| Environmental pollutants are ubiquitous in our daily lives,and people are exposed to those through eating,drinking,breathing,etc.Ovary is an important reproductive-endocrine organ for female mammals,and its main functions are follicular development and steroidogenesis.Ovaries are susceptible to various chemical substances in environment,which can induce ovarian inadequacy and endocrine disorders,etc.Pentachloronitrobenzene(PCNB)is a seed dressing agent and soil bactericide widely used in agricultural production.PCNB can be absorbed by plants and accumulated in animals,posing a threat on food safety.Our previous studies found that PCNB exposure for 7 days affected normal functions of ovary.However,the effect on ovarian function of PCNB with relative long-term exposure has not been reported.In this study,the interferential effect and its mechanism was investigated about the effect of PCNB exposure for 28 days on ovarian reproductive endocrine function.Three-week old female SD rats were randomly divided into three groups and intragastric administrated every day for 28d,including high dose group with 100mg/kg PCNB(n=10),low dose group with 50mg/kg PCNB(n=10),control group with same volume of corn oil(n=10).The results showed that,the estrus period of the rats in 50mg/1kg PCNB group was significantly shortened compared with the control group(P<0.05),and the proestrus and estrus period of rats in 100mg/kg PCNB group were shortened(P<0.05),suggesting that PCNB exposure may cause estrous cycle disturbance in rats.Follicles are the basic functional units of ovary.To study effect of PCNB exposure on follicle development,Hematoxylin and Eosin(H&E)Staining was performed for follicle counting.The results indicated that,the growth follicles of rats in 50mg/kg PCNB group had a downward trend compared with control group,yet showing no statistically significant difference.The proportion of growth follicles in 100mg/kg PCNB group decreased significantly(P<0.05).The result of follicle counts suggested that PCNB exposure can affect normal development of follicles.AMH belongs to the transforming growth factor-beta(TGF-?)super-family.It can inhibit the recruitment of primordial follicles and the growth of primary follicles,and its expression can reflect ovarian reserve.Immunohistochemistry(ICH)and Western blot(WB)were used to detect the expression of AMH in rat ovarian tissues.ICH result showed that the positive signal was mainly located in the granulated cells of the secondary follicles and small antral follicles.The result of WB showed that expression level of AMH in ovarian tissue was reduced by PCNB.Main steroid hormones synthesized by ovary are progesterone and estrogen,which are important for functional maintaining in female mammals.The levels of Progesterone and E2 were detected after PCNB treatment by electrochemiluminescence.The result demonstrated that progesterone is significantly increased(P<0.05)in 50 and 100mg/1kg PCNB group compared with control group,and E2 was decreased significantly in both two experience group(P<0.05).Furthermore,progesterone level in 50mg/kg PCNB group was slightly higher than of 100mg/kg PCNB group.Protein levels of Steroidogenic acute regulatory protein(StAR),Cytochrome P450scc enzyme(P450scc)and Aromatase in ovarian tissue were detected to study whether PCNB affected protein expression levels of steroidogenesis related genes.Results of WB showed that PCNB upregulated the protein expression of StAR in both experimental groups,but the difference between control group and experimental groups has no statistical significance(P>0.05).Furthermore,no significant change in the protein expression of P450scc after PCNB treatment in both two group was detected(P>0.05).The expression of the key enzyme StAR and P450scc in 50mg/kg PCNB group was slightly higher than that of 100mg/kg PCNB group,which was consistent with the changes in serum hormone levels,suggesting that both StAR and P450scc may play a role in operating the progesterone level caused by PCNB exposure.Aromatase is a key enzyme of estrogen synthesis,and the result of WB showed that PCNB significantly decreased aromatase levels in rats' ovarian tissues,which was consistent with the decrease in serum estradiol levels,indicating that PCNB may decreased estradiol levels by downregulating aromatase protein expression in ovarian tissues.RNA-Seq results show that the differentially expressed genes in rat ovarian tissue before and after PCNB treatment are mainly related to hormone receptor binding,hormone activity,ect.The differentially expressed genes are mainly enriched in the MAPK signaling pathway.Our study demonstrates that the continuous exposure to PCNB for 28 days interfered the estrus cycle of rats,and reduced the proportion of growing follicles and the level of AMH in ovary.In addition,PCNB affects the production of progesterone and estradiol in rats,and it may be related with the protein expression of steroidogenesis enzymes in ovarian tissue.The MAPK signaling pathway may play a role in the effects of PCNB on ovarian function. |
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