Effect Of Bisphenol A On Androgen Transformation In Ovarian Granulosa Cells

Polycystic ovary syndrome(PCOS)is a multi-factor endocrine disease but the specific pathogen is still uncertain.Hyperandrogenism,the leading cause of clinical phenotype and the main cause of infertility,is one of the three criteria of PCOS.Increased synthesis of androgen or declined conversion of androgen could lead to elevated level of androgen in the body.The conversion of androgens to estrogens is known as aromatization,while aromatase is the rate-limiting enzyme for this process.Aromatase is encoded by CYP19A1,and the expression of aromatase in granulosa cells is regulated by ovarian-specific promoter and could be affected by variety of factors.As know,Steroidogenic Factor 1(SF1)gene could positively regulate the expression of CYP19A1 gene by binding on its promoter.Studies showed that SF1 gene was involved in the development and in the function of the pituitary gland,of the adrenal gland and of the gonads.Its mutation leads to severe defects in sexual organ formation and its abnormal expression was associated with changes in its DNA methylation profile and with endometriosis,the major cause of infertility in women,but the reason causing methylation was unknow.Bisphenol A(BPA),a kind of estrogenic substances,is a member of endocrine disrupting chemicals(EDCs).Studies showed that the contain of BPA in blood and urine of PCOS patients was significantly higher than others,and was positive correlated with the content of testosterone and androstenedione.Also,many researches have shown that environmental endocrine disruptors,including BPA,could not only modulate metabolic and reproductive hormonal effects through classical estrogen/androgen receptor-mediated signaling pathways,but also cause changes in epigenetics,including DNA methylation.In our study,using the KGN cell lines,we aimed to explore whether BPA could decrease the expression of CYP19A1 gene and the aromatase by increasing the level of methylation of SF1 gene thus affect the conversion of androgens to estrogens.The effect of bisphenol A on the androgen transformation of granulosa cells was studied by adding Bisphenol A to culture medium of granulosa cell lines(KGN).The mRNA level of CYP19A1 and SF1 as well as aromatase protein level in KGN cell lines after BPA treatment were examined by RT-PCR and Western Blot.The results showed that the level of mRNA and protein of these genes under the influence of BPA were significantly decreased.After we overexpressed SF1 gene,the impact of BPA had been partially eased.In addition,MSP experiments confirmed that BPA could increase the methylation level of SF1 gene methylation.We also used radioimmunoassay to detect the estradiol(E2)and testosterone(T)content in the supernatant of KGN cell culture medium after BPA treatment.The results showed that the ratio of E2 and T in the supernatant was decreased because of the influence from BPA.This phenomenon was partially reversed by subsequent interference SF1.In order to verify our results in PCOS patients,the ovarian granulosa cells of PCOS patients were collected by our laboratory,the expression of CYP19A1 and SF1 mRNA in granulosa cells was detected by RT-PCR.The results showed that the transcriptional level of these two genes was decreased in PCOS patients.Using MSP we confirmed that the methylation level of SF1 was increased in PCOS patients.Our study demonstrated that BPA may affect the transcription of androgen in granulosa cells by increasing the methylation level of SF1 and we proved our results on clinical patients.This may be one of the causes of hyperandrogenism,and may eventually lead to the occurrence and development of polycystic ovary syndrome.Our research provided a new thought and possibility for the study of pathogenesis of PCOS,while providing a potential target for the treatment and medication of the disease.