Tubb4b (tubulin β-4b chain) is essential for cell growth and development as a microtubule network protein. Previous studies have shown that TUBB4B affects mouse pronucleus migration, but the gene function has yet to be elucidated. To study TUBB4B-related functions in mouse reproductive development, we designed a single sgRNA in chromosome 2 and generated a knockout spermatogonia cell line of the β-tubulin isoform Tubb4b by the CRISPR/Cas9 system. Tubb4b-KO spermatogonia recognized abnormal lysosomal membranes and cell morphology defects. Compared to control mouse spermatogonia, the proliferation rate was significantly slower and cycling stagnated in the G1/0 population. Although spermatogonia lacking TUBB4B have abnormal divisions, they are not lethal. We detected the mRNA levels of the cell-regulating cyclins CyclinsD1, CyclinsE, Cdk2, Cdk4, P21, Skp2 and the cell growth factors C/EBP α, C/EBP β, and G-CSF in the spermatogonia of Tubb4b-KO and found that the expressions of CyclinsD1, Skp2 and cell growth factors were significantly reduced. Further analysis revealed that 675 genes were expressed differently after Tubb4b deletion and were enriched in negative regulation of cell population proliferation (GO:0008285), negative regulation of cell cycle G2/M phase transition (GO:1902750), and positive regulation of cell death (GO: 0010942). We also found that there is a common gene Cdkn1a (P21) in these three GO pathways related to cell proliferation and cell cycle, and both quantitative analysis and transcriptome sequencing results showed that the expression of this gene was up-regulated in Tubb4b knockout cells. This implies that Tubb4b may be involved in the division of spermatogonia with multiple cell cycle regulatory proteins. Overall, these data indicate that Tubb4b has a specific role in regulating spermatogonia proliferation and cell cycle.

Male germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells. Porcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis. We identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs). Collectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.