The Role Of SET Protein In Spermiogenic And Its Mechanism

Background and Objectives:The mature spermatozoon produced from the testis is the precondition of male fertility.The process from the spermatogonial stem cells to mature spermatozoa is constant,which can be divided into the mitotic,meiotic and postmeiotic(spermiogenic)phases.At the physiological status,FSH acts to stimulate spermatogonial proliferation and entry into meiosis and testosterone acts to ensure completion of meiosis and spermiogenesis,both play synergistic effects.Besides,the other hormones or local factors can also regulate spermatogenesis.The local regulating mechanism of spermatogenesis is still obscure.Spermiogenesis refers to the haploidic round spermatids undergo dramatic morphological changes,including cytoplasmic dropping and the form of compact nuclear chromatin,and transform into spermatozoa.During which,hyperacetylated nuclear histones are replaced by more basic transition proteins,and ultimately protamines to facilitate denser packaging of the paternal genome into the sperm head.The compated nuclear can protect the genome from the deleterious materials from female genital tract.BRDT(bromodomaintestis-specific)recognize the replaced hyperacetylated histones and induce its evicted,which would then be degraded through the action of spermatoproteasome or/and yet unknown proteolytic systems.The acetylized histone is mainly regulated by HATs and HDACs,local factors in addition.Observing the mechanism of spermatogenesis and spermiogenesis has important significance in the cause of male infertility.Since previous studies have demonstrated that more than half of male infertility patients showed oligospermia,asthenospermia,azoospermia and teratozoospermia.The cause is not clear and the core mechanism is abnormal spermatogenesis and spermiogenesis.SET is a multifunctional protein that plays an important role in the regulation of cell cycle,DNA replication,nucleosome assembly,chromatin modification,transcription,cell proliferation,apoptosis and so on.It is also called the inhibitor of acetyltransferase(INHAT)and is the component of the INHAT complex,which can inhibit histone acetylation.Our previous study showed that SET protein is mainly expressed in spermatogonia and spermatocytes,with low expression in spermatid.What’s more,SET protein promoted the spermatogonium proliferation.Therefore,we propose this hypothesis that SET,as a local factor,regulate spermatogenesis and spermiogenesis.To confirm this hypothesis,with further investigations of mechanisms of spermatogenesis and spermiogenesis,we applied GC-2 spd cell line to carry out some experiments.We observed the expression and subcellular location of SET protein in GC-2 spd;focused on the effects of SET protein in regulating the spermatocyte proliferation by knockdown the SET protein;further studied the potential mechanisms in SET regulating histone acetylation during spermiogenesis.Material and Methods:(1)The GC-2 spd cell was cultured in vitro,the immunofluorescence was used to identify two markers of spermatocytes,LDHC(lactate dehydrogenase C)and TESK1(Testis-Specific Kinase 1).(2)Constructed AdH1-siRNA/NS and AdH1-siRNA/SET adenovirus.Immunofluorescence,Western blot and Real time RT-PCR were used to prove the expression validity.(3)The cells were transfected with AdH1-siRNA/SET to knockdown the expression of SET protein,AdH1-siRNA/NS as control.Cells were in vitro cultured for 24h,48h,72h,cells number was used to detect the cell proliferation.(4)AdH1-siRNA/NS and AdH1-siRNA/SET adenovirus were transfected in GC-2 spd cells for 72h.Western blot and Real time RT-PCR was used to detect the expression levels of SET protein,Ac-histone,histone acetyltransferases and histone deacetylases,HAT Activity Assay Kit and HDAC Activity Assay Kit to assess the HAT and HDAC activity.(5)The combination of SET and H4 or SET and H3 were detected by an immunoprecipitation kit from the protein extracted from in vitro GC-2 spd cells.(6)Statistical methods.The SPSS 16.0 software was used for statistical analysis.Data were presented as mean±standard deviation(Means ± SD).P<0.05 was considered significant difference.Results:(1)Immunofluorescence confirmed the positive expression of Two markers of spermatocytes,LDHC and TESK1.Therefore,GC-2 spd cell line was used in our study as spermatocyte.(2)SET protein was expressed in both cytoplasm and nucleus of GC-2 spd cell.AdHl-siRNA/NS and AdH1-siRNA/SET adenovirus were successfully constructed in our laboratory.The expression of SET protein in the cytoplasm and nucleus of the cells transfected with AdHl-siRNA/SET was significantly decreased,the expression levels of SET was inhibited by 70～80%(P<0.05).So,these adenovirus vectors with high efficient were used our study.(3)In GC-2 spd treated with AdH1-siRNA/SET,knockdown of SET protein significantly decreased the cell number,inhibited the cell proliferation(P<0.05).(4)After knockdown of SET protein,the acetylation level of H3 and H4 was significantly increased(P<0.05)by 2-3 fold.However,there were no significantly differences in the mRNA and protein expressions of HATs and HDACs,the same with HAT activity/HDAC activity.(5)Immunoprecipitation showed that SET protein combined with H3 and H4 in the protein extracted from in vitro GC-2 spd cells.Conclusion:(1)SET protein was expressed in both cytoplasm and nucleus of spermatocytes.The knockdown the expression of SET protein inhibited the cell proliferation,which indicated that SET regulated spermatogenesis by promoting spermatocytes proliferation.(2)SET protein inhibited the acetylation of histone H3 and H4 by combining and masking the lysine acetylation sites in spermatid,thus possibly inhibiting the spermiogenesis.(3)This study demonstrates that SET,as a local factor,is involved in the regulation of spermatogenesis and spermiogenesis.This study enhance our knowledge on the regulating mechanisms of spermatogenesis and spermiogenesis and provide us a theoretical basis of cause for male infertility,especially in oligospermia,asthenospermia,azoospermia and teratozoospermia.