Study On Function Mechanism Of Mitochondria Signal Pathway In The Germ Cell Apoptosis Induced By Heat Stress In Boars

Objective:Mammalian spermatogenesis was regulated by a variety of factors,including hormones and growth factors.Heat stress had detrimental effects on the spermatogenesis and sperm quality in boars.But the molecular mechanism has not yet well understood.The previous study of experimental animal rat found that the germ cells apoptotic induced by heat stress.Mitochondria signal pathway is the key apoptotic pathway for heat-induced male germ cell death cell.The aims of present study were determine whether or not heat stress starting germ cell apoptotic by mitochondria signal pathway,thus affect the spermatogenesis.Methods:The present study designed boar testicular heat stress model.Mature boars(Landrace)were used in the study.Parts of the experimental animals were subjected to elevated ambient temperature(37～40 ℃ for 3 h daily for 7 days and 42 days or 40～42℃for 1 h daily for 7 days and 42 days)as experimental group.Other animals were kept in the normal house(20～27℃)as control group.After experiment treatment,all boars were castrated and the testes were harvested.Parts of testis tissue were snap frozen in liquid nitrogen,and then used to extract total RNA and protein.Parts of testis tissue were fixed in Glutaraldehyde solution,used to prepare transmission electron microscopy sections.Parts of testes tissue were fixed in Bouin’s solution,used to prepare paraffin sections.TUNEL assay was used for the detection of apoptotic germ cells.QRT-PCR、Western blotting and immunohistochemistry were used to analysis the expression of Bcl-2,Bax,Cyt-C,Apaf-1,Caspase-9,Caspase-3.Result:1 Effect of environmental heat stress on the germ cell apoptosisTUNEL showed that spermatocytes and spermatids were sensitive to heat stress.Apoptotic signals increased under heat stress conditions compared with the control.In contrast,extensive numbers of TUNEL-positive cells were found in the heat-stress boars,including spermatocytes,elongated and round spermatids.2 Effect of environmental heat stress on the electron microscopic structure of testisHeat stress affected the germ cell nucleus and mitochondria ultrastructure.The results showed that heat treatment resulted in germ cell chromatin clumping and edge set,and mitochondria closed to the nuclear and swell,etc.3 Effect of heat stress on the expression of apoptosis regulating gene and proteinThe results of QRT-PCR and Western Blotting showed that mRNA and protein expression of Bax,Cyt-C,Apaf-1,Caspase-9 and Caspase-3 increased significantly in heat treatment compared with the control.The expression of anti-apoptotic protein(Bcl-2)increased,but failed to inhibit the increase of germ cell apoptosis.4 Immunohistochemical showed that a redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia,spermatocytes and spermatids obtained in the heat treated group.In control and experimental groups,Bcl-2 was expressed in most seminiferous tubule cells with preferential expression in the nucleus and in the cytoplasm close to the luminal surface of differentiating spermatids.Heat treatment resulted in a more diffuse cytoplasmic localization of Cyt-C.Immunoreactivity-positive of Apaf-1 was increased in spermatocytes and some Sertoli cells after heat treatment.Heat stress increased the expression levels of Caspase-9 in post-meiosis germ cells and Sertoli cells.Heat stress resulted in some Sertoli cells Caspase-3 positive staining and Caspase-3 positive with nucleus staining increased.Conclusion:Environment heat stress induced that germ cell apoptotic rate increased and resulted in the expression change of mitochondria-depended pathway related protein in boar testis.Mitochondria signal pathway involved in the regulation of germ cell apoptosis induced by heat stress in the boar.