TCF3 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cells by Targeting PODXL

Abstract

Spermatogonial stem cells (SSCs) are the initial cells for the spermatogenesis. Although much progress has been made on uncovering a number of modulators for the SSC fate decisions in rodents, the genes mediating human SSCs remain largely unclear. Here we report, for the first time, that TCF3, a member of the basic helix-loop-helix family of transcriptional modulator proteins, can stimulate proliferation and suppress the apoptosis of human SSCs through targeting podocalyxin-like protein (PODXL). TCF3 was expressed primarily in GFRA1-positive spermatogonia, and EGF (epidermal growth factor) elevated TCF3 expression level. Notably, TCF3 enhanced the growth and DNA synthesis of human SSCs, whereas it repressed the apoptosis of human SSCs. RNA sequencing and chromatin immunoprecipitation (ChIP) assays revealed that TCF3 protein regulated the transcription of several genes, including WNT2B, TGFB3, CCN4, MEGF6, and PODXL, while PODXL silencing compromised the stem cell activity of SSCs. Moreover, the level of TCF3 protein was remarkably lower in patients with spermatogenesis failure when compared to individuals with obstructive azoospermia with normal spermatogenesis. Collectively, these results implicate that TCF3 modulates human SSC proliferation and apoptosis through PODXL. This study is of great significance since it would provide a novel molecular mechanism underlying the fate determinations of human SSCs and it could offer new targets for gene therapy of male infertility.

Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

“…To establish the human SSC line, the human SV40 large T antigen was overexpressed in human primary GPR125-positive undifferentiated spermatogonia [ 41 ]. Immortalized human SSCs retained many primary cell properties and were positive for several SSC markers, such as PLZF, GFRA1, UCHL1, and THY1 [ 34 ]. Cultures of the human SSC line were done in DMEM/F12 (Gibco, Grand Island, NY) with 10% FBS (Gibco) and 100 unit/mL streptomycin/penicillin (Invitrogen, CA, USA) at 34℃ in a 5% CO 2 atmosphere.…”

“…MiR-1908-3p promotes human SSC proliferation by targeting and degrading KLF2 mRNA [ 33 ], while CARF influences the self-renewal and proliferation of both human and mouse SSCs via the WNT signaling pathway [ 21 ]. In addition, we have previously demonstrated that the transcription factor TCF3 enhances SSC proliferation and inhibits apoptosis by promoting the transcription of genes such as PODXL [ 34 ]. However, the developmental regulatory mechanisms of human spermatogonial stem cells require additional research.…”

SPOC domain-containing protein 1 regulates the proliferation and apoptosis of human spermatogonial stem cells through adenylate kinase 4

“…Immortalized human SSCs maintained many properties of their primary cells and expressed many markers of primary SSCs including GFRA1, RET, and promyelocytic leukemia zinc finger (PLZF). They did not express testicular endosomal cell markers such as SRY-box transcription factor 9[ 15 ]. The immortalized human SSCs were grown at 34 °C with 50 mL/L CO 2 in an incubator, and the culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY, United States) supplemented with 100 mL/L fetal bovine serum (FBS; Gibco).…”

“…According to the operating manual, the BCA Kit was utilized to detect total protein concentration. Twenty micrograms of total protein were taken from each sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, as previously described[ 15 ]. Detailed antibody information is listed in .…”

“…Human SSCs have also been modulated by RNF144B through the Fc epsilon receptor II/neurogenic locus notch homolog protein 2/HES1 pathway[ 14 ]. In addition, we previously reported that transcription factor 3 is specifically localized in the nucleus of human SSCs, and promotes human SSC proliferation by regulating podocalyxin-like protein 1 expression[ 15 ]. However, the regulatory mechanisms of SSCs are poorly understood.…”