Effects Of Trichostatin A On Sika Deer Ovarian Granulosa Cells Proliferation And Histone Acetylation

The granulosa cells(GCs)play an important role in animal reproduction,and they not only regulate follicular development,but also provide nutrition and signal transduction for oocytes.A variety of epigenetic modifications happen during the process of granulosa cells,and there is a close contact between histone acetylation and granulosa cells steroid hormone secretion and cell cycle distribution.Trichostatin A(TSA),the tumor inhibitor and the histone deacetylase inhibitor(HDACi),can stimulate the acetylation of cell,and improve the normal gene sequence.TSA has been widely used in granulosa cells of porcine and cattle,but few studies have look into the effects of TSA on sika deer cells.In this study,we chose the ovary granulosa cells of sika deer as the research object,while a series of experiments were taken in order to find out the effects of TSA on the granulosa cells proliferation,apoptosis,cell cycle distribution and histone acetylation.We tried to ascertain the histone acetylation modification mechanism in the process of sika deer granulosa cell proliferation.The contents of the study were as follows: 1.The cell separation,in vitro culture and identification of granulosa cells:After superovulation treatment with FSH,we separated the GCs from follicular fluid by follicular puncture,and then the granulosa cells were cultured in DMEM/F12 Nutrient Mixture supplemented with 10% Foetal bovine serum(FBS)and 1% penicillin/streptomycin.We identified granulose cells by HE staining and FSHR immunofluorescence.The experimental results showed that,the granulosa cells grew well in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin.After HE staining,we found that the cell morphology was complete,present polygon,the cytoplasm was pink,and the nuclear was blue,which was similar to the description of the other livestock granulosa cells,therefore we judged that the cells were sika deer granulosa cells.In laser confocal microscope,we found that FSHR positive rate > 90%,which meant the granulosa cells purity was over 90%,and they could be used the follow-up experiments.2.The effects of TSA on the granulosa cell proliferation activity and the synthesis of steroid hormones:GCs were treated with varying concentrations of TSA(0,10,50,100,and 500 nM)for 48 h,and then we detected the cell proliferation activity by CCK,while the concentrations of estradiol and progesterone in the culture supernatant were detected by ELISA.The results showed that TSA significantly inhibited the proliferation of GCs compared with the control group(p<0.05),while significant differences were observed among all groups(p<0.05).These results suggested that TSA inhibited sika deer GCs in a dose-dependent manner,with stronger effects elicited by high doses of TSA.ELISA results showed that the secretion of estradiol was increased at the dose of 50 n M and 10 nM,and the secretion was the highest at the dose of 50 nM(p<0.05).Though the secretion of estradiol was decreased when TSA was 100 nM,it was also higher than the control group(p<0.05).There was no significant difference between 0 nM and 500 nM(p>0.05).Progesterone testing found that when the concentration of TSA was 10 nM,50 nM and 100 nM,the difference was not significant(p>0.05),but the secretion of progesterone was higher than the control group(p < 0.05).The secretion of progesterone was lower than the control group(p<0.05)when TSA was 500 nM,which meant that high doses of TSA could inhibit the secretion of progesterone.3.The effects of TSA on GCs apoptosis and cell cycle distributions:We evaluated GC apoptosis and cell cycle distributions of TSA-treated GCs(0,10,50,100,and 500 nM)for 48 hours with flow cytometry using an Annexin V-FITC/PI Apoptosis Assay Kit and a Cell Cycle Detection Kit,respectively.And,we detected the mRNA expression of apoptosis-related proteins(BCL-XL?BAX?GLUT3?GLUT8)and cycle-related proteins(Cyclin D2?CDK4)by qRT-PCR.The results showed that the rate of GC apoptosis increased gradually with increasing TSA concentrations,and the apoptosis rate was significantly higher in cells treated with 500 nM TSA compared with other TSA concentrations(p<0.05).The results indicated that high dose of TSA may strongly induce granulosa cell apoptosis.The cell cycle analysis revealed that TSA had no dramatic effects on G2 phase.However,TSA appeared to significantly increase G1 phase but inhibit S phase at concentrations greater than 10 nM(p<0.05).The results of qRT-PCR showed that the mRNA expression of GLUT3?GLUT8?BCL-XL?Cyclin D2 and CDK4 presented a decreasing trend(p<0.05)with the increase of TSA concentrations,while the mRNA expression of BAX was increased(p<0.05).4.The effects of TSA on GCs histone acetylationWe detected the mRNA expression of four epigenetic modifying enzymes(DNMT3a?DNMT1?HAT1?HDAC1)by q RT-PCR,and detected the acetylation of histone H3(Lys9)(H3K9)and H4(Lys8)(H4K8)by the immunofluorescence and western blotting.QRT-PCR results revealed that the expression levels of these epigenetic modifying enzymes(DNMT3a?DNMT1 and HAT1)exhibited the same trends.Specifically,the expression levels of these genes first increased with increasing TSA concentrations,but then dropped significantly,with the highest expression at a dose of 50 nM TSA(p<0.05).However,the expression level of HDAC1 was decreasing progressively.The results of immunofluorescence and western blotting were similar,and they showed that the acetylation of H3K9 and H4K8 was significantly elevated(p<0.05)with increasing TSA concentrations and were extremely significant(p<0.05)at a dose of 500 nM.We also found that the acetylation of H4K8 was higher than that of H3K9 at identical TSA doses.To conclude,the reserach demonstrated that TSA inhibited the proliferation of sika deer granulosa cells in vitro,promoted the synthesis and secretion of steroid hormones at moderate concentration,contributed to the histone acetylation of sika deer granulosa cells.High dose of TSA could stimulate the apoptosis of granulose cells,and make the cell cycle blocked in the G0/G1 phase.This study would become a foundation for studyding the effects of TSA on sika deer oocytes in vitro maturation.