The Expression, Role, And Mechanism Of BMP4 In Human Sertoli Cells And Its Association With Azoospermia And Seminoma

Objective: Azoospermia and seminoma, which are often accompanied by spermatogenesis disorder, are the major causes of male infertility. Besides the deficiency of male germ cells themselves, the abnormality of testis microenvironment or niche often results in spermatogenesis disorder. Sertoli cells are the key component of the testicular niche. It has recently been reported that there are variant number, morphology, maturity, and biological function of Sertoli cells in patients with abnormal spermatogenesis. Therefore, it is of great significance and interest to identify novel factors affecting the growth and function of human Sertoli cells. It has been demonstrated that BMP4 plays important roles in regulating the proliferation and differentiation of rodent spermatogonia. However, it remains unknow about the function and molecular mechanism of BMP4 in controlling human Sertoli cells. In this study, we have for the first time explored the expression, role, and signaling pathways of BMP4 in regulating adult human Sertoli cells and its association with non-obstructive azoospermia(NOA) and seminoma patients.Methods: Various approaches, including reverse transcription polymerase chain reaction(RT-PCR), Western blots, immunocytochemistry and immunohistochemistry, were utilized ro examine the expression of BMP4 and its multiple receptors in human Sertoli cells and testicular tissues at m RNA and protein levels. With the addition of BMP4 or its antagonist noggin, or BMP4 small interfering RNA(siRNA), CCK-8 cell proferation assay, BrdU incorporation, PCNA staining, RT-PCR and Western blots were employed to determine the effect and signaling pathways of BMP4 on regulating human Sertoli cell proliferation and function. Testis tissue arrays were used to disclose the association of BMP4 abnormal expresssion with Sertoli cell-only syndrome and maturation arrest in spermatogonia or spermatocytes as well as seminoma. All data were presented as mean ± SEM from at least three independent experiments and analyzed by Student’s t-test or one-way ANOVA with the appropriate post-hoc tests(Dunnet’s test or Turkey’s multiple comparison) using Prism(version 5, GraphPad Software), and P < 0.05 was considered statistically significant.Results: We first isolated human Sertoli cells with high purity and viability. Our data revealed that BMP4 and its three receptors, including ALK3, ALK6, and BMPRII, were present in human Sertoli cells, indicating that BMP4 may play a role in human Sertoli cells. CCK-8 cell proliferation and BrdU incorporation assays showed that BMP4 promoted DNA synthesis and proliferation of Sertoli cells. In contrast, BMP4 antagonist noggin and BMP4 knockdown reduced the division of Sertoli cells. Moreover, BMP4 knockdown inhibited the synthesis of fibroblast growth factor 2(FGF2), stem cell factor(SCF), claudin 11 and zonula occludens 1(ZO-1) but enhanced p27Kip1 transcription. BMP4 activated Smad1/5 but not PI3K/AKT or ERK pathway, and upregulated inhibitor of DNA binding/differentiation 2(ID2) and ID3 transcripts, whereas noggin counteracted these increases. Significantly, tissue arrays disclosed that overexpression or absence of BMP4 may be associated with Sertoli cell-only syndrome and maturation arrest in spermatogonia or spermatocytes stages and seminoma.Conclusion: BMP4 was identified as the first autocrine factor that regulates the proliferation and proteins’ synthesis of human Sertoli cells via Smad1/5 and ID2/3 and its abnormality is associated with human non-obstructive azoospermia and seminoma patients. This study thus provides novel insights into molecular mechanism underlying adult human Sertoli cell growth and offer new targets for gene therapy of male infertility and testis tumor.