Effects Of Fluoride On Cytoskeleton And Its Related Proteins By Sertoli Cells In Rat Testes

[Objective]Fluoride as a well-known environmental pollutants,seriously affecting the human and animal production and life.A large number of studies have shown that fluoride has an adverse effect on the reproductive health of male animals.The reproductive toxicity of fluoride is mainly reflected in the damage to sperm.In the process of spermatogenesis,testicular support cells can form a blood testis barrier,so that sperm from exogenous and endogenous toxic substances against the sperm cells to ensure the smooth generation.However,whether the fluoride can induce blood testicular barrier and structural damage rarely reported.Therefore,the aim of this study was to investigate the effect of fluoride on the cytoskeleton of the testis and its related proteins,and to lay a theoretical foundation for further elucidating the reproductive toxicity mechanism of fluoride.[Methods]In this experiment,different concentrations of sodium fluoride under the influence of the support of cell culture.The morphological observation and purity of the cells were detected with the HE and the immuniohistochemical analysis of GATA4 and WT1.The effect of different concentrations of sodium fluoride on the relative viability of testicular cells was examined by CCK-8.Through the confocal laser scanning microscopy,the effect of NaF on the cytoskeletal proteins of a-tubulin and F-actin structure were observed.The effect of NaF on the expression of cytoskeleton and its related proteins RAI14,Palladin,ARP3,ARPC2,?-tubulin,ARP2,ARPC3 and ARPC4 were assessed by QRT-PCR,then RAI14,Palladin,ARPC2,ARP3 and a-tubulin were assessed by Western blot.[results]1.Sertoli cells identification results.The morphological observation of cells were detected by HE staining.The purity of the sertoli cells were more than 95%which was evaluated by the immunohistochemical analysis of GATA4 and WT1.2.The effects of NaF on the cytoskeleton of sertoli cells.According to the results of the CCK-8,the concentration of NaF in subsequent experiments were 0mg/L,0.1mg/L,lmg/L,10mg/L and the exposure time was 24h.The results showed that the damage of a-Tubulin and F-actin were more serious with the increasing concentration of NaF.3.The results of QRT-PCR suggested that.compared with the control group,the mRNA levels of ARP3 and ARPC3 didn't change significantly in the exposure groups.However,the mRNA levels of Palladin,ARPC2,a-Tubulin,ARP2 and ARPC4 had different levels of decline.The Palladin,ARPC2 and ARPC4 reduced significantly in the group of 10mg/L NaF(P<0.05);the a-Tubulin and ARP2 reduced markedly in the treatment groups compared with the control group(P<0.05,P<0.01).In contrast,the mRNA levels of RAI14 increased markedly in the treatment groups compared with the control group(P<0.05,P<0.01).4.The results of Western bloting showed that,the protein content of Palladin did not change significantly compared with the control group.The protein expression of ARPC2,ARP3 and a-tubulin showed a significant decrease(P<0.01,P<0.001),while the protein expression of RAI14 increased markedly in the group of 10mg/L NaF group(P<0.01).[conclusion]The reproductive toxicity of NaF may influence the structure of sertoli cell cytoskeleton damage will affect the blood testosterone barrier.Fluoride causes testicular support cytoskeleton and its associated proteins RAI14,Palladin,ARP3,ARPC2,ARP2,ARPC3,ARPC4 and a-tubulin abnormalities,this will directly affect the process of sperm production,due to lack of essence,less refined,or even serious damage of the male reproductive organs.