Expression pattern of PARP-1 during folliculogenesis in mice under genistein exposure
[Objective]This experiment aims to explore the correlation between PARP-1 signaling and its mediated PARylation modification level in the promotion of follicular development and inhibition of follicular atresia by genistein,and further enrich the molecular mechanism of genistein and other estrogen-like substances interfering with ovarian follicular development in female mammals.It will provide the scientific guidance for the development of high-yield feed formulas for breeding livestock.[Method]This study used 21-day-old Kunming mice as experimental animals,and supplemented with 25 mg/kg,50 mg/kg,and 100 mg/kg genistein by intraperitoneal injection,once a day at fixed points for 7 consecutive days,their ovarian tissues and serum were collected.Immunohistochemical method was used to detect the expression and localization characteristics of PARP-1 protein in ovarian tissues,and Western blot was used to quantify the expression of PARP-1 protein and pADPr process in the tissues.Taking apoptosis indicator protein Cleaved-caspase3,estradiol(E2)level,steroid synthase CYP19A1 and HSD17B1 into consideration,the comprehensive analysis of PARP-1 signaling and its mediated PARylation modification signaling involved in genistein inhibiting follicular atresia and promoting follicular development process was conducted.[Result]Immunohistochemical staining showed that PARP-1 positive staining mainly occurred in the healthy granulosa cell nuclei and oocytes in primordial follicles,primary follicles,secondary follicles,and antral follicles,followed by theca cells,but it was not expressed in the apoptotic cells stained with Cleaved-caspase3.Western blot analysis showed that the expression of PARP-1 cleavage fragment(89 ku)in the 50 mg/kg genistein treatment group was significantly decreased compared with that of the control group(P＜0.05),but the expression of PARP-1 full-length(116 ku)was not significantly different among the various dose groups(P＞0.05).Secondly,the expression level of pADPr in the 50 mg/kg genistein treatment group was significantly lower than that of the control group,the 25 mg/kg and 100 mg/kg dose groups in the three regions of＜15 ku,25 ku and 35-45 ku.(P＜0.05).The expression level of pADPr in the 100 mg/kg dose group was significantly lower than that in the control group and the 25 mg/kg dose group only in the 25 ku region(P＜0.05).The expression of cleaved-caspase3 in the genistein treatment groups at doses of 25 mg/kg and 50 mg/kg showed a dose-dependent decrease compared with that of the control group,and the difference was significant in the 50 mg/kg dose group(P＜0.05).The level of E2 also showed a dose-dependent increase and significant difference compared with that of the control group(P＜0.05),but there was no significant change in the expression of estrogen synthesis enzymes CYP19A1 and HSD17B1(P＞0.05).[Conclusion]PARP-1 widely exists in healthy granulosa cells,follicular cells,and theca cells in the mammalian ovary,and it may mediate intracellular activities such as energy metabolism,apoptosis,and steroid hormone synthesis by catalyzing pADPr synthesis,which may participate in the interference of estrogen-like substances such as genistein in the process of ovarian follicle development.
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