The Mechanism Of Metformin Inhibiting Testosterone-induced Endoplasmic Reticulum Stress In Ovarian Granulosa Cells

Background and Objective: Polycystic ovary syndrome(PCOS)is the most common endocrine disease in women of reproductive age with major symptoms of hyperandrogenism,polycystic ovaries or oligo-ovulation.Metformin is widely used in PCOS patients especially those with metabolic problems.Excessive testosterone induces endoplasmic reticulum(ER)stress and unfolded-protein-response(UPR)activation in human cumulus cells,leading to their apoptosis.This study is designed to study whether metformin inhibits ER stress activated by excessive androgen in mouse granulosa cells(GCs)and cumulus oocytes complexes(COCs)in vivo and in vitro.Methods: Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our assisted reproductive center.The expression of ER stress sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients.Meanwhile,a dihydrotestosterone(DHT)-induced mouse model of PCOS was constructed.The ovaries,denuded oocytes,GCs,and COCs from the DHT-treated PCOS mouse models were collected to measure ER stress related genes and proteins.DHT-induced mouse models fed with or without metformin were alsoconstructed to determine the inhibitory effects and specific mechanism of metformin on ER stress.Besides,primary cultured mouse GCs and COCs were treated with testosterone,metformin,p38 MAPK inhibitor,or p38 MAPK small interfering RNA.The activation of ER stress induced by testosterone and the inhibitory effects of metformin on ER stress and UPR activation were analyzed.Their correlation with phosphorylated p38 MAPK was also explored.Finally,the effects of the activation of ER stress on GCs and COCs in relation to luteinizing hormone(LH)responsiveness were also examined.Results: The testosterone levels in follicle fluid from PCOS patients were significantly higher than that from control patients.ER stress gene markers GRP78,CHOP,XBP1 s,and ATF4 and UPR sensor proteins p-IRE1?,p-EIF2? and GRP78 were increased in the cumulus cells of PCOS patients.ER stress gene markers and UPR sensor proteins were also increased in ovaries,GCs and COCs of DHT-treated PCOS mice compared with those of control mice.The activation of ER stress in the cumulus cells of PCOS patients as well as ovaries from PCOS mice was determined.However,COCs and GCs from mice fed with metformin had lower expression level of ER stress related genes and proteins.Meanwhile,metformin was also able to inhibit ER stress activation which was induced by testosterone in primary cultured GCs and COCs.The phosphorylation of p38 MAPK participated in the androgen induced ER stress activation.Metformin inhibits ER stress via the suppression of p38 MAPK phosphorylation.The occurrence of ER stress can change the response of GC and COC to LH,leading to excessive expression of downstream genes.Metformin,an inhibitor of ER stress or p38 MAPK small interfering RNA can partially inhibit the abnormal expression of downstream genes.Conclusions: As a conclusion,metformin could decrease ER stress induced by testosterone in mouse COCs and GCs through p38 MAPK pathway.Our study provides a new mechanism of metformin in the treatment of PCOS patients.