A Preliminary Study Of CIRBP Regulating ERK And Sam68 Induced Spermatogenic Apoptosis During Testicular Heat Stress

OBJECTIVE: To evaluate the expression of the CIRBP and Sam68 in the mouse testis and mouse primary spermatocytes,and the possible molecular mechanism of the CIRBPinduced apoptosis of primary spermatocyte after heat shock.METHODS:(1)First,in the animal experiments,for the experiments groups,the mouse scrotum was treated with a heat shock at 43°C in a water bath for 30 min,and there was no treatment in the control group.The testicular tissues were collected after 3h and 12 h.(2)Second,in the cells experiments for heat treatment,the GC-2spd cells were divided into control group,heat treatment for 30 min group and 60min;control group without any intervention,those cells were collecte after 12 h,24h,48 h.(3)Third,in the cells experiments for RNAi,the si RNA for knocking down the expression of the CIRBP was used to transfecte GC-2spd cells,and the cells were collected after 12 h,24h and 48 h after transfection.For the above parts,RT-PCR was applied to evaluate the m RNA expression of the gene CIRBP and Sam68;the western blot techniques was applied to evaluate the protein expression of CIRBP,Sam68,ERK1/2 and p ERK1/2.The apoptosis for GC-2spd with heat treatment or transfected with si RNA was also detected by using Annexin-V(PI)staining and flow cytometry.RESULTS: At the experiments with heat shock on mouse testicular,qPCR and western blot results showed that the expression levels of CIRBP and Sam68 were significantly decreased after heat shock(p<0.05),and with the time prolonging afetr heat shock,the expression of CIRBP was gradually decreased.Western blot results showed at 12 h after heat,the expression of p-ERK1/2 was significantly decreased with a statistical difference(p<0.05),but there was no significant changes in the expression of ERK1/2,p-ERK1/2 at3 h after heat.At GC-2spd cells with heat shock for 30 min group and heat shock for 60 min group,real-time PCR results showed that CIRBP and Sam68 m RNA expression at 12 h after heat shock all had no significant statistical difference(p>0.05),at 24 h,m RNA expression of CIRBP and Sam68 were significantly decreased(p<0.05)with heat for 60 min compared with the control group,at 48 h,the m RNA of CIRBP and Sam68 were statistically different(p<0.05)in two hot groups,but there was no significant difference between those groups(p>0.05).Western blot results showed that at 48 h after heat shock,the level of CIRBP and Sam68 protein in the two heat shock groups were statistically different(p<0.05),but there was no significant difference between the groups(p>0.05).In the cell interfect model,expression of CIRBP m RNA and protein was significantly reduced with increasing si RNA concentration(p<0.05);m RNA expression of Sam68 was statistically significant only at 48 h after transfection and si RNA concentration was 50 n M(p<0.05),its protein had no obvious change after transfection;ERK1/2 protein expression almost did not change;the expression level of p ERK1/2 protein decreased after transfection,but only in the transfection concentration of 50 n M group Statistically significant(p<0.05).CONCLUSION: After heat shock,CIRBP may regulate the expression of Sam68 at the transcriptional level,thereby regulating post-transcriptional gene expression and initiating the downstream mechanisms to induce the apoptosis of spermatogenic cells.In addition,during the heat shock,CIRBP may also activate the Ras/Raf/MEK/ERK cascade signaling pathway by regulating the activation and phosphorylation of ERK1/2,and posibally regulate the apoptosis of spermatogenic cells.