The Interference Effects Of Tributyltin And Bisphenol A On The Systhesis Of Steroid Hormones In Ovarian Granulosa Cells

Polycystic ovary syndrome(PCOS)is a common endocrine disorder among women of reproductive age,and its incidence rate is about 6%?10%worldwide.Rare ovulation or ovulation,androgen hyperplasia or high androgen blood disease,ovarian polycystic are the common clinical features of PCOS.Most studies show that PCOS is mainly caused by genetic factors and environmental factors,but its accurate pathogenesis is still unclear and needs farther in-depth study.Numerous studies have shown that environmental endocrine disruptors(EDCs)are one of the important pathogenic factors in women with PCOS.EDCs mainly include environmental androgen disruptors,environmental estrogen disruptors,environmental progesterone disruptors,environmental thyroid hormone disruptors and so on.The ovaries are the main organs that maintain the endocrine homeostasis and secondary sexual characteristics of the female body by secreting hormones.Granulosa cells are closely related to ovarian development and hormone secretion.At the same time,granulosa cell changes may be an intrinsic mechanism in patients with PCOS.In this study,tributyltin(TBT)and bisphenol A(BPA)were selected as the representative of environmental androgens and environmental estrogens,respectively.Based on SD rats samples which were exposed to TBT(10 ng/kg/d,100 ng/kg/d),BPA(50?g/kg/d)in our preliminary study,HE section staining of ovaries in SD rats were used to observe the proliferation of ovarian granulocytes,and ovarian gene microarray in SD rats was analysized by DAVID tool to explore the potential target genes for PCOS.Human ovarian granulosa cells(KGN)were used in this research;the concentrations of TBT(0.1 ng/L,1 ng/L,10 ng/L),BPA(0.5 ?g/L,5 ?g/L,50?g/L,500?g/L)were set to explore the toxic effects on KGN,and to compare the differences mechanism of PCOS caused by TBT or BPA.This study will provide theoretical basis and new ideas for clinical treatment of PCOS.The results are as follows:HE staining showed that ovarian granulosa cells had proliferative effect in TBT or BPA-exposed rat compared with control rats.The DAVID tool was used to dig and analyze the rats' gene chip results in our previous research.The results showed that TBT group had positive regulation on testosterone,glycolipid biosynthesis,and inflammatory response.Whereas,BPA exposure mainly affected Toll-like receptor signaling pathway,lipid transport,I-?B phosphorylation,and phosphorylation of RNA polymerase ? C-terminal domain.These biological processes are all related to the occurrence and development of PCOS.The differentially expressed genes Dhx40,Epm2aipl,Fam92a1,Map3K7,Slc30a9,SET,Sytl4 and Fam160a1 were randomly selected from the gene microarray of rat ovaries for RT-PCR verification in KGN cells exposed to TBT and BPA.The gene expression in KGN cells was basically consistent with the trend of ovary tissue in rats.The proliferation of KGN cells was detected by dual fluorescence staining with EdU and DAPI,and it was found that the proliferation effect of KGN cells was consistent with the result in rats.By using transmission electron microscope(TEM),both TBT and BPA affected the structure and morphology of KGN cells,inculding the nucleus enlargement,and endoplasmic reticulum expansion and so on.In the high concentration group,BPA 50 ?g/L and 500 ?g/L significantly increased nuclear size and expanded endoplasmic reticulum.TBT and BPA exposure affected the hormone secretion of KGN cells.Compared with the control group,TBT exposure increased the testosterone(T)secretion;descended the progesterone(P)secretion;did not affect the estradiol(E2)secretion.This is consistent with the clinical symptoms of high androgen and low progesterone in PCOS patients,as well as the results in serum of rats in preliminary study.After BPA exposure,the secretion of progesterone and estradiol in KGN cells were decreased,while there was no significant change in testosterone,but the ratio of estradiol and testosterone was decreased significantly.TBT and BPA exposure to KGN cells affect the gonadal steroid hormone synthesis pathway,and they affect the final hormone secretion by influencing on the key metabolic enzyme genes in the pathway.Both TBT and BPA decreased the STAR gene which is related to progesterone synthesis.POR gene related to testosterone and estradiol synthesis was increased significantly.CYP1A1 and CYP1B1 related to estradiol degradation were significantly decreased.At the same time,BPA also significantly decreased the HSD17B1,HSD17B2 and HSD17B3 genes related to testosterone synthesis,while TBT had no significant difference effect on these three genes.TBT and BPA also affected the expression of cytokine synthesis related genes at protein level in KGN cells.In the FSHR/GS/AC pathway,TBT significantly decreased the protein expression level of AC and CYP19A,CYP19A is the key enzyme for estrogen synthesis,while BPA significantly increased the protein expression level of AC and had no significant effect on CYP19A.The protein expressions of GS in BPA groups were significantly lower than that in control.In addition,TBT and BPA also down-regulated the enzymes related to estradiol degradation,CYP1A1 and CYP1B1,which were consistent with the trend of mRNA level.GONAL-f(Recombinant human follicle stimulating hormone,Follicle-stimulating hormone,FSH),a commonly used drug in clinical practice,is often used in the treatment of PCOS to promote patients for normal ovulation.In this study,KGN cells exposed to TBT and BPA,and then stimulated by FSH.It was found that testosterone,progesterone,estradiol and the E2/T ratio were recovered after stimulation.Therefore,FSH can provide a new strategy for alle'viating pcos-like symptoms caused by EDCs interference.In summary,both TBT and BPA can cause the proliferation effect of ovarian granulosa cells,resulting in the disturbance of ovarian granulosa cytokines,and cause the typical PCOS symptoms,decrease the E2/T ratio and progesterone secretion.Further,we found that TBT and BPA had an effect on steroid hormone synthesis pathway in ovarian granulosa cells.They all regulated progesterone level by down-regulating the expression of STAR gene,up-regulating the expression of POR gene which could cause the increase of androgen secretion,and down-regulating the expression of estradiol-related genes CYP1A1 and CYPJB1.However,TBT and BPA have different effects on the expression of genes related to testosterone synthesis.TBT had no significant effect on HSD17B1,HSD17B2 and HSD17B3 genes related to testosterone synthesis,while BPA significantly down-regulated their expression levels.In addition,TBT exposure indirectly regulated the decrease of CYP19A related to estradiol synthesis by inhibiting the expression of protein in downstream pathway of FSHR/AC/GS.Meanwhile,FSH stimulated the KGN cells which exposed to TBT and BPA before,can rescue the secretion of hormones to the normal level,which indicated that FSH has a protective effect on EDCs-induced PCOS symptoms.This study demonstrated that typical EDCs TBT and BPA can cause PCOS symptoms,and clarified their potential molecular mechanism of regulating abnormal hormone secretion through affecting steroid hormone synthesis pathway respectively,providing a new research idea for alleviating reproductive toxicity induced by environmental pollutants.