Role And Mechanism Of Tri-ortho-cresyl Phosphate (TOCP) Induced Injury Of Ovary Granulosa Cells In Female Mice

Objectives: The present study focuses on the toxicity and impact of TOCP on the ovarian granulosa cells in mouse,and explores the mechanism of TOCP inducing female animal reproductive toxicity.Methods: 1、In vivo: Twenty-four adult female mice were randomly divided into four groups,each 6.The female mice were administrated with different concentrations of TOCP(0,100,200,400 mg TOCP/kg/day)for 14 d.All mice exposed to TOCP for 14 days were sacrificed.Ovarian and uterus coefficient were assessed with the relative value ovarian and uterus and body weight,respectively.Elisa was used to detected the level of serum estrogen in mouse.Ovaries were fixed in formalin.HE staining was utilized to observe the effect of with different concentrations TOCP on ovary in mice.Immunohistochemistry was used to identify the expression of NTE in the mice ovary.2、In vitro: Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP and LPC for 48 h.The concentration of TOCP: control groups(0 mmol/L TOCP),low concentration groups(0.125 mmol/L TOCP),high concentration groups(0.25 mmol/L TOCP)and the highest concentration groups(0.5mmol/L TOCP).Cells count was utilized to observe the effect of TOCP on mouse granules cells.Flow cytometry(FCM)and RT-PCR was used to investigate the impact of TOCP on the cycle of mouse granulosa cells.Annexin V-FITC/PI double staining and Hoechst staining was utilized to measure the effect of TOCP on the apoptosis of mouse granulosa cells.Western blot was used to investigate the effect of TOCP on the autophagy of mouse granulosa cells.Western blot was used to investigate the effect of TOCP on the level of NTE protein in the mouse granulosa cells.RT-PCR was used to investigate the effect of TOCP on the level of NTE m RNA in the mouse granulosa cells.1.HE staining was utilized to observe the effect of with different concentrations TOCP on ovary in mice.2.Immunohistochemistry was used to identify the expression of NTE in the mice ovary.3.Elisa was used to detected the level of serum estrogen in mouse.4.Cells count was utilized to observe the effect of TOCP on isolated mouse granules cells from 14-day-old female Kunming mouse.5.Flow cytometry(FCM)and RT-PCR was used to investigate the impact of TOCP on the cycle of mouse granulosa cells.6.Annexin V-FITC/PI double staining and Hoechst staining was utilized to measure the effect of TOCP on the apoptosis of mouse granulosa cells.7.Western blot was used to investigate the effect of TOCP on the autophagy of mouse granulosa cells.8.Western blot was used to investigate the effect of TOCP on the level of NTE protein in the mouse granulosa cells.RT-PCR was used to investigate the effect of TOCP on the level of NTE m RNA in the mouse granulosa cells.Results: 1.The female mice were administrated with different concentrations of TOCP for 14 d.Compared with the control group,the high and the highest concentration TOCP groups significantly decreased the weight coefficient of ovary(P<0.05),while the low concentration TOCP groups not(P>0.05).The different concentrations of TOCP count not significantly decreased the weight coefficient of uterus(P>0.05).2.Compared with the control group,receiving the highest concentration TOCP groups,the granulosa cells has been disordered arrangement in mice ovary.While in the low concentration and high concentration TOCP groups,no morphologic changes of arrangement of the granulosa cells were noted.3.Elisa show that the level of serum E2 was significantly inhibited by TOCP in a dose-dependent manner(P>0.05).4.Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP for 48 h,cell viability was observed with Cell count assay.Compared with the control group,cell viability of mouse granules cells was significantly inhibited by TOCP in a dose-dependent manner(P<0.05).5.Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP for 48 h,the cells were treated with different concentrations of TOCP for 48 h.Flow cytometry found that TOCP had no effect on the cycle of mouse granules cells.The RT-PCR assay showed that there was no change in m RNA levels of P21,P27,P53 and Cyclin D1 between control group and TOCP-treated group.6.Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP for 48 h.Annexin V-FITC/PI double staining showed that,compared with the control group,TOCP had no significant effect on apoptosis of mice granules cells.Hoechst staining showed that TOCP had no significant effect on apoptosis of mice granules cells.7.Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP for 48 h.Western blot showed that,compared with the control group,TOCP significantly increased the protein levels of autophagic protein Atg5 and Beclin1,and the proportion of LC3-II/LC3-I in mice granules cells.8.Immunohistochemisty showed that NTE was expressed in granulosa cells of mice ovary.9.Isolated granulosa cells from 14-day-old female Kunming mice were treated with different concentrations of TOCP for 48 h.RT-PCR and Western blot showed that TOCP significantly inhibited NTE protein and m RNA level in granules cells of mice.Conclusion: 1.TOCP lead to the mouse ovarian granulosa cells damage,and reduce the secretion of estradiol of granule cells.2.NTE in mouse ovarian tissue expression,and the TOCP inhibit the activity of granule cells NTE;3.TOCP can induce autophagy in mouse ovarian granulosa cells and inhibit its activity.