated in both plasma and follicular fluids of PCOS women. Based on MR, a potential direct, causal role for BCAA metabolism was revealed in the pathogenesis of PCOS, and PPM1K was detected as a vital driver. Ppm1k-deficient female mice had increased BCAA levels and exhibited PCOS-like traits, including hyperandrogenemia and abnormal follicle development. A reduction in dietary BCAA intake significantly improved the endocrine and ovarian dysfunction of Ppm1k-/- female mice. Knockdown of PPM1K promoted the conversion of glycolysis to pentose phosphate pathway and inhibited mitochondrial oxidative phosphorylation in human granulosa cells. Ppm1k deficiency-impaired BCAA catabolism causes the occurrence and development of PCOS. PPM1K suppression disturbed energy metabolism homeostasis in the follicular microenvironment, which provided an underlying mechanism of abnormal follicle development. This study was supported by the National Key Research and Development Program of China (2021YFC2700402, 2019YFA0802503), the National Natural Science Foundation of China (81871139, 82001503, 92057107), the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-001), Key Clinical Projects of Peking University Third Hospital (BYSY2022043), the China Postdoctoral Science Foundation (2021T140600), and the Collaborative Innovation Program of Shanghai Municipal Health Commission (2020CXJQ01).

Chitosan oligosaccharide improves ovarian granulosa cells inflammation and oxidative stress in patients with polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is the most common reproductive endocrine disorder among women of reproductive age, which is one of the main causes of anovulatory infertility. Even though the rapidly developed assisted reproductive technology (ART) could effectively solve fertility problems, some PCOS patients still have not obtained satisfactory clinical outcomes. The poor quality of oocytes caused by the abnormal follicular development of PCOS may directly contribute to the failure of ART treatment. Ovarian granulosa cells (GCs) are the most closely related cells to oocytes, and changes in their functional status have a direct impact on oocyte formation. Previous studies have shown that changes in the ovarian microenvironment, like oxidative stress and inflammation, may cause PCOS-related aberrant follicular development by impairing the physiological state of the GCs. Therefore, optimizing the ovarian microenvironment is a feasible method for enhancing the development potential of PCOS oocytes. In this study, we first detected the expression of inflammatory-related factors (TGF-β1, IL-10, TNFα, IL-6) and oxidative stress-related factors (HIF-1α and VEGFA), as well as the proliferation ability and apoptosis level of GCs, which were collected from control patients (non-PCOS) and PCOS patients, respectively. Subsequently, human ovarian granulosa cell line (KGN) cells were used to verify the anti-inflammatory and anti-oxidative stress effects of chitosan oligosaccharide (COS) on GCs, as well as to investigate the optimal culture time and concentration of COS. The optimal culture conditions were then used to culture GCs from PCOS patients and control patients. The results showed that GCs from PCOS patients exhibited obvious inflammation and oxidative stress and significantly reduced proliferation and increased apoptosis. Furthermore, COS can increase the expression of anti-inflammatory factors (TGF-β1 and IL-10) and decrease the expression of pro-inflammatory factors (TNFα and IL-6), as well as promote the proliferation of GCs. Moreover, we found that COS can reduce the level of reactive oxygen species in GCs under oxidative stress by inhibiting the expression of HIF-1α and VEGFA and by suppressing the apoptosis of GCs induced by oxidative stress. We find that inflammation and oxidative stress exist in the GCs of PCOS patients, and COS can reduce these factors, thereby improving the function of GCs.

The Gengnianchun recipe attenuates insulin resistance-induced diminished ovarian reserve through inhibiting the senescence of granulosa cells.

Insulin resistance (IR) is found in patients with polycystic ovary syndrome (PCOS), but the effects and mechanisms of IR on diminished ovarian reserve (DOR) remain unclear. This study set out to investigate the effects of IR on ovarian reserve; to explore the effects of high concentrations of insulin on the function of ovarian cells in vitro; and to validate the hypothesis that the Gengnianchun recipe (GNC) helps to attenuate DOR caused by IR through reducing the senescence of granulosa cells. Estrus cycle, follicle count, and sex hormone levels were detected to evaluate ovarian function in mice with IR caused by feeding a high-fat diet (HFD). In addition, KGN cells (human granulosa cell line) were treated with high concentrations of insulin. The staining for senescence-associatedβ-galactosidase (SA-β-gal), cell cycle, and expression levels of mRNA and gene proteins related to cell aging were detected in KGN cells treated with high concentrations of insulin. Mice treated with an HFD were fed metformin, GNC, or saline solution for 6 weeks by oral gavage. HOMA-IR, the area under the curve (AUC) of the oral glucose tolerance test (OGTT), levels of fasting blood glucose (FBG), and fasting serum insulin (FINS) were examined to confirm the IR status. Then estrus cycle, follicle count, and sex hormone levels were detected to evaluate ovarian function. Expression levels of mRNA and gene proteins related to cell aging were detected in the ovarian tissue of mice in each group. The results demonstrated that IR reduced murine ovarian reserves, and high doses of insulin caused granulosa cells to senesce. There was a considerable improvement in HFD-induced IR status in the metformin (Met) and GNC treatment groups. In addition, the expression levels of aging-associated biomarkers were much lower in GNC mice than Met mice; and both the latter groups had considerably lower levels than the HFD group. Moreover, higher follicle counts in different stages and shorter diestrus in the Met or GNC groups compared to the HFD group indicated that ovarian aging could be largely reversed. This work showed that: IR impaired ovarian reserve; high concentrations of insulin induced granulosa cell aging; and GNC attenuated ovarian function through inhibiting IR-induced cell aging.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

Protective effect of L‑carnitine against oxidative stress injury in human ovarian granulosa cells.

Granulosa cells (GCs) are important for supporting and nourishing oocytes during follicular development and maturation. Oxidative stress (OS) injury of GCs can lead to decreased responsiveness of follicles to follicular stimulating hormone (FSH), which will accelerate ovarian senescence and adversely affect oocyte and embryo quality. Since L-carnitine has been previously reported to exert strong antioxidant activity, the present study aimed to explore the possible effects of L-carnitine on OS injury and FSH receptor (FSHR) expression in ovarian GCs, results of which may be of significance for GCs protection. In the present study, OS was induced in vitro in KGN cells by treatment with H2O2. KGN cells were cultured and divided into the following four groups: Blank, OS, and 40 and 80 µmol/l L-carnitine pre-treatment groups. In the OS group, cells showed nuclear pyknosis, mitochondria swelled irregularly whilst featuring fractured cristae. In addition, cell viability, ROS levels, superoxide dismutase levels, glutathione levels, malondialdehyde levels, the mitochondrial membrane potential and FSHR expression, as determined by Cell Counting Kit-8 (CCK-8), 2,7-dichloro-dihydrofluorescein diacetate, spectrophotometry, ELISA, spectrophotometry, JC-1 and western blot analyses, respectively, were all significantly different in the OS group compared with those in the control group. However, malonaldehyde levels, reactive oxygen species levels and the apoptosis rate according to flow cytometry were all significantly increased compared with those in the control. Compared with those in the OS group, the morphology of cells and mitochondria in the L-carnitine pre-treatment groups were improved, whilst cell viability and the expression of FSHR were significantly increased but oxidative stress injury was decreased. The present results suggest that L-carnitine can protect the cells from OS damage induced by H2O2, enhance antioxidant activity whilst suppressing the apoptosis of GCs, in addition to preserving FSHR expression in GCs under OS. Therefore, the present study revealed that the introduction of L-carnitine in clinical medicine or dietary supplement may protect GCs, improve follicular quality and female reproductive function.

Resveratrol improves ovarian state by inhibiting apoptosis of granulosa cells

Aim Among the natural polyphenolic compounds, resveratrol (RES) is known for reducing the effects of declining reproductive power through resisting senility, anti-oxidant and anti-inflammatory, while the molecular mechanism of RES in human ovaries is unclear. We aimed to evaluate the most likely mechanisms of RES against apoptosis induced by H2O2 in human ovary granulosa cells. Methods Ovarian granulosa cells from infertile women (≤35 years old) were collected. Those patients defined as polycystic ovary syndrome (PCOS), poor ovarian responder (POR) and Endometriosis were excluded. Then they were randomly divided into control group, model group and the treatment group. Cellular apoptosis was analyzed by flow cytometer method. The related protein and mRNA expressions were detected by western blot and RT-PCR. Results Apoptosis rates of the treatment group containing RES with concentrations of 1 μM and 10 μM were significantly decreased (p < 0.001). Western blot results demonstrated that the proteins levels of transforming growth factor-β (TGF-β), Bax and Caspase 9 were decreased, and Bcl-2 was increased under RES treatment, while the protein levels of Caspase 8, Caspase 3, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) expressed no significant difference. The results by RT-PCR of follicle and ovarian development related mRNA factors were consistent with that of western blot assay. Conclusion In conclusion, the present study provides the evidence that RES may affects apoptotic factors to protect human ovarian state.

Negative effects of endocrine disruptor bisphenol A on ovarian granulosa cells and the protective role of folic acid.

Bisphenol A (BPA) is a widely produced chemical, mostly used in the production of polycarbonate plastics, and can act as an endocrine disruptor. This paper focuses on the different effects of BPA on ovarian granulosa cells. Bisphenol A (BPA) is an endocrine disruptor (ED), widely used as a comonomer or an additive in the plastics industry. It can be found in food and beverage plastic packaging, epoxy resins, thermal paper and other common products. To date, there have only been several experimental studies to have examined how BPA exposure affects human and mammalian follicular granulosa cells (GCs) in vitro and in vivo; the collected evidence data show that BPA negatively affects the GCs by altering steroidogenesis and gene expression, inducing autophagy, apoptosis and cellular oxidative stress through reactive oxygen species production. Exposure to BPA can also lead to abnormally constrained or elevated cellular proliferation and can even reduce cell viability. Therefore, research on EDs such as BPA is important as it provides some important insights into the causes and development of infertility, ovarian cancer and other conditions related to impaired ovarian and GC function. Folic acid, a biologic form of vitamin B9, is a methyl donor that can neutralize the toxic effects of the BPA exposure and is, as a common food supplement, an interesting option for researching its protective role against ubiquitous harmful EDs such as BPA.

Carnosol inhibits KGN cells oxidative stress and apoptosis and attenuates polycystic ovary syndrome phenotypes in mice through Keap1-mediated Nrf2/HO-1 activation.

Excessive oxidative stress and apoptosis of ovarian granulosa cells lead to abnormal follicular development and ovulation disorders in polycystic ovary syndrome (PCOS). Carnosol is a plant-derived polyphenol that has been proven to exhibit several cell protective effects. In this study, we established hyperandrogenic PCOS models both in vitro and in vivo. In the humanovariangranulosacellline,KGN cells, decreased viability and mitochondrial membrane potential, and upregulated reactive oxygen species (ROS) level and apoptosis induced by DHT were partly reversed by carnosol. Western blotting results showed that carnosol treatment inhibited the DHT-activated mitochondrial apoptotic pathway by activating nuclear factor-erythroid 2-related factor (Nrf2)/heme oxygenase 1 (HO-1). Knockdown of Nrf2 by transfecting with siRNA or inhibiting HO-1 by zinc protoporphyrin (ZnPP) blocked the protective effects of carnosol. Computational modeling and pull-down assay results confirmed the direct binding of carnosol to kelch-like ECH-associated protein 1 (Keap1). In vivo results showed that the intraperitoneal administration of carnosol (50 and 100 mg/kg) improved estrous cycle disorders, polycystic ovary, and decreased elevated androgen in the PCOS mice. In summary, Carnosol has an effective role in inhibiting oxidative stress and apoptosis in DHT-treated KGN cells and protecting against mouse PCOS phenotypes through the Keap1-mediated activation of Nrf2/HO-1 signaling.

MicroRNA-1298-5p in granulosa cells facilitates cell autophagy in polycystic ovary syndrome by suppressing glutathione-disulfide reductase.

The aim of this study was to investigate the effect and mechanism of action of miR-1298-5p in polycystic ovary syndrome (PCOS). Granulosa cells were isolated from follicular fluid of patients with PCOS and healthy women, and the expression of miR-1298-5p and glutathione-disulfide reductase (GSR) mRNA in these cells was evaluated using reverse transcription-quantitative polymerase chain reaction (qRT-PCR). Clinical data were obtained from all subjects, and reproductive hormones and endocrine indices were assayed to analyze the correlation between miR-1298-5p and clinicopathological characteristics of patients with PCOS. Following transfection with the miR-1298-5p mimic or inhibitor and/or pcDNA3.1-GSR, LC3 immunofluorescence and transmission electron microscopy were used to evaluate autophagy in the COV434 human granulosa cell line. Additionally, western blotting was performed to detect LC3-II, Beclin 1, and p62 protein levels in COV434 cells. The interaction between miR-1298-5p and GSR was also examined. A PCOS rat model was established and injected with the miR-1298-5p antagomir, followed by measurement of body and ovary weights, histological examination, and autophagosome observation. The protein expression levels of GSR, LC3-II, Beclin 1, and p62 were determined in rat ovaries. miR-1298-5p was expressed at a high level, and GSR was downregulated in granulosa cells from patients with PCOS. In COV434 cells, miR-1298-5p inversely mediated GSR expression, and miR-1298-5p mimic transfection promoted autophagy, whereas GSR overexpression blocked miR-1298-5p mimic-promoted autophagy. In PCOS rats, miR-1298-5p inhibition reduced autophagy and alleviated abnormalities in follicular development. Overall, miR-1298-5p enhances autophagy in granulosa cells by downregulating GSR, thereby affecting PCOS development.

Malvidin-3-O-Glucoside Ameliorates Cadmium-Mediated Cell Dysfunction in the Estradiol Generation of Human Granulosa Cells.

Cadmium (Cd) is a frequent environmental pollutant associated with biological toxicity that can harm female reproduction. Anthocyanins have been reported to reduce the toxicity of Cd. In the present study, the protective effects and underlying mechanisms of malvidin-3-O-glucoside (M3G) against the toxicity of Cd on female reproduction in KGN cells (human ovarian granulosa-like tumor cells) were investigated. After treating cells with 10 µmol/L cadmium chloride, the results showed that M3G lessened Cd-induced KGN cell cytotoxicity better than malvidin and malvidin-3,5-O-diglucoside. Additionally, M3G significantly decreased the Cd-induced generation of reactive oxygen species, inhibited the Cd-induced arrest of the G2/M phase of the cell cycle, and increased estradiol (E2) production. According to transcriptomic results, M3G reduced the abnormal expression of genes that responded to estrogen. Additionally, M3G promoted the endogenous synthesis and secretion of E2 by controlling the expression of CYP17A1 and HSD17B7. The current findings indicated that M3G is of great potential to prevent Cd-induced female reproductive impairment as a dietary supplement.

Oxytocin enhances progesterone production with upregulation of BMP-15 activity by granulosa cells

To elucidate the reproductive role of oxytocin (OXT) in ovarian steroidogenesis and its functional interaction with bone morphogenetic proteins (BMPs), the effects of OXT on ovarian steroidogenesis were investigated by utilizing primary culture of rat granulosa cells and human granulosa KGN cells. Here we revealed that the OXT receptor was expressed in both rat and human granulosa cells and that OXT treatment significantly increased follicle-stimulating hormone (FSH)- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat and human granulosa cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA expression of CYP11A1 and HSD3B2 induced by FSK in human granulosa cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the transcription of ID1 induced by BMP-15, but not those induced by BMP-6, in human granulosa cells. It was also revealed that OXT treatment upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by human granulosa cells. Collectively, it was shown that OXT accelerates ovarian progesterone synthesis with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis (186 words).

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells.

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC.