Effect Of Heat Stress On Germ Cell Apoptosis And Expression And Localization Of Glucose Transporter In Boar Testes

Objective:Heat stress severely damages normal spermatogenesis in mammalian,but its mechanism remains unclear.Recently,some studies showed that heat stress results in germ cell apoptosis in testes of rat,mouse,dog and rhesus monkey.To our knowledge,there were few researches on the effect of heat stress on boar germ cell.Previously,our research group has studied the germ cell apoptosis of boar in environmental heat stress condition.In the study,the model of local testicular heating was established in boar,and the germ cell apoptosis and expression of related proteins were detected.Finally,the model was successfully established.Whether the heat stress affects the expression of glucose transporter(GLUT),then the energy uptake and metabolism of germ cells were affected.This study investigated the expression levels of glucose transporter(major GLUT in testes:GLUT1,GLUT2,GLUT3,GLUT5,GLUT8)in models of environmental heat stress and local testicular heating in boar.Methods:The model of local testicular heating was established in adult boar(Landrace).The boars of environmental heat stress group were kept in a thermally-controlled hot house(37-40 0C,7 or 42 days,3 hours per day).The self-made thermo-controlled 42 ? blanket was used in local testicular heating group(42 ? for 1 hour).Parts of boars were assigned as control and kept in normal temperature house(21-25 ?).After heat treatment,the boars were surgically harvested.The testes tissues of each boar were divided into two parts.The testes of part one were frozen in liquid nitrogen for extraction of total RNA and protein.Then the expression of mRNA and proteins of related genes were detected by Western blot analyses and quantitative real-time polymerase chain reaction(qRT-PCR),respectively.The testes tissues of part one were fixed in Bouin's solution for paraffin sections.Then germ cell apoptosis and expression and localization of related proteins were detected by TUNEL assay and immunohistochemical analyses,respectively.Results:1.TUNEL assay in local testicular heating groupThe results showed that the number of TUNEL-positive cells was increased(round and elongated spermatids)in local testicular heating group compared with the control group.The heat stress resulted in stronger Bax immunoreactivity in spermatogonia and spermatocyte,and Bax redistributed from a cytoplasmic to a perinuclear localization.In control and local testicular heating groups,Bcl-2 was localized in cytoplasm and nucleus.The heat stress resulted in stronger Bcl-2 immunoreactivity in elongated spermatid.The results of qRT-PCR and Western blotting showed that the Bax and Bcl-2 mRNA significantly increased in the local testicular heating group compared with the control2.Expression and localization of GLUT in heat stress condition(1)The effect of heat stress on expression of GLUT mRNA and proteinThe results of qRT-PCR and Western blotting showed that GLUT1 mRNA and protein expression levels in the environmental heat stress groups(short-term and long term)did not have significantly differences compared with control group.In the environmental heat stress group(short-term and long term),GLUT2 mRNA and protein expression levels significantly increased compared with control group.In the environmental heat stress group,the expression levels of GLUT3,GLUT5 and GLUT8 mRNA and protein significantly decreased compared with control group.After local testicular heating,the expression levels of mRNA and protein of GLUT1,GLUT2,GLUT5 and GLUT8 were significantly increased,but the expression of GLUT3 mRNA and protein did not have significantly differences compared with control group(2)The effect of heat stress on GLUT localizationThe results showed that GLUT1 localized in spermatocyte and round spermatid.GLUT2,GLUT3 and GLUT5 were localized in all types of germ cell.GLUT8 was localized in spermatocyte,round and elongated spermatidGLUT1 immunoreactivity in the environmental heat stress groups(short-term and long term)did not have significantly differences compared with control group.After local testicular heating,GLUT1 immunostaining was stronger.In the environmental heat stress and local testicular heating groups,GLUT2 immunostaining was stronger compared with control.After environmental heat stress,GLUT3 staining became weaker than control;however,GLUT3 staining in local testicular heating group did not have significantly differences compared with control.In the environmental heat stress groups,GLUT5 and GLUT8 staining became weaker than control.After local testicular heating,GLUT5 and GLUT8 staining were stronger than control groupConclusion:After heat treatment,the number of germ cell apoptosis was increased in boar testes,and the expression of Bax and Bcl-2 were increased.Heat stress resulted in changes in the expression of glucose transporter in the boar testes,changes in glucose uptake of germ cell,then normal spermatogenesis was affected.