Role Of ERK1/2 Signaling Pathway In Heat-stimulated Lactate Production In Immature Boar Sertoli Cell

Heat stress has negative effects on animal health and reproductive function.During the course of evolution,mammals form a regulatory mechanism to prevent male infertility from inhibiting excessive apoptosis of germ cells.Lactate SCs secreted provides nutrition for developing germ cells,and enhnaces the capacity of germ cells resisting apoptosis.Extracellular signal regulated kinase 1 and 2(ERK1/2)signaling pathway and HSP70 play important roles in survival of cells after heat stress.However,it is unclear whether heat stimulated activation of ERK1/2 and iccrease of HSP70 promote lactate production of SCs.In this present study,heat stress was inudced by a 43 ? incubator for 30 min in immature boar SCs.Using the model,we explored the role of ERK1/2 on heat-stimulated lactate production and the relationship between phosphorylated ERK1/2 and the expression of HSP70 via addition of ERK1/2 inhibitor and siRNA.Immature boar SCs(21-day-old)were isolated,purified and cultured under the condition of 32 ? in 5% humidified atmosphere.Heat stress was induced in a 43 ? incubator for 30 min,and SCs were cultured at 32 ? in 5% humidified atmosphere for different periods(0,1,2,4,6 h).Cell viaility was detected using CCK-8 kit.Quantified PCR was used to quantify the mRNA levels of lactate production related molecules and HSP70.Western blotting was used to detect the protein levels of lactate production related proteins and HSP70.Cell and medium was collected to detect lactate production using a commercial kit.Furthermore,SCs were pretreated with U0126(a specific inhibitor of ERK1/2,2 h)and different siRNAs(6 h)to make sure the role of ERK1/2 on the expressions of HSP70,GLUT3,LDH A,LDH activity,and lactate production.These results were as followed:(1)Within 6 h after heat stress,the level of ERK1/2 phosphorylation was higher than that in the control group.With the extension of recovery time,it decreased gradually.From 4 h after heat stress,the level of ERK1/2 phosphorylation had no significant difference compared with that in the control group(P>0.05).The HSP70 protein levels and lactate production increased gradually within the 6 h after heat stress(P<0.01).Thus,2 h after heat stress was chosen to be the recovery time for the subsequent experients.(2)Different concentrations of U0126 had effect on cell viability.With the increase of concentrations of U0126,cell viability gradullay reduced.U0126(1?M)inhibited the phosphorylation of ERK1/2 in immature boar SCs significantly(P<0.01),but had no effects on cell viability(P>0.05).(3)Synthetic ERK1 siRNA had no effect on mRNA expression levels of ERK1(P>0.05),while ERK2 si RNA inhibited mRNA expression levels of ERK2 signifigcantly(P<0.01).Therefore,ERK2 siRNA was selected in the following experiments.(4)Compare the the control group,heat stress increased protein and mRNA expression levels of HSP70,GLUT3,and LDH A and lactate production,and enhanced LDH activity.U0126 and ERK2 siRNA inhibited the trend changes.In summary,heat stress(43 ?,30 min)enhances lactate production by activating ERK1/2 signaling pathway in immature boar SCs.Moreover,activation of ERK1/2 signaling pathway increases the protein and mRNA expression level of HSP70.Then,HSP70 increases the protein and m RNA expression levels of GLUT3 and LDH A and enhanced the LDH activity,resulting in lactate production.This study reveals the role of ERK1/2 signaling pathway in heat-stimulated lactate production and provide preclinical evidence that ERK1/2 agonist and HSP70 may be used as method to reduce heat-induced damage in male reproductive system.