Effect On INSL3 Regulating The Proliferation And Apoptosis Of Mouse Gubernacular Cells Through PKA/ERK Signaling Pathway

Objective:Recent studies have shown that ectopic testis was accompanied with gubernacular dysplasia.Therefore,the factors affecting the development of the gubernaculum have drawn more and more attention.Insulin-like Factor 3(INSL3)was considered to be the key factor affecting the transabdominal descent of testis,stimulating gubernacular hyperplasia and regulating its growth and development.The purpose of this study was to test the effects of INSL3 on the proliferation and apoptosis of mouse gubernacular cells and the activation of downstream PKA and ERK pathways by in vitro culture of mouse gubernacular cells.At the same time,the effects of INSL3 on the proliferation and apoptosis of gubernacular cells after PKA/ERK signaling pathway was blocked.It might help to reveal the mechanism how INSL3 affected testicular descent and development to understand the effects of PKA and ERK signaling pathways on the proliferation and apoptosis of mouse gubernacular cells.Materials and Methods:The gubernacula were removed from 3-day-old mice and cultured.The subcultured cells were randomly divided into four experimental groups(INSL3,INSL3+ PKA enzyme inhibitor(H89),INSL3+ ERK enzyme inhibitor(PD98059),INSL3+H89+PD98059),and control group.CCK8 was used to detect the cell proliferation in each groups in different timepoints(24h,48 h,72h).After 48-hour treatment,western blotting was used to detect the changes in the expression of cell proliferation markers Ki67,PCNA,apoptosis-related proteins Bax,Caspase3 and Bcl-2,as well as the phosphorylation of PKA and ERK.Results:1.The mice gubernacula were dissected and obtained.In vitro cultured mice gubernacular cells grew in the form of fibroblasts,with excellent homology and high survival rate after passage.2.The cell proliferation rate of INSL3 group was faster than that of the control.The proliferation difference appeared after 24 h culture(P<0.05),and the difference was more obvious after 48 h and 72 h culture(P<0.001).The proliferation trend of INSL3+H89 group,INSL3+PD98059 group and INSL3+H89+PD98059 group was consistent,and the proliferation rate of those groups were slower than that of INSL3 group,and the difference appeared at 24h(P <0.05),and the difference was more obvious at 48 h and 72 h.(P<0.01).INSL3+PD98059group,INSL3+H89+PD98059group inhibited the most significantly(P<0.001).3.Western Blot analysis showed that:3.1 Proliferation-related proteins: Compared with the control group,the expression levels of Ki67 and PCNA were increased in INSL3 group,with statistical differences(P <0.01),the expression levels of INSL3+H89 group,INSL3+PD98059group and INSL3+H89+PD98059 group were significantly decreased compared with INSL3 group,with statistical difference(P<0.05).Ki67 expression of INSL3+H89+PD98059 group was decreased compared with that of INSL3+H89group and INSL3+PD98059 group,respectively,and the difference was statistically significant(<0.01),while there was no significant difference in the expression of PCNA.3.2 Apoptosis-related proteins: Compared with the control group,the expression levels of Bax and Caspase3 in INSL3 group were decreased,while the expression levels of Bcl-2 were increased,with statistical differences(P<0.05).Compared with INSL3 group,INSL3+H89 group,INSL3+PD98059 group and INSL3+H89+PD98059 group,the expression levels of Bax and Caspase3 were increased,while the expression levels of Bcl-2 were decreased,with statistical differences(P<0.05).Caspase3 expression level of INSL3+H89+PD98059 group was significantly decreased compared with that of INSL3+H89 group and INSL3+PD98059 group,with statistical difference(P <0.05).Compared with INSL3+H89 group,Bcl-2 expression was decreased and Bax expression was increased in INSL3+H89+PD98059 group,with statistical differences(P<0.05).There was no significant difference in Bcl-2 and Bax expression between INSL3+H89+PD98059 group and INSL3+PD98059 group(P>0.05).3.3 Related signaling pathway proteins: Compared with the control group,p-PKA/PKA and p-ERK/ERK ratios in INSL3 group were increased,with statistical differences(P<0.01);p-PKA/PKA and p-ERK/ERK of INSL3+H89 group,INSL3+PD98059 group and INSL3+H89+PD98059 group were decreased to different degrees compared with INSL3 group,with statistical difference(P <0.01),the p-PKA/PKA ratio of INSL3 +H89+PD98059 group decreased compared with INSL3 +H89 group and INSL3 +PD98059 group,and the difference was statistically significant(P<0.05),the p-ERK/ERK ratio of INSL3+H89+PD98059 group decreased significantly compared with that of INSL3+H89 group,and the difference was statistically significant(P<0.05),there was no significant difference compared with INSL3+PD98059 group.(P> 0.05).Conclusion:1.INSL3 could promote the proliferation and inhibit the apoptosis of mice gubernacular cells.2.PKA and ERK cellular signaling pathways were involved in the proliferation promotion and anti-apoptotic effects of INSL3 on gubernacular cells.3.INSL3 could promote the phosphorylation of ERK and PKA proteins in mouse gubernacular cells,suggesting that INSL3 regulated the proliferation and apoptosis of micegubernacular cells by activating PKA and ERK signaling pathways.