Maternal Long-term Daily Dose Exposure To Propylparaben On Ovarian Function In Offsprings And Its Mechanisms

Objective:To explore the effects of maternal long-term daily dose exposure to propylparaben（PrPB）on follicular development and its mechanism in offsprings.Methods:Animal experiment:According to the PrPB exposure pattern in human,6-week-old C57BL/6j female mice were randomly divided into a blank control group（Control）,a low-dose PrPB exposure group(7.5 mg·kg bw^-1·d^-1,which simulated daily exposure dose of human)and a high-dose PrPB exposure group(90 mg·kg bw^-1·d^-1,which simulated the maximum safe dose of human).We prepared special fodder containing PrPB and fed female mice until the mice were 12weeks old.Then female mice mated with normal male mice.PrPB exposure was continued during pregnancy and lactation until the offspring mice were weaned.Then,the offspring mice were fed with normal fodder.We monitored the offsprings’body weight to evaluate the effect of PrPB exposure on the general health.H&E stained ovarian paraffin sections were used to count ovarian follicles.To evaluate the ovarian function of female offspring,we monitored the estrous cycles,and detected serum sex hormone levels（FSH,E2,P4）.Immunohistochemical experiments were performed on ovarian tissue sections to analyze the expression of microtubule-associated protein light chain 3（LC3B）,p62/sequestosome-1and Caspase-3,and explore the possible mechanisms.Cell experiment:The ovarian granulosa cells（GCs）of the 25-28 days old mice were collected by mechanical separation method.The growth state of granulosa cells was observed and the growth curve was plotted after the intervention of gradient PrPB concentration.Combining literature and the cell growth status in our experiment,GCs were divided into DMSO control group,50μM group and 100μM group.We explored the effects of PrPB and its molecular mechanism by flow cytometry apoptosis ratio detection,western blot and immunofluorescence.The granulosa cells were treated with the autophagy inhibitor3-methyladenine（3-MA）to investigate whether 3-MA could reverse the autophagy-promoting effect of PrPB.Results:（1）The birth weight of offsprings decreased in PrPB-esposed groups,which means that maternal long-term daily dose exposure to PrPB may affect embryonic development;（2）Maternal long-term daily dose exposure to PrPB affected the ovarian endocrine function in offsprings.The proportion of disturbed estrous cycles in PrPB-exposed groups increased significantly;（3）The number of ovarian antral follicles in PrPB-exposed groups decreased significantly（p<0.05）,suggesting that maternal long-term daily dose exposure to PrPB could affect the ovarian reserve of the offspring female mice;（4）Immunohistochemical experiments showed that the PrPB-exposed groups exhibited higher levels of autophagy and apoptosis in the offsprings’ovaries（p<0.05）;（5）PrPB intervention affected the growth of mice granulosa cells in vitro.The proliferation of granulosa cells decreased in PrPB-exposed groups;（6）In in vitro primary mouse granulosa cell culture,exposure to PrPB can induce cell apoptosis and autophagy;（7）Autophagy inhibitor 3-MA treatment could reverse the effect of increased autophagy induced by the PrPB.Conclusions:After maternal long-term daily dose exposure to PrPB,the offsprings’birth weight decreased,the female offsprings’ovarian reproductive endocrine function decreased and the follicles developed abnormally.Our study suggested that PrPB promoted autophagy and apoptosis in ovarian granulosa cells,leading to abnormal follicular development and decreased ovarian reserve,thereby affecting the ovarian reproductive endocrine function of offspring female mice.