BMP6 Regulates Proliferation And Apoptosis Of Human Sertoli Cells Via Smad2/3 And Cyclin D1 Pathway And DACH1 And TFAP2A Activation

Objective: Infertility is one of the most serious diseases affecting 10%-15% of the couples worldwide,and half of them are attributed to male factors.Azoospermia comprises approximately 15% of male infertility.Spermatogenesis is composed of three main stages,including mitosis of spermatogonia,meiosis of spermatocytes,and spermiogenesis of spermatids,which is regulated precisely by the testicular microenvironment.Any abnormality in the process of spermatogenesis may result in dyszoospermia and eventual male infertility.It remains to be an unsolved and key issue to get genetic offspring for males wishing to have babies.Sertoli cells,as unique and the most important somatic cells within seminiferous tubule,play essential roles in regulating normal spermatogenesis.Testicular microenvironment or niche is composed of Sertoli cells,blood vessels,and growth factors.Sertoli cells can secrete some important growth factors,e.g.,GDNF and SCF,which regulate the self-renewal and differentiation of spermatogonial stem cells.Sertoli cells are the main component of blood-testis barrier,which provides a stably internal microenvironment for spermatogenesis.Moreover,recent studies have demonstrated that Sertoli cells can be reprogramed to become multipotent neural stem cells by defined factors,reflecting that Sertoli cells have great applications in regenerative medicine.Any changes in the quantity and quality of Sertoli cells may lead to the dyszoospermia.Bone morphogenetic protein 6(BMP6)belongs to the member of the transforming growth factor-?(TGF-?)superfamily,and it plays an important role in mediating the self-renewal and differentiation of various kinds of stem cells,as evidenced by the following facts: i)BMP6 promotes the proliferation of germline stem cells(GSCs),and conversely,it prevents the differentiation of GSCs;ii)BMP6 stimulates the division of mesenchymal stem cells(MSCs)and hematopoietic stem cells(HSCs);iii)BMP6 regulates neural stem cell(NSC)fate determinations in a time-dependent manner;and iv)the deficiency of BMP6 may cause cancer,skeletal deformities,heritable and metabolic disorders,and cardiovascular disease.However,the expression,function,and mechanism of BMP6 in regulating the fate determinations of human Sertoli cells remain elusive.Therefore,this study was designed with the following Aims: i)to identify the expression of BMP6 and its receptors in human Sertoli cells and human testes;ii)to explore the biological roles of BMP6 in regulating proliferation and apoptosis of human Sertoli cells;and iii)to probe signaling transduction pathways of BMP6 in the regulation of human Sertoli cells.Methods: Human Sertoli cells were isolated and purified from testis tissue of OA patients using a two-step enzymatic digestion and followed by the differential plating.The viability of freshly isolated cells was evaluated by trypan blue exclusion assay,and the identity and the purity of isolated human cells was assessed using immunocytochemistry.Furthermore,RT-PCR and Western Blots were utilized to determine whether BMP6 and its multiple receptors,including ACVR1,MBPR1 A,BMR1B,BMPR2,were expressed in human Sertoli cells.Immunohistochemistry was used to reveal the cellular location of BMP6 and its multiple receptors in human testis.The effects of exogenous BMP6 on human Sertoli cells in culture were measured by CCK-8,EDU incorporation assay,ELISA assay,and Annexin-V/ PI flow cytometry.Meanwhile,RNA interference(RNAi)was employed to probe the roles of endogenous BMP6 in human Sertoli cells in vitro.The secretion of SCF in Sertoli cells treated by BMP6 or BMP6 si RNA-1 was measured by ELISA.Meanwhile,Western blots was utilized to compare the expression levels of ZO1,OCLN,SCF,GDNF,AR and AMH in human Sertoli cells without or with transfection BMP6 si RNA treatment.The levels of phosphorylation(phos)-ERK/ERK,phos-AKT,phos-Smad2/3/Smad2/3,phos-Smad1/5/8/Smad1/5/8,as well as cell cycle proteins including cyclin A,cyclin B1,cyclin D1,CDK2,and cyclin E,were compared to explore the molecular mechanisms of BMP6 in regulating human Sertoli cells.Finally,the influence of DACH1 and TFAP2 A transcription factors on the proliferation of human Sertoli cells was conducted using RNAi and CCK-8 assay.RT-PCR and Western blots were further utilized to compare the expression levels of BMP6 gene and protein in Sertoli cells of OA and NOA patients.Results: The viability of freshly isolated cells was over 98%,as measured by trypan blue exclusion assay.Immunocytochemistry demonstrated that more than 96% of the isolated cells were positive for WT1,SOX9,GDNF,SCF,ZO1,OCLN,and BMP4,markers of Sertoli cells,reflecting that the purity of these cells was more than 96%.RT-PCR and Western blots further verified that the isolated cells were human Sertoli cells.RT-PCR,immunocytochemistry and Western blots revealed that BMP6 and its multiple receptors,including ACVR1,BMPR1 A,BMPR1B,ACVR2 A,ACVR2B,and BMPR2,were present in Sertoli cells.Immunohistochemical staining further showed that BMP6 was present in human Sertoli cells but not male germ cells,while its receptors were detected in human Sertoli cells and some types of human male germ cells(e.g.,spermatogonia and spermatocytes).CCK-8 and EDU incorporation assay demonstrated that BMP6 promoted the proliferation of Sertoli cells,whereas the apoptosis was reduced using Annexin-V/ PI assay.On the other hand,CCK-8 and EDU incorporation assays demonstrated that the proliferation of Sertoli cells was reduced after silencing BMP6,while the apoptosis was increased by BMP6 si RNA-1 using Annexin-V/PI assay.ELISA displayed that the secretion of SCF in Sertoli cells was increased by BMP6 and decreased by BMP6 si RNA-1.The levels of ZO1,SCF,GDNF,and AR proteins were reduced by BMP6 si RNA-1,while OCLN and AMH were not significantly changed after silencing BMP6 in Sertoli cells.The levels of phos-smad2/3 and cyclin D1 were enhanced by BMP6 in Sertoli cells and decreased by BMP6 knockdown.The transcripts of DACH1 and TFAP2 A were increased by BMP6 and reduced by BMP6 si RNA-1.Finally,the proliferation of human Sertoli cell was decreased when silencing DACH1 and TFAP2 A respectively,suggesting that BMP6 regulates the proliferation and apoptosis of human Sertoli cells via activation DACH1 and TFAP2 A.RT-PCR and Western blots showed that the levels of BMP6 gene and protein were higher in human Sertoli cells from OA patients compared to hypospermatogenesis,MA,and SCO patients.Conclusions: BMP6 and its multiple receptors are present in human Sertoli cells,and BMP6 acts via an autocrine pathway in human Sertoli cells,while it regulates the fate determinations of human male germ cells through a paracrine manner.BMP6 promotes the proliferation and DNA synthesis but suppresses the apoptosis of adult human Sertoli cells.BMP6 stimulates the secretion of ZO1,SCF,GDNF,and AR proteins in human Sertoli cells.In summary,BMP6 regulates proliferation,DNA synthesis,and apoptosis of human Sertoli cells via Smad2/3 and cyclin D1 pathway and DACH1 and TFAP2 A activation.Since BMP6 may regulate the spermatogenesis via affecting Sertoli cells,this study thus provides new insights into molecular mechanisms underlying human Sertoli cell development and the etiology of male infertility.