Tubulin TUBB4B Is Involved in Spermatogonia Proliferation and Cell Cycle Processes

• Published in: Genes 2022-06, Vol.13 (6), p.1082
• Main Authors: Feng, Meiying, Wang, Kai, Fu, Shuying, Wei, Hengxi, Mu, Xiaokun, Li, Li, Zhang, Shouquan
• Format: Article
• Language: English
• Subjects: Cancer; CCAAT/enhancer-binding protein; Cell cycle; Cell death; Cell division; Cell proliferation; Chromosome 2; CRISPR; Cyclin-dependent kinase inhibitor p21; Cyclins; Cytology; Cytoskeleton; Flow cytometry; Genetic aspects; Genomes; Germ cells; Granulocyte colony-stimulating factor; Growth factors; Motility; Phase transitions; Physiological aspects; Pronucleus; Protein expression; Proteins; Regulatory proteins; Skp2 protein; Sperm; Spermatogenesis; Spermatogonia; Transcriptomes; Tubulin; Tubulins
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes13061082

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.