Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although ...the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin serine/threonine kinase) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.
Microplastics (MPs) considered as a new persistent environmental pollutant could enter into the circulatory system and result in decrease of sperm quantity and quality in mice. However, the effects ...of Polystyrene MPs (PS MPs) on the ovary and its mechanism in rats remained unclear. In this present study, thirty-two healthy female Wistar rats were exposed to different concentrations of 0.5 µm PS MPs dispersed in deionized water for 90 days. Using hematoxylin-eosin (HE) staining, the number of growing follicles was decreased compared to the control group. In addition, the activity of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were decreased while the expression level of malondialdehyde (MDA) was increased in ovary tissue. Confirmed by immunohistochemistry, the integrated optical density of NLRP3 and Cleaved-Caspase-1 had been elevated by 13.9 and 14 in granulosa cells in the 1.5 mg/kg/d group. Furthermore, compared to the control group, the level of AMH had been decreased by 23.3 pg/ml while IL-1β and IL-18 had been increased by 32 and 18.5 pg/ml in the 1.5 mg/kg/d group using the enzyme-linked immune sorbent assay (ELISA). Besides, the apoptosis of granulosa cells was elevated measured by terminal deoxyribonucleotide transferase-mediated nick end labeling (TUNEL) staining and flow cytometry. Moreover, western blot assays showed that the expressions of NLRP3/Caspase-1 signaling pathway related factors and Cleaved-Caspase-3 were increased. These results demonstrated that PS MPs could induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/Caspase-1 signaling pathway maybe triggered by oxidative stress. The present study suggested that exposure to microplastics had adverse effects on ovary and could be a potential risk factor for female infertility, which provided new insights into the toxicity of MPs on female reproduction.
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•Polystyrene microplastics could enter the rat ovary and be taken in by granulosa cells.•Polystyrene microplastics induce pyroptosis and apoptosis of ovarian granulosa cells.•Polystyrene microplastics induce pyroptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway.
MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional ...knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.
A central dogma of mammalian reproductive biology is that the size of the primordial follicle pool represents reproductive capacity in females. The assembly of the primordial follicle starts after ...the primordial germ cells (PGCs)-derived oocyte releases from the synchronously dividing germline cysts. PGCs initiate meiosis during fetal development. However, after synapsis and recombination of homologous chromosomes, they arrest at the diplotene stage of the first meiotic prophase (MI). The diplotene-arrested oocyte, together with the surrounding of a single layer of flattened granulosa cells, forms a basic unit of the ovary, the primordial follicle. At the start of each estrous (animal) or menstrual cycle (human), in response to a surge of luteinizing hormone (LH) from the pituitary gland, a limited number of primordial follicles are triggered to develop into primary follicles, preantral follicles, antral follicles and reach to preovulatory follicle stage. During the transition from the preantral to antral stages, the enclosed oocyte gradually acquires the capacity to resume meiosis. Meiotic resumption from the prophase of MI is morphologically characterized by the dissolution of the oocyte nuclear envelope, which is generally termed the "germinal vesicle breakdown" (GVBD). Following GVBD and completion of MI, the oocyte enters meiosis II without an obvious S-phase and arrests at metaphase phase II (MII) until fertilization. The underlying mechanism of meiotic arrest has been widely explored in numerous studies. Many studies indicated that two cellular second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play an essential role in maintaining oocyte meiotic arrest. This review will discuss how these two cyclic nucleotides regulate oocyte maturation by blocking or initiating meiotic processes, and to provide an insight in future research.
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•Polystyrene microplastics could enter into the ovary of rat.•Polystyrene microplastics cause ovary fibrosis via Wnt/β-Catenin signaling pathway.•Polystyrene microplastics cause ...granulosa cells apoptosis of ovary.•Polystyrene microplastics could result in decrease of ovarian reserve capacity.
Microplastics (MPs) are receiving increased attention as a harmful environmental pollutant. Studies have investigated that MPs have reproductive toxicity, but the mechanism is little known. Here, we aimed to investigate the effects of polystyrene microplastics (PS-MPs) on ovary in rats and the underlying molecular mechanisms. in vivo, thirty-two female Wistar rats were exposed to 0.5 μm PS-MPs at different concentrations (0, 0.015, 0.15 and 1.5 mg/d) for 90 days. And then, all animals were sacrificed, ovaries and blood were collected for testing. in vitro, granulosa cells (GCs) were separated from rat ovary and treated with 0、1、5、25 μg/mL PS-MPs and reactive oxygen species (ROS) inhibitor N-Acetyl-l-cysteine (NAC) respectively. Our results showed that PS-MPs could enter into GCs and result in the reducing of growing follicles number. And the Enzyme-linked immunosorbent assay (ELISA) manifested that PS-MPs could obviously decrease the level of anti-Müllerian hormone (AMH). In addition, PS-MPs induced oxidative stress, apoptosis of GCs and ovary fibrosis evidenced by assay kits, flow cytometry, immunohistochemistry, Masson’s trichrome and Sirius red staining. Moreover, the western blot assay manifested that PS-MPs exposure significantly increased the expression levels of Wnt/β-Catenin signaling pathways-related proteins (Wnt, β-catenin, p-β-catenin) and the main fibrosis markers (transforming growth factor-β (TGF-β), fibronectin, α-smooth muscle actin (α-SMA). Additionally, the expression levels of Wnt and p-β-catenin, apoptosis of GCs decreased after NAC treatment. In summary, polystyrene microplastics cause fibrosis via Wnt/β-Catenin signaling pathway activation and granulosa cells apoptosis of ovary through oxidative stress in rats, both of which ultimately resulted in decrease of ovarian reserve capacity.
It has been found that long noncoding RNA HOTAIR, microRNA‐130a (miR‐130a) and insulin‐like growth factor 1 (IGF1) expression are associated with ovarian cancer, thus, we hypothesised that the ...HOTAIR/miR‐130a/IGF1 axis might associate with endocrine disorders and biological behaviours of ovarian granulosa cells in rat models of polycystic ovary syndrome (PCOS). PCOS rat models were established by injection of dehydro‐isoandrosterone, followed by treatment of si‐HOTAIR, oe‐HOTAIR, miR‐130a mimics or miR‐130a inhibitors. Serum hormonal levels were determined to evaluate endocrine conditions. The effect of HOTAIR and miR‐130a on activities of isolated ovarian granulosa cells was assessed, as well as the involvement of IGF1.In the ovarian tissues and granulosa cells of PCOS rat models, highly expressed HOTAIR and IGF1 and poorly expressed miR‐130a were identified. In response to oe‐HOTAIR, serum levels of E2, T and LH were increased and serum levels of FSH were reduced; the proliferation of granulosa cells was reduced and apoptosis was promoted; notably, expression of miR‐130a was reduced while expression of IGF1 was increased. The treatment of si‐HOTAIR reversed the situation. Furthermore, the binding of HOTAIR to miR‐130a and targeting relationship of miR‐130a and IGF1 were confirmed. LncRNA HOTAIR up‐regulates the expression of IGF1 and aggravates the endocrine disorders and granulosa cell apoptosis through competitive binding to miR‐130a in rat models of PCOS. Based on our finding, we predict that competitive binding of HOTAIR to miR‐130a may act as a novel target for the molecular treatment of PCOS.
Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in child‐bearing‐age women. It is characterized by ovulation dysfunction, polycystic ovaries, and hyperandrogenism. ...Inflammation is likely to be a crucial contributor to the pathogenesis of PCOS. However, the association between the inflammatory cytokines and the development of PCOS has not been reported. To explore the relationship between the inflammatory cytokines and PCOS, alterations of serum proteins in dehydroepiandrosterone (DHEA)‐induced PCOS rats were screened by protein array, and the concentration of IFN‐γ was further measured by using enzyme‐linked immunosorbent assay (ELISA). DHEA‐induced PCOS rats had a decreased level of IFN‐γ compared with the control rats, which was restored partly in flutamide (an androgen receptor antagonist)‐treated rats. Moreover, the level of IFN‐γ in serum of patients with PCOS was also lower than that in healthy women. Using the ovarian granulosa cells (KGN), we demonstrated that DHEA downregulated the expression and secretion of IFN‐γ in dose‐ and time‐dependent manners, which could be restored to some extent by treating with flutamide. Furthermore, flutamide ameliorated the inhibitory effect on cell proliferation and promotive effect on cell apoptosis by DHEA. The results also revealed that IFN‐γ promoted the proliferation but inhibited the apoptosis of KGN cells, which was suppressed by DHEA via activating the downstream PI3K/AKT signaling pathway. Taken together, these results showed that DHEA inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells through downregulating the expression of IFN‐γ which could be restored by flutamide, and IFN‐γ may serve as a potential inflammatory biomarker for PCOS detection.
To explore the relationship between the inflammation cytokines and PCOS, alterations of serum proteins in dehydroepiandrosterone (DHEA)‐induced PCOS rats were screened by protein array, and the concentration of IFN‐γ was further measured by using the sandwich enzyme‐linked immunosorbent assay. DHEA‐induced PCOS rats had a decreased level of IFN‐γ compared with the control rats, which was restored partly in flutamide‐treated rats. Using the KGN cell line, we demonstrated that the DHEA downregulated the expression and secretion of IFN‐γ, which could be restored to some extent by treating with flutamide. As the results showed, DHEA inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells through downregulating the expression of IFN‐γ which could be restored by flutamide, and IFN‐γ may serve as a potential inflammation biomarker for PCOS detection.
Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte ...maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 μM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative stress released exosomes enriched with mRNA of Nrf2 and candidate antioxidants. Subsequent co-incubation of StressExo with cultured granulosa cells could alter the relative abundance of cellular oxidative stress response molecules including Nrf2 and antioxidants CAT, PRDX1 and TXN1. The present study provide evidences that granulosa cells exposed to oxidative stress conditions react to stress by activating cascades of cellular antioxidant molecules which can also be released into extracellular environment through exosomes.
Reactive oxygen species (ROS) play important roles in follicular development and survival. Granulosa cell death is associated with increased ROS, but the mechanism of granulosa cell death induced by ...ROS is not clear. In order to define the molecular link between ROS and granulosa cell death, COV434, human granulosa tumor cells, were treated with H2O2. Compared to control cells, H2O2 induced granulosa cell death in a dose- and time-dependent manner. H2O2 induced an increase in Bax, Bak and Puma, and a decrease in anti-apoptotic molecules such as Bcl-2, Bcl-xL and Mcl-1. Both knockdown of Puma and overexpression of Bcl-xL could inhibit H2O2-induced granulosa cell death. These results suggest that suppression of Puma and overexpression of anti-apoptotic Bcl-2 family members could improve granulosa cell survival. To explore the mechanisms responsible for these findings, ROS in granulosa cells treatment with H2O2 were measured. The results showed that ROS was increased in a H2O2 dose- and time-dependent manner at the earlier time point. In addition, H2O2 induced an increase in Nrf2 and phosphorylation of JNK and p53. SP600125, an inhibitor of JNK, inhibits H2O2-induced phosphorylation of JNK and p53, and granulosa cell death. Antioxidant N-acetylcysteine (NAC) dose-dependently prevents H2O2-induced granulosa cell death. Furthermore, NAC also prevents phosphorylation of JNK and p53 induced by H2O2. Taken together, these data suggest that H2O2 regulates cell death in granulosa cells via the ROS-JNK-p53 pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be useful for future clinical applications.
Excessive fluoride intake has a strong female reproductive toxicity, which can result in follicular developmental dysplasia and decrease oocytes developmental potential. The underlying mechanisms of ...fluoride-induced mitochondrial dysfunction in ovarian granulosa cells remain largely unknown. In this study, the ultrastructure changes of mitochondria and DNA damage in ovarian granulosa cells were observed under transmission electron microscope and TUNEL staining. Then, the ATP content and ROS level in granulosa cells were measured. The expression of mitochondrial fusion proteins and mitochondrial respiratory chain complexes, including OPA1 and Mfn1, and NDUFV2, SDHA and CYC1, in the ovarian tissues were measured by immunohistochemistry, Western blot and Quantitative real-time PCR analyses. The expression of ATP5j and ATP5h in the ovarian tissues was also measured. Results show that fluoride treatment considerably damages mitochondrial ultrastructure and enhances the apoptosis of granulosa cells. The ATP content greatly decreased, whereas the ROS level increased after fluoride treatment. The expression level of Mfn1 in the ovarian tissue was up-regulated, whereas OPA1 expression had no significant change. The expression levels of NDUFV2, SDHA and CYC1 were considerably up-regulated, and the expression of ATP5j and ATP5h were down-regulated after fluoride treatment. In summary, the damage in the mitochondrial ultrastructure, ATP content decrease, ROS level increase and the abnormal expression of OPA1, Mfn1, NDUFV2, SDHA, CYC1, ATP5j and ATP5h in ovary tissue are closely associated with fluoride-induced mitochondrial dysfunction, which might be responsible for the follicular developmental dysplasia and the potential decrease in oocyte development induced by fluoride in female mice.
•Fluoride damaged mitochondrial ultrastructure in ovarian granulosa cells.•Fluoride induced mitochondrial fusion disorder.•Fluoride interfered with the expression of mitochondrial respiratory chain complex.•Fluoride decreased ATP content and increased ROS levels.•Fluoride induced mitochondrial dysfunction in ovarian granulosa cells.
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