Expression Analysis Of Spermatogenesis-related Gene Spata3in Mice

Objective:To analyze the expression characteristics of mouse spermatogenesis-related gene spata3and to reveal its role during spermiogenesis.Methods:1. Semi-quantitative RT-PCR was used to demonstrate the tissue-specific expression patterns of spata3;2. Real-time quantitative PCR was used to show the stage-specific expression patterns of spata3;3. Prokaryotic expression vector pGEX6p-1-spata3was constructed by gene cloning and was induced to express fusion GST-SPATA3by IPTG in Rosetta. GST-SPATA3fusion protein was purified by affinity chromatography and was confirmed by Western blot analysis. Anti-SPATA3polyclonal antibody was prepared by immunizing New Zealand white rabbits with the purified GST-SPATA3fusion protein. Antibody titer was determined by ELISA and its specificity was detected by Western blot.4. SPATA3protein distributions in testicular seminiferous tubules was detected by immuno-histochemical staining with anti-SPATA3polyclonal antibody;5. SPATA3protein sub-cellular distributions in spermids was examined by Multiple indirect immunofluorescence staining;6. Co-immunoprecipitation technique was applied to analyze the relationship between SPATA3and a-tubulin.Results:1. Semi-quantitative RT-PCR analysis showed that spata3was highly expressed in mouse testis.2. Real-time quantitative PCR analysis demonstrated that spata3was expressed abundantly at later stage during mouse spermatogenesis and probably participated in spermiogenesis.3. DNA sequencing and Restriction enzyme pattern analysis demonstrated that spata3was cloned correctly into prokaryotic expression vector. SDS-PAGE analysis showed that the fusion GST-SPATA3protein was successfully induced and Western blot analysis demonstrated that the anti-GST Monoclonal antibody can recognize the recombinant protein. ELISA results showed that high titer(1:102,400) of sera against GST-SPATA3fusion protein was harvested and Western blot showed that the anti-sera could specifically recognize SPATA3protein.4. Immunohistochemical staining showed that SPATA3protein located around the round and elongating spermids in mouse testicular seminiferous tubules.5. Multiple indirect Immunofluorescence staining showed that SPATA3protein was located around the spermid nucleus surface along with the manchette.6. Immunoprecipitation complexes between SPATA3and a-tubulin could not be detected by Co-immunoprecipitation in vitro.Conclutions:1. Spata3is a spermatogenesis-related gene which was specifically expressed in mouse testis and was transcribed highly in round and elongating spermatids.2. SPATA3proteins locate around nucleus surface along with the manchette in round and elongating spermids. It implies that SPATA3might take part in spermiogenesis during mouse spermatogenesis.3. Anti-SPATA3polyclone antibody can completely meet the need of protein level study and provide a vital basis for further research the function of spata3.4. We could not detect immunoprecipitation complexs formation in vitro between SPATA3and a-tubulin by Co-immunoprecipitation analysis in the present studuy. Other methods will be applied to study the interaction between SPATA3and a-tubulin...