The Effects Of Zearalenone On The Cytoskeletal Structure And The Secretion Of Functional Proteins ABP In Rat Sertoli Cells

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin produced by several fungi of the fusarium which exists widely in the animal feed and human food. Many researches show that ZEA has the reproductive, genotoxic, and immunotoxic toxicity and carcinogenicity. ZEA can inhibit the secretion of testosterone and affect the production and maturation of sperm in male animals, which results in the feminization and the decrease of sex drive. Sertoli cells play a key role in the process of spermatogenesis. so far, however, the male reproductive toxicity caused by ZEA especially on the molecular level has not been defined clearly. On basis of the establishment of the vitro models of sertoli cells ZEA, this study detected the effects of ZEA on the cytoskeletal structure and the secretion of related functional protein of Sertoli cells to discuss the toxic mechanism of ZEA on a molecular level.In this experiment, the primary sertoli cells were isolated by means of the enzyme digestion method with trypsin and collagenase. The survival rate and the purity of cells were detected with the heip of the trypan blue, oil red O and the immunohistochemical analysis of FSHR. The virus concentration and infected time of ZEA were determined by the MTT assay. Through the confocal laser scanning microscopy, the effect of ZEA on the cytoskeletal proteins of a-tubulin, F-actin, and the nucleus structure in sertoli cells were observed. We examined the ultrastructural effects of ZEA on the mitochondrion, golgi apparatus and endoplasmic reticulum using the transmission electron microscopy. The effects of ZEA on the expression of ABP, FSHR, vimentin, transferrin, and N-cadherin of the sertoli cells were assessed by QRT-PCR, Western blot and ELISA. The results are as follows:1. The isolation and identification of sertoli cells. The sertoli cells can be effectively isolated by using trypsin and collagenase to digest the testicular tissue in two steps. The survival rate of cells was higher than 95% detected by the trypan blue. The purity of the Sertoli cells was more than 95% which was evaluated by the immunohistochemical analysis of FSHR and oil red O.2.The effects of ZEA on the the cytoskeleton and ultrastructure of the sertoli cells. According to the results of the MTT, the concentration of ZEA used in subsequent experiments was 0 μg/mL、 5 μg/mL、10 μg/mL、20μg/mLand the exposure time was 24h. The results showed that the damage of a-tubulin、F-actin and nucleus were more serious with the increasing concentration of ZEA. The nucleus in the exposed group became crescent, dented and fragmental observed though the confocal laser scanning microscopy. The observation of the ultrastructure indicated that ZEA could damage the internal structure of sertoli cells including the shedding and the disappearance of the mitochondria ridge, the vague appearance of the edema and rupture of the endoplasmic reticulum, the vacuoles appearing in the cytoplasm and the breakage of fat droplets.3.The effects of ZEA on the secretion of sertoli cells. The results of QRT-PCR suggested that, compared with the control group, the mRNA levels of FSHR did not change significantly in the exposure groups. However, the RNA levels of ABP, Vimentin, N-cadherin had different levels of decline. The ABP reduced significantly in the 10 and 20 μg/mL groups (P<0.05) and the vimentin and N-cadherin reduced significantly in the group of 20 μg/mL ZEA (P<0.05). In contrast, the mRNA of transferrin increased markedly in the treatment groups compared with the control group(P<0.05;p< 0.01). The result of Wester-blot indicated that, compared with the control group, the expression of ABP, FSHR, vimentin, transferrin, and N-cadherin decreased significantly in the 10 and 20 μg/mL ZEA groups (P<0.05; p< 0.01, respectively). The results of ELISA showed that, the secretion of Inhibin P reduced dramatically in the 10 and 20 μg/mL ZEA groups.compared with the control group and the transferrin reduced significantly in the 20 μg/mL ZEA groups (P<0.05).In conclusion, the reproductive toxicity of ZEA may influence the structure of BTB since it can damage the cytoskeletal structure of the sertoli cells. ZEA can inhibit the sertoli cells from secreting proteins of ABP, FSHR, transferrin, Inhibin-β. The abnormal expression of these functional proteins will directly influence the sperm regulation of male animals and the nutrition supply.