Interaction Of SPATA1 With The Cilia Transporter IFT20 And Its Expression And Localization During Spermatogenesis

Objective:To further verify the interaction between the spermatogenesis-related protein SPATA1 and the flagella transporter IFT20,and to explore the expression,localization and role of SPATA1 in spermatogenesis.Method:IFT20 and SPATA1 protein expression plasmids were transfected into CHO cells,and their colocalization was detected by immunofluorescence.At the same time,IFT20/Flag and SPATA1/GFP were co-transfected into COS-1 cells,and cell lysates were co-immunoprecipitated to further verify the interaction between IFT20 and SPATA1.The SPATA1/pEGFP-N2 plasmid was constructed,and this plasmid and the control plasmid pEGFP-N2 were transfected into COS-1 cells respectively.The cell lysate was subjected to Western blotting to detect the specificity of the SPATA1 polyclonal antibody.Using SPATA1 and IFT20 as primary antibodies,immunofluorescence staining was used to observe the expression and localization of SPATA1 and IFT20 in testicular spermatogenic cells.The wild-type mouse epididymis sperm was collected,and the expression of SPATA1 in mature sperm was detected by Western blotting and immunofluorescence.The total protein of testes of Ift20 knockout mice and control mice were extracted,and testicular tissue sections were prepared.Western blotting and immunofluorescence were used to detect the effect of IFT20 knockout on the expression and localization of SPATA1in testis tissue.Result:Intracellular protein localization experiments showed that IFT20 and SPATA1were partially colocalized in CHO cells to form clusters.When COS-1 cells were co-transfected with IFT20/Flag and SPATA1/GPF expression plasmids,Flag antibody precipitated both IFT20 and SPATA1 simultaneously.Western blotting showed that the prepared SPATA1 antibody could specifically recognize the protein.Immunofluorescence staining of mouse testis tissue and epididymal mature sperm showed that SPATA1 protein signal was detected in the acrosome of round spermatids,but the expression of this protein was not found in mature sperm.IFT20 was also localized to the acrosome of round sperm cells.There was no significant effect of IFT20 deletion on SPATA1 protein expression in testis compared to control mice(Ift20^flox/+;Stra8-Cre).Immunofluorescence results showed that SPATA1 expression and localization were similar in Ift20 knockout mice and control mice during IV-XII of spermatogenesis.In the step 12 of spermatogenesis,the sperm cell nuclei of the Ift20 knockout mice did not normally elongate,and SPATA1 was not distributed along the acrosome.Conclusion:SPATA1 forms a protein complex with IFT20 and may be involved in sperm acrosome formation by regulating IFT20-mediated protein trafficking.