Sigma-1 Receptor Is Involved In Diminished Ovarian Reserve Possibly Via Influencing Endoplasmic Reticulum Stress-mediated Granulosa Cell Apoptosis

Diminished Ovarian Reserve(DOR)refers to the reductions in oocyte quantity and quality,those women' ability to produce effective oocytes is impaired.DOR is found in approximately 10% of infertile women and it represents a major challenge in women's reproductive health.Previous studies on DOR mainly focused on finding effective indicators that could predict and evaluate ovarian function.However,there are few studies about the exact pathogenesis of DOR.There are lots of external factors affecting ovarian reserve,such as age,body mass index,menarche age,menstrual cycle,genetic factors and internal factors like infection,gynecologic surgery,radiotherapy,chemotherapy,psychological and environmental factors.A great number of studies of DOR reported that follicular atresia during the progression of DOR is primarily induced by granulosa cells apoptosis.Ovarian granulosa cells apoptosis is the fundamental initiating factor for the pathogenesis of DOR,many research teams have focused on it.Sigma non-opioid intracellular receptor 1(Sigma-1 receptor),a non-opioid transmembrane protein,is located on mitochondrial membranes and the endoplasmic reticulum(ER),and expresses in a broad range of tissues.Previous studies have demonstrated that Sigma-1 receptor is a degenerative disease-related protein via protein-protein interactions.In recent years,a series of studies have found that Sigma-1 receptor has protective effects in several progressions of diseases,such as celluar signal transduction,mitochondrial functions,oxidative stress,cell apoptosis and tumor cells proliferation.Different Sigma-1 receptor agonists or antagonists may regulate functional cells via relevant pathways and affect the disease.Sigma-1 receptor is a ligand-regulated membrane chaperone protein associated with ERS.Ovary is the most sensitive organ to aging.There are few studies about the relationship between Sigma-1 receptor and ovarian reserve.We speculated that Sigma-1 receptors may participate in female ovarian degenerative diseases progressions,affects ovarian granulosa cells apoptosis,and plays a protective effect in the process of DOR.Part ? The expression of Sigma-1 receptor in ovarian and therelationships between Sigma-1 receptor levels and ovarian reserveResearch purposes To detect the expression of Sigma-1 receptor in ovarian tissues,plasma,follicular fluid and granulosa cells,and to analyse the relationships between the Sigma-1 receptor levels in plasma/follicular fluid and ovarian reserve.Research methodsIn immunohistochemistry part,we choosed mature female Kunming white mice and normal women' ovaries as study subjects,and investigated the expression of Sigma-1 receptor in human and mice ovarian tissues.Patients who underwent IVF treatment with the gonadotropin-releasing hormone antagonist(GnRH-ant)protocol were chosen as target patients.We divided those patients into normal ovarian reserve(NOR)group and DOR group.In the retrieved oocyte day,we collocted the first tube of clear follicular fluid and extracted granulosa cells by lymphocyte separation liquid method.The levels of Sigma-1 receptor in plasma,follicular fluid were determined by enzyme-linked immunosorbent assay(ELISA)and Sigma-1 receptor in granulosa cells were determined by flow cytometry(FCM).The correlations between Sigma-1 receptor expression in plasma/follicular fluid and clinical data were analyzed.ResultsSigma-1 receptor was widely expressed in human and mice ovarian tissue.Positive immunostaining was observed in the cytoplasm or nucleus or both,and is expressed significantly in luteinized granulosa cells.Sigma-1 receptor protein level was decreased in the serum,follicular fluid,and granulosa cells of women with DOR.There were negative correlations of Sigma-1 receptor concentration in follicular fluid/serum with basal Follicle-stimulating hormone(FSH)and maternal age.Sigma-1 receptor level in follicular fluid/serum was positively correlated with serum anti-mullerian hormone(AMH),retrieved oocytes,and available embryos.ConclusionsLow expression of Sigma-1 receptor is correlates with DOR.Sigma-1 receptor may be involved in the regulation of ovarian function.Its expression levels may reflect the patients' ovarian response.Sigma-1 receptor might be a novel potential ovarian reserve biomarkers.Part ? The effects of Sigma-1 receptor on granulosa cell apoptosis and its possible mechanismResearch purposesFrom the perspective of the apoptosis of ovarian granulosa cells,to explore the relationships between Sigma-1 receptor and DOR pathogenesis.Research methodsFCM was used to detect the apoptosis index of granulosa cells in different ovarian reserve patients.Real time quantitative polymerase chain reaction(qRT-PCR)assays showed the mRNA levels of the ERS and apoptosis-related genes in granulosa cells.We constructed SIGMAR1 knockdown/overexpressing KGN cell lines to explore the effects of Sigma-1 receptor on ERS and apoptosis-related gene expression levels.ResultsDecreasing Sigma-1 receptor expression was accompanied by increased apoptosis rate in the GCs of women with DOR.Compared with NOR group,the expression levels of ERS-related genes BIP,CHOP,ATF4 and ATF6 were increased significantly in DOR patients' granulosa cells.SIGMAR1 knockdown/overexpressing plasmids were constructed successfully.SIGMAR1-knockdown KGN cells and DOR patients' granulosa cells showed similar ERS and apoptosis-related gene expression levels.SIGMAR1-knockdown KGN cells and SIGMAR1 overexpressing KGN cells showed the opposite gene expression levels.ConclusionsThe Sigma-1 receptor is associated with ERS-induced apoptosis and it may regulate granulosa cell apoptosis by affecting the ATF6/ATF4-CHOP-ERS signaling pathways.Part ? DHEA affect endoplasmic reticulum stress apoptosis pathway in ovarian granulosa cells by Sigma-1 receptor agonist-like effectsResearch purposesTo investigate the possible mechanism of endogenous Sigma-1 receptor agonist dehydroepiandrosterone(DHEA)in ovarian granulosa cells apoptosis.In order to provide a new explanation for DHEA improves DOR patients' treatment outcomes.Research methodsThapsigargin(TG)was used to induce ERS in KGN cells.KGN cells with ERS were cultured with a classical Sigma-1 receptor agonist PRE-084 or DHEA.Then we detected apoptotic rates,ERS and apoptosis-related genes expression levels.The effectiveness of DHEA in DOR patients' treatment was discussed with clinical data.Results1?mol/L TG could induce ERS in KGN cells effectively.After TG-induced treatment,cell apoptosis rate was significantly increased.PRE-084/DHEA-TG-induced KGN cells showed similar experimental results.Compared with TG-induced KGN cells,PRE-084/DHEA-TG-induced KGN cells apoptosis rates were decreased significantly,and the expression levels of BIP,CHOP,ATF4 and ATF6 mRNA were decreased.The clinical data showed supplementation with DHEA in DOR patients could improve their pregnancy outcomes.ConclusionsActivation of Sigma-1 receptor by DHEA may regulate the granulosa cells' apoptosis by ATF4/ATF6-CHOP-ERS pathway.Sigma-1 receptor could be a new target for DOR patients' therapy.