BPDE Eeposure Affects Luteinization Of Ovarian Granulosa Cells

Objective:Benzo?a?pyrene?B?a?P?is a chemical environmental carcinogen widely present in people's production and living environment.It has been reported that B?a?P exposure in early pregnancy affects the embryo implantation process,impairs the endometrium decidualization,showing obvious reproductiv etoxicity.Benzo[a]pyrene-7,8-diol-9,10-epox-ide?BPDE?is an endocrine disruptor and ultimate carcinogen of B?a?P.It can not only interfere with normal pregnancy and lead to fetal growth restriction and abortion,but also cause chromosomal abnormalities in the offspring and induce fetal birth defects.The ovarian corpus luteum regulates the process of embryo implantation by secreting progesterone and estrogen.The ovarian granulosa cells are an important steroid-secreting cell.After ovulation,residual granules in follicular cells are transformed into luteinizing granulocytes under the stimulation of LH,continue to secretion of progesterone to maintain pregnancy.In this study,mouse ovarian granulosa cells?KK-1?were luteinized by hCG.We investigate the effect and mechanism of the B?a?P metabolite BPDE on the luteinizing function of granulosa cells.Methods:?1?Cell culture and treatments:KK-1 cells were treated with different concentrations of hCG?0?0.1?0.5?1.0 IU/ml?to induce luteinization.The result showed that 1.0 IU/ml hCG could successfully induce luteinization.Then 1.0 IU/ml hCG was added into different concentrations of BPDE?0,0.25,0.5,1.0,2.0,4.0?mol/L?to treat the cells for 24h at 37?with 5%CO₂.The mRNA levels of 3?-HSD,17?-HSD,P450SCC and protein of StAR were detected to verify whether BPDE affected luteinization of granulosa cells.?2?The level of oxidative stress in granulosa cells was determined by the CM-H₂DCFDA method.MMP was examined by measuring the fluorescent intensity of JC-1 in the mitochondria according to the manufacture's protocol.?3?TUNEL was used to detect the apoptosis of granulosa cells.Apoptosis related genes BAX,BCL2 and Cyt C were detected by immunofluorescence.?4?Western blot was used to detect the expression of melatonin receptor MT1,MT2.?5?The expression of GAS1 in granulosa cells was detected by immunofluorescence,western blot and qRT-PCR.?6?Animals and treatments:The pregnant mice were divided into B?a?P treatment group and control group randomly.The mice in the treatment group was given 0.2 mg/kg body weight B?a?P by gastric administration from the first day of gestation?D1?.The mice in the control group was given the same volume of corn oil.The mice were killed on D4and D7 of pregnancy and the ovarian tissues were collected.Immunofluorescence,western blot and qRT-PCR were used to detect the expression of GAS1 in the ovaries of D4 and D7 day mice.Results:?1?BPDE exposure down-regulated the level of steroid hormone synthesis-related molecules in granulosa cells,confirming that BPDE damages the luteinization of mouse ovarian granulosa cells.?2?BPDE exposure increased oxidative stress levels in KK-1 cells.?3?BPDE exposure induced KK-1 cell apoptosis.?4?BPDE exposure reduced the expression of melatonin receptors in KK-1 cells?5?Melatonin receptor agonists alleviated BPDE-induced granulosa cell apoptosis.?6?B?a?P and BPDE affected the expression of GAS1 in vivo and in vitro respectively.Conclusion:The research showed that BPDE exposure impaired luteinization of mouse granulosa cells and affected the expression of steroid hormone synthesis-related molecules.During this process,the oxidative stress of luteinized granulosa cells was increased and apoptosis was induced.In addition,BPDE exposure can also affected the expression of melatonin receptors.Supplementation of melatonin receptor agonist alleviated BPDE-induced oxidative stress and apoptosis.Besides,GAS1may be involved in BPDE-induced ovarian dysfunction,but its mechanism needs to be further explored.