The Researches On The Apoptosis Mechanism Of Manganism On Cock Sertoli-germ Cell In Vitro Culture

Manganese is a metal which is widely used in the industry. Manganese affect the normal physiological function when it is excessive accumulation in the body. In recent years, with the development of the reproductive toxicology and reproductive biochemistry, more and more attention has been paid to the reproductive system damage of manganese. Most of the researches about reproductive toxicity of manganese mainly focused on human and mouse not on poultry. In the study, cock Sertoli-germ cell was taken as a model, cultivated in the DMEM for 24 h with the final concentration of MnCl2 which was 0, 2, 3, 4 mmol·L-1 respectively. To discuss the apoptosis mechanism of manganism on cock Sertoli-germ cell, we detected the cell viability, the changes of apoptotic morphology, DNA fragmentation, mitochondrial transmembrane potential, expression of Cytochrome c in cytolymph and the experssion quantity alteration of Bak, Bcl-x, Caspase-3 mRNA. The results showed as follows:1 Utilizing tapan blue staining, WST-1 assay and dinitrophenylhydrazone (DNPH) chromometry to assess the effect of manganese to the viability of cock Sertoli-germ cell. The result showed that MnCl2 could inhibit cell viability, demage the capability of adherence and the sticky interaction between the spermatogemc cells and the support cells. It revealed that manganese had toxic effort to cock Sertoli-germ cell;2 Sertoli-germ cell growth condition was detected by inverted microscope. Morphology changes were detected by HE staining, AO/EB double fluorescence coloration, transmission electron microscope, which indicated that cock Sertoli-germ cell apoptosis was induced by MnCl2 accompanied with dose-effect relation. Utilizing agarose gel electrophoresis and TUNEL to detect cell DNA damage. The results showed that MnCl2 could damage the DNA of Sertoli-germ cell accompanied with dose-effect relation. It indicated that causing cell DNA damage may be one mechanism of cell apoptosis induced by MnCl2;3 Mitochondrial transmembrane potential was detected by flow cytometer, expression of Cytochrome c (Cyt-c) in cytolymph was detected by western blott, and the activities of Caspase-9, 3 and the activities of Mitochondrial Respiratory Chain ComplexⅠ,Ⅱ,Ⅲ,Ⅳwere examined by spectrophotography. Expression quantity alteration of Bak, Bcl-x, Caspase-3 mRNA were examined by real-time fluorescence quantitative PCR. The results showed that mitochondrial transmembrane potential was significantly decreased, expression of Cyt-c in cytolymph was increased, the activities of Caspase-9,3 and the activity of Mitochondrial Respiratory Chain ComplexⅠ,Ⅱ,Ⅲ,Ⅳwere increased. The expression of Bak mRNA was reduced while the expression of Bcl-x, Caspase-3 mRNA was decreased. It indicated that MnCl2 could induce cell apoptotic through mitochondrial dysfunction.In conclusion, MnCl2 could damage the function of Mitochondrial, regulate the the expression of Bak, Bcl-x mRNA, release Cyt-c from Mitochondrial into cytolymph, activate Caspase Cascade, effort Mitochondrial energy metabolism, induce Sertoli-germ cell apoptosis. One reproduction toxicity mechanism of manganese might be mediated by Mitochondria- Cytochrome c apoptosis pathway.