Study On Mechanism Of Bmi1 Action In Maitaining Male Reproductive Function

Bmil(B cell-specific MLV integration site-1)is a member of the PcG(Polycomb group)family which plays a key role in self-renewal of multiple normal adult stem cells.Bmi1 knockout mice exhibit growth retardation and premature aging.However,It is still unknown that whether Bmil deletion will disrupt spermatogenesis We will conduct this research by using Bmil knockout mice model,and observe effects of Bmil deletion on spermatogenesis,in addition,we will deeply explore the molecular mechanisms of Bmi1 loss induced impaired spermatogenesis.Firstly,We used RT-PCR,immunofluorescence and immunohistochemistry technique to detect Bmil relative expression and distribution in testis.Results showed that:compared to the heart,liver,spleen,kidney and other organs,the highest expression of Bmi1 is in testis,and about 140 times to liver,in addition,Bmi1 almost located in all kinds of spermatogenic cells,besides,Bmil was also highly expressed in Sertoli cells.These results suggest that Bmil may play an important role in spermatogenesis.To clarify the effect of Bmi1 on male reproduction,we used Bmi1 knockout mice.We analyzed the difference of reproductive phenotype between WT and Bmil-/-male mice by using mating experiment,detection of testicular size,weight,CASA sperm count and HE staining.Results showed that:compared to WT mice,Bmil-/-male mice were completely sterile,and showed significantly reduced testis size,decreased testis weight,disorganized seminiferous tube structure and decreaed maturation sperms.These results demonstrated that deletion of Bmil cause male infertility and severely spermatogenesis defect.To determine whether Bmi1 loss induced spermatogenesis defect due to decreased testosterone,we used Western-blot,RT-PCR and serology,flow cytometry technique to analyze serum testosterone levels,the expression levels of testosterone synthesis related enzyme 3 β HSD,17 β HSD and Leydig cells population in WT and Bmil-/-male mice.Results showed that:compared to WT mice,the serum level of testosterone the expression of testosterone synthesis related enzyme 3 β HSD and 17β HSD and Leydig cells population were significantly decreased in Bmil-/-male mice.These results indicated that the reduced testosterone level of Bmi1 null mice may attribute to decreased Leydig cells population and the expression of testosterone related enzymes.To determine the effect of Bmi1 deficiency on spermatogenic cells apoptosis and proliferation,we used immunohistochemistry and TUNEL staining method to analyze spermatogonial cell proliferation,apoptosis and spermatocyte numbers in WT and Bmi1-/-mice.Results showed that:compared to WT mice,Bmi1-/-mice showed decreased Ki67 positive cells significantly,increased TUNEL positive cells,and decreased number of spermatocytes.These results demonstrated that deletion of Bmil induced apoptosis and inhibited proliferation of spermatogonial cells,and resulted in decreased spermatocytes.To determine whether Bmi1 loss will cause sperm maturation disorder,we used HE,Mito-tracker staining,electron microscopy and RT-PCR method to analyze sperm morphology,sperm abnormality rate,the expression of sperm deformation related genes in testis,mitochondrial content of sperm sheath.Results showed that:compared with WT male mice,Bmi1-/-mice showed more numbers of head abnormalities sperms,defect nuclear transformation and acrosome formation,decreased expression of sperimiogenesis related genes tnpl,tnp2,prml,prm2,h1fnt,speml,and less mitochondria content in sperm sheath.These results demonstrated that Bmil play a critical role in spermiogenesis such as nuclear condensation,formation of acrosome and mitochondrial sheath formation.Bmil is expressed in Sertoli cells.To determine the effect of Bmil deficiency on Sertoli cells,we used immunohistochemical staining to analyze cell numbers and morphology of Sertoli cell in WT and Bmil-/-mice.Results showed that:There were no difference in numbers of Sertoli cell in WT and Bmi1-/-mice,however,Bmi1-/-mice showed the sertoli cytoplasm degradation.These results demonstrated that Bmi1 plays an important role in maintaining the intact of Sertoli cell cytoplasm.To determine whether Bmi1 loss induced spermatogenesis defect due to the dysfunction of spermatogonial stem cell(SSC),we used immunofluorescence and flow cytometry to analyze SSC numbers,population,proliferation and DNA damage.Results showed that:compared to WT mice,Bmil loss increased the numbers and population of SSC in testis and have no effect on SSC proliferation and DNA damage.These results suggested that Bmil deletion does not affect the SSCs pool.To determine whether Bmi1 loss will inhibit SSC "start on" after toxin induced spermatogenic damage,we used busulfan to damage the male reproductive system of our experimental mice.We used HE and immunofluorescence technique to analyze the reproductive phenotype,the proliferation and DNA damage of spermatogenic cells(SSCs included)and the numbers of SSCs in WT,Bmi1-/-,busulfan treated WT and Bmi1-/-male mice.Results showed that:compared to busulfan treated WT mice,Bmi1-/-mice showed more severe damaged seminiferous tube structure,more spermatogenic cells DNA damage and decreased spermatogenic cells proliferation in 2 weeks after busulfan treatment,However,the numbers,proliferation and DNA damage of SSCs in Bmil-/-mice had no significant difference;In 3 weeks after busulfan treatment,only the inner cells left including SSC in some seminiferous tube of Bmil-/-mice,and intact seminiferous epithelium exited in WT mouse.what’s more,Bmil KO mice show more numbers of SSCs than that of WT mice.These results demonstrated that Bmi1 loss is more sensitive to reproductive toxin,however there are no significant difference in SSCs numbers between WT and Bmil KO mice.Bmil can not only supress p16 and p19 gene expression at transcariptional level but also play a critical role in antioxiant defence.To explore whether the defective spermatognesis of Bmil null mice is associatied increased proteins levels in p16,p19 pathways and elvated reactive oxygen,we used immunohistochemistry,Western-blot,Real-time PCR,flow cytometry and biochemical testing to analyze p16 protein,proteins in p19-p53 pathways,oxidative stress and DNA damage related molecular in WT and Bmi1-/-mice.Results showed that:compared to WT mice,the expression p19,p53,p21and p16 protein up regulated in Bmi1-/-mice.Bmil-/-mice showed higher levels of oxidative stress and DNA damage.These results suggested that the Bmil deletion intrigued increased expression of p16,activation of the p19-p53 signaling pathway,oxidative stress and DNA damage in testis.To determine whether p16,p53,oxidative stress play a critical role in mediating Bmi1 loss induced spermatogenesis defect,we generated p16 or p53 with Bmi1 double knockout mice,and treated Bmil-/-mice with antioxidants NAC or PQQ.We analyzed the difference of reproductive phenotype among WT,Bmil-/-,double KO and NAC or PQQ treated Bmil-/-male mice by using detection of testicular size,weight,CASA sperm count,HE and immunohistochemical staining Results showed that:compared with Bmil-/-mice,p16-/-Bmi1-/-,p53-/-Bmi1-/-and NAC or PQQ treated Bmi1-/-mice showed significantly increased testis size,testis weight and sperm counts.What’s more,the seminiferous tube structure get to be normal;p16-/-Bmi1-/-,p53-/-Bmi1-/-and NAC or PQQ treated Bmil-/-showed significantly reducedγ H2A.X positive cells,increased PCNA positive cells and decreased TUNEL positive cells.These results demonstrated that p16,p53,and oxidative stress play a critical role in mediating Bmil loss induced spermatogenesis disorder.To determine the interaction among oxidative stress,p16 and p19-p53 pathway,we used Western-blot technique to analyze the expression of p16,p19,p53 and p21 in WT,Bmi1-/-,p16-/-,p53-/-,p16-/-Bmi1-/-,p53-/-Bmi1-/-and NAC or PQQ treated Bmil-/-,mice.Results showed that:NAC or PQQ treatment can significantly reduce the expression of p19,p53,p21 and p16 protein in testis of Bmil-/-mice;compared to WT mice,increased the expression of p19 and p53 protein were observed in p16-1-mice and higher level p16 protein was in p53-/-mice;compared to Bmi1-/-mice,p16-/-Bmi1-/-mice show more higher level of p19 and p53,accordingly,there was higher level of p16 protein in p16-/-Bmi1-/-mice.These results indicated that the improved spermatogenesis of p16 or p53 deletion to Bmil deficiency mice are at least partly suppressed,sepratly by up regulation of molecular in p19-p53 pathway or p16 proteins;The corrected reproduction phenotype for antioxidants NAC or PQQ supplementation to Bmi 1 null mice,may attribute to drectly reduce the high level of ROS and indirectly decrease p16,p19,p53 and p21 proteins of Bmi1 deficiency mice;This research mainly focused on the effect of Bmil deletion on spermatogenesis,and deeply explore the molecular mechanisms for Bmil lossinduced spermatogenesis disorder and will provide targets and new experimental basis for clinical treatment of male infertility.