Expression On Tissues Of Male Mice For Spata1 And Its Relationship With Intraflagella Transport Protein 20

Objective: To observe the expression of SPATA1 in different tissues of male mice and the location of SPATA1 in cells,and explore the interaction between SPATA1 and Intraflagellar transport protein 20.Method: The RNA was extracted from eight tissues of heart,liver,spleen,lung,kidney,brain,muscle and testis of adult male mice.The expression of Spata1 in different tissues was detected by RT-PCR method.The Spata1 coding region was amplified by PCR based on cDNA from mice testis tissue to construct recombinant plasmids of Spata1/p ET-28 a,Spata1/p EGFP-N2 and Spata1/p GADT7.SPATA1 antibody were prepared from Spata1/p ET-28 a plasmid by protein purification.The expression of SPATA1 protein in different tissues and different stages of testicular tissue was observed by Western-blot method in mice.The Spata1/p EGFP-N2 recombinant plasmids were transfected into COS-1 cells and APRE19 cells respectively.The expression of fusion protein was detected by Western-blot method and the localization of SPATA1-GFP in APRE19 cells was detected by Confocal Laser Scanning Microscopy.The mixed germ cells from mouse testis were extracted to detect the localization of SPATA1 in the testis of normal male mice by immunofluorescence staining.The interaction between SPATA1 and IFT20 was determined by yeast two-hybrid experiment.Western-blot method was used to detect the expression of SPATA1 protein in the testis of IFT20 deficient mice.Result: RT-PCR results showed that Spata1 was expressed in testis and brain.Western-blot experiments showed that SPATA1 was expressed in testis,brain,heart and kidney of mice,and the molecular weight of protein was 52 k D.The expression of SPATA1 protein was first visible at postnatal day 24(PD24)and increased signifcantly at PD30,when germ cells enter the third stage of spermatogenesis,that is sperm elongation stage.The molecular weight of SPATA1-GFP fusion protein was 78 k D,which was distributed in the cytoplasm of APRE19 cells.Immunofluorescence staining experiments revealed that SPATA1 was located in the acrosome of germ cells.Yeast two hybrid experiment showed that there were colonies formated of SPATA1/IFT20 on the culture plate lacking three amino acids(Leu,Trp,His).SPATA1 protein was also detected in the testis of Ift20 KO mice.Conclusion: Three groups of Spata1 recombinant plasmids were successfully constructed and SPATA1 antibody was prepared.SPATA1 was abundantly expressed in the testis,and SPATA1 was located in the acrosome of the sperm cells and in the cytoplasm of the cells in vitro.There was a direct interaction between SPATA1 and IFT20,but the absence of IFT20 protein in testis tissue could not affect the expression of SPATA1 protein.