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

Abstract

Background: Spermatogonial 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. Methods: SSCs 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.Results: Flow 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. Conclusions:The 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.

Characteristics of piRNAs and their comparative profiling in testes of sheep with different fertility

“…Previous studies showed that these pathways were involved in the regulation of testicular growth and development ( Chambard et al, 2007 ), specifically playing important roles in proliferation, mitosis, and meiosis of germ cells ( Godet et al, 2008 ; Nicol and Guiguen, 2011 ; Jiang et al, 2013 ; Kerr et al, 2014 ) and proliferation and lactate production of Sertoli cells ( Riera et al, 2003 ; Lucas et al, 2012; Galardo et al, 2013 ; Yao et al, 2015 ; Ni et al, 2019 ) during both early and late spermatogenesis. When the above pathways were disturbed, the proliferation ability of SSCs ( Lee et al, 2007 ; Yao et al, 2020 ) and Sertoli cell ( Niu et al, 2015 ; Wang et al, 2015 ; Wang et al, 2017 ) would be inhibited. In addition, a study on asthenozoospermia population indicated that differentially expressed miRNAs in seminal plasma samples between patients with asthenospermia and healthy men were significantly associated with PI3K-Akt signaling pathway, MAPK signaling pathway and HIF-1 signaling pathway ( Liang et al, 2022 ), which were consistent with our findings.…”

“…When the above pathways were disturbed, the proliferation ability of SSCs (Lee et al, 2007;Yao et al, 2020) and Sertoli cell (Niu et al, 2015;Wang et al, 2015;Wang et al, 2017) would be inhibited. In addition, a study on asthenozoospermia population indicated that differentially expressed miRNAs in seminal plasma samples between patients with asthenospermia and healthy men were significantly associated with PI3K-Akt signaling pathway, MAPK signaling pathway and HIF-1 signaling pathway (Liang et al, 2022), which were consistent with our findings.…”

Genome-wide identification and expression profiling analysis of Wnt family genes affecting adipocyte differentiation in cattle

“…Then, β-catenin binds to the transcription factor T-cell factor/lymphoid enhancing factor (LEF1/TCF) family. This activates the transcription of target genes that regulate embryo development, tissue regeneration and cell proliferation and differentiation 2 , 3 .…”