GDF-8 Mediates Aromatase Expression In Human Granulosa Cells And The Molecular Mechanism Involved In The Development Of PCOS

Growth differentiation factor 8(GDF-8),also known as Myostatin,is a member of the Transforming growth factor-?(TGF-?)superfamily.GDF8 is widely involved in various physiological functions of the body,such as regulating skeletal muscle growth,regulating protein metabolism,lipid metabolism,glucose metabolism and bone occurrence.GDF8 also plays an important role in female reproductive system and is expressed in human uterus and placenta.it may be involved in the occurrence of uterine leiomyoma and regulate the proliferation and migration of placental trophoblastic ectodermal cells.Recent studies have shown that GDF8 plays a very important physiological role in the regulation of ovarian reproductive activity,and is involved in the regulation of cumulus dilatation and steroid hormone production.Polycystic ovary syndrome(PCOS)is one of the most common endocrine diseases leading to female reproductive dysfunction and endocrine and metabolic disorders.According to Rotterdam criteria,the incidence is about 20%,accounting for 30% or 60% of anovulatory infertility,which is one of the most common diseases causing female infertility.The main clinical manifestations were ovulation disorder,hirsutism,sores,obesity and metabolic disorder;ultrasound showed polycystic ovary;and its main serological manifestation was hyperandrogen.At present,the pathogenesis of PCOS is not completely clear,some studies believe that high androgen is the main factor causing various clinical symptoms and metabolic disorders of PCOS.Androgen metabolism disorder can lead to the increase of androgen,and androgen production can be regulated by many factors,such as insulin growth factor,renin and so on.Aromatase(AROM),also known as estrogen synthase,is a member of CYP19A1 cytochrome P450 superfamily and a key enzyme to promote the transformation of androgen to estrogen.it has been found that AROM is poorly expressed in granulosa cells of PCOS patients.This may be a key factor in the occurrence of hyperandrogen symptoms and ovulation disorders in PCOS.Previous studies have shown that GDF-8 is involved in the regulation of the expression of various key enzymes in steroid hormone production in human granulosa cells.For example,steroid hormone synthesis acute regulatory protein,a key enzyme of progesterone production,and Pentraxin-3,which can regulate follicular cumulus dilatation,and so on.In 2012,Chen MJ et al.found that the concentration of GDF-8 in serum of PCOS patients was higher than that of normal people,suggesting that GDF-8 may be involved in the occurrence and development of PCOS.Some other studies have also shown that the low expression of AROM in granular cells of PCOS patients may be the key factor leading to the occurrence of hyperandrogen symptoms and ovulation disorder in PCOS.However,the relationship between GDF-8 and the occurrence and development of PCOS and the specific regulatory mechanism of GDF-8 on AROM in human granulosa cells are still unclear.Therefore,the purpose of this study was to investigate whether GDF-8 regulates the production and molecular mechanism of AROM in human luteinized granulosa cells and its role in the occurrence and development of PCOS hyperandrogens.ObjectiveTo explore the molecular mechanism of GDF-8 regulating AROM expression in human granulosa cells and its role in the occurrence and development of PCOS hyperandrogens.MethodsIn this study,human primary granulosa cells were used as a model to investigate the regulatory role of GDF-8 in the expression of AROM and whether GDF-8 is involved in the occurrence of hyperandrogenemia in PCOS through AROM.After granulosa cells were treated with GDF-8 for 3 h,6 h,12 h and 24 h,and granulosa cells were treated with different concentrations of GDF-8,the expression of AROM at RNA and protein levels was observed.To investigate whether the regulatory effect of GDF-8 on AROM is time-dependent and dose-dependent.Reverse transcription real-time quantitative PCR(RT-qPCR)and Western Blot were used to detect the expression of RNA and protein in human granulosa cells.In order to verify whether GDF-8 is involved in the regulation of AROM by activating the upstream signaling pathway of ALK4/5,granulosa cells were pretreated with SB431542,a specific inhibitor of ALK4/5,to observe whether GDF-8 could inhibit the regulation of AROM.In order to verify whether the downstream signaling pathway of Smad2/3 is involved in the regulation of AROM by GDF-8,we detected the regulatory effect of GDF-8 on Smad2/3 phosphorylation in granulosa cells.In order to further verify the key role of Smads signaling pathway in the regulation of AROM by GDF-8,granulosa cells were transfected with Smad4 SiRNA in advance to knock out the expression of endogenous Smad4.To observe the regulatory effect of GDF-8 on mRNA and protein levels of AROM.The concentration of GDF-8 in follicular fluid of non-POCS patients and PCOS patients was detected by Enzyme-linked immunosorbent assay(ELISA).Granulosa cells of non-POCS patients and PCOS were treated with GDF-8 under the same conditions.the changes of AROM expression at RNA and protein levels were observed,and the regulatory effect of GDF-8 on AROM in granulosa cells of PCOS patients was explored.ResultsIn human granulosa cells,GDF-8 can promote the expression of AROM in a time-and dose-dependent manner.SB431542,an inhibitor of ALK4/5 pathway receptor in granulosa cells,was found can inhibit the promoting effect of GDF-8 on AROM,suggesting that GDF-8 is involved in the regulation of AROM by activating the upstream pathway of ALK4/5.After granulosa cells were treated with GDF-8,the level of phosphorylated Smad2/3 in granulosa cells was significantly increased,suggesting that GDF-8 promotes AROM production through downstream signaling pathway of Smad2/3.By using SiRNA of Smad4 to knockout the expression of endogenous Smad4 in granulosa cells,the results showed that the regulatory effect of GDF-8 on AROM was inhibited,suggesting that GDF-8 regulates AROM production through the classical Smads signaling pathway.ELISA was used to detect the concentration of GDF-8 in follicular fluid of non-POCS patients and PCOS patients.the results showed that the concentration of GDF-8 in follicular fluid of patients with PCOS was significantly higher than that of patients without PCOS.The granulosa cells of PCOS patients were treated with GDF-8 under the same conditions.the results showed that GDF-8 significantly inhibited the expression of AROM mRNA and protein in granulosa cells of PCOS patients.Conclusion1.In human granulosa cells,GDF-8 can promote the expression of AROM mRNA and protein in a time-and dose-dependent manner.2.The regulation of AROM expression by GDF-8 occurs by activating ALK4/5 receptor,which in turn activates the classical Smad pathway.3.The high expression of GDF-8 and the inhibition of AROM expression in PCOS patients may be related to the occurrence of serum hyperandrogen in PCOS patients.