Effects And The Mechanism Of Bmp4/Smad Pathway On The Differentiation Of Spermatogonial Stem Cells

Objective and significance Spermatogonial stem cells (SSCs) are single cells located on the basement membrane of the seminiferous tubules. Like other adult stem cells, the SSCs are able to self-renew to maintain a stem cell pool in the testis, and also are able to differentiate to produce spermatozoa eventually. In recent years, because SSCs play critical roles in the spermatogenesis mechanism research, the treatments of infertility, and the hereditary diseases, the establishment of transgenic animal and pluripotent stem cells, revealing the molecular regulation mechanism of SSCs self-renewal and differentiation, became the hot spots of the reproductive biology and the stem cell biology research.Bone morphogenesis protein4(Bmp4) is an important member of the transforming growth factor-beta (TGF-β) superfamily. In human embryonic development, Bmp4is a critical signaling molecule required for the early proliferation and differentiation of the embryo. Bmp4knockout mice undergo early gestational death due to the developmental defects of the mesoderm.Bmps transduce signals by binding to complexes of type Ⅰ and Ⅱ serine/threonine kinase receptors. Ligand blinding induces phosphorylation of the receptors, which then activate canonical signaling via receptor Smad(R-Smads)1,5and8. R-Smads are phosphorylated at the C-terminus by activated type Ⅰ receptor. They then complex with Smad4(Co-Smad), triggering nuclear translocation. It has been showed that the Bmp4/Smad signaling pathway is involved in stem cell self-renewal and differentiation such as embryonic stem cells, nerve stem cells. However, little is known about the function of the Bmp4/Smad signaling pathway on the differentiation of spermatogonial stem cells and its underlying mechanism.In the present study, mouse spermatogonial stem cells were cultured in vitro, immunofluorescence staining, quantitative real-time PCR and RNA interference were used to detect the effects of exogenous Bmp4on the SSCs differentiation, and the expression of sohlh2and c-kit, two critical regulators of SSCs differentiation. To explore Bmp4/Smad signaling pathway in the function of SSCs differentiation, and its mechanism provides a new theoretical and experimental basis for the future of male infertility treatment.Methods1. SSC culture and identification After48h culture, SSCs on the MEF feeder layer were identified for the expression of PLZF and Oct4, two known SSC specific markers, by immunofluorescence staining.2. Bmp4treatment The culture SSCs were divided into three groups: normal control culture medium (con group), culture medium with50ng/mL Bmp4(Bmp4group), culture medium with50ng/mL Bmp4and2u mol/L inhibitor(Bmp4+inhibitor group). After48h treatment, the protein expression of Sohlh2, C-kit, Smad and PLZF in three groups were assayed by immunofluorescence staining. The mRNA levels of sohlh2, c-kit, oct4and PLZF were detected by quantitative real-time PCR.3. Sohlh2siRNA transfection The transfected SSCs were divided into four groups:non-specificity siRNA+con group, non-specificity siRNA+Bmp4group, sohlh2siRNA+con group and sohlh2siRNA+Bmp4group. After24h transfection, the mRNA levels of sohlh2, c-kit, oct4and PLZF were analyed by quantitative real-time PCR. Results1. The morphologic results showed that, more than ten cells formed colony, the cells were round,relatively uniform in the cell size, with large nucleus, small cell body,and high nuclear/cytoplasm ratio. Immunofluorescence staining results showed that, cells in the colonies were PLZF and Oct4expressing cells. These results demonstrate that the cultured cells in the colonies are SSCs.2. Immunofluorescence staining results showed that, the Sohlh2, C-kit and Smad expression mainly distributed in the margine of the SSC colonies, the PLZF positive cells was observed in the center of the SSC colonies. In the Bmp4group, the positive cells and protein levels of sohlh2and c-kit were significantly more than the Con group and the Bmp4+Bmp4inhibitor group.There was no significant difference between the Con group and the Bmp4+Bmp4inhibitor group. The expression of smad in nuclei of the SSCs in the Bmp4group was significantly more than that of the other two groups. The PLZF expression deceased significantly in the Bmp4group. Quantitative real-time PCR results showed that, compared with the Con group, the mRNA levels of PLZF and oct4were down-regulated, and the mRNA levels of sohlh2and c-kit were up-regulated in the Bmp4group, the difference was significance (P<0.05). There was no significant difference for gene expression between the Con group and the Bmp4+Bmp4inhibitor group.3. Sohlh2siRNA transfection, the quantitative real-time PCR results showed, compared with the Bmp4+non-specific siRNA group, the c-kit mRNA level decreased, the mRNA levels of PLZF and oct4increased in the Bmp4+sohlh2siRNA group, the difference was significant (P<0.05); Compared with the con+non-specific siRNA group, the c-kit mRNA level increased, the mRNA levels of PLZF and oct4decreased in the Bmp4+non-specific siRNA group, the difference was significant (P<0.05). Conclusion1. Bmp4induced the differentiation of mouse SSCs in vitro.2. Bmp4signals through Smad pathway to up-regulate the expression of sohlh2, then stimulate the expression of c-kit to drive SSCs differentiation.