The effect of regulating the Wnt signaling pathway on the proliferation and differentiation of spermatogonial stem cells.

Abstract

BackgroundSpermatogonial stem cells and organ engineering research has raised new hope in infertility treatment. Spermatogenesis is a complex physiological process. To observe the proliferation ability and differentiation tendency of mice spermatogonial stem cells (SSCs), to study the effect of regulating the Wnt signaling pathway on the proliferation and differentiation of SSCs, and to provide a valuable basis for the clinical application of SSCs.MethodsSSCs were isolated and cultured by immunomagnetic separation. Cell surface markers were identified by flow cytometry. Axin1 was chosen as the target gene to inhibit fibrosis of SSCs by inhibiting the activity of Wnt signaling pathway. Axin-siRNA interference vector was constructed and transfected into spermatogonial stem cells. Cultured SSCs were randomly divided into six groups: control group, SSCs + TGF-β group, SSCs + DKK1 group, SSCs + Axin-RNAi group, SSCs + TGF-β + DKK1 group, SSCs + TGF-β + Axin-RNAi group. Proliferation of SSCs in each group was detected by MTT assay. Immunofluorescence, western blot and real time polymerase chain reaction analysis were used to detect protein expression in the Wnt/β catenin signaling pathways and the molecular markers of fibroblasts in SSCs.ResultsFlow cytometry analysis confirmed that the cultured SSCs were of high purity. MTT assay showed there was no significant difference between Axin-siRNA transfected and non-transfected cells. The proliferation ability was significantly increased in the SSCs + TGF-β group, however, it was retarded in SSCs + Axin-RNAi group. The results of immunofluorescence and western blot analysis showed that the expression levels of the Wnt signaling pathway proteins were relatively inhibited after Axin-siRNA was applied. Real-time polymerase chain reaction showed that the expression levels of the molecular markers of fibroblasts were close to the normal control group.ConclusionsThe Axin-siRNA constructed in this study specifically inhibited Wnt/β-catenin signal pathway activation, then inhibited the differentiation of SSCs into fibroblasts, which provides a valuable basis for the clinical application of SSCs.