Cloning of a new testis-enriched gene C4orf22 and its role in cell cycle and apoptosis in mouse spermatogenic cells

Spermatogenesis is a complicated and dynamic cellular differentiation process mainly regulated by genes, steroid hormones and environmental factors. Although a number of genes involved in spermatogenesis have been identified, there are still a lot of genes underlying spermatogenesis remained unexplained. Here, a novel gene C4orf22, also known as 1700007G11Rik or Cfap299 was identified from mouse testis. C4orf22 protein contains 233 amino acid residues and is highly conserved in metazoan species. C4orf22 mRNA was predominantly expressed in mouse testis and increased from 2-week-old testes to 8-week-old testes during the developing testes by RT-PCR and qRT-PCR. Immunohistochemical analysis indicated that C4orf22 protein was mainly distributed in the cytoplasm of spermatogonia and primary spermatocytes, which was further confirmed by C4orf22-tagged with GFP in the GC-1 and GC-2 cells. Over-expression of pEGFP-C3-C4orf22 significantly inhibited GC-1 cells apoptosis and promoted cell cycle progression with an increase in the cell number of S and G2 phase. Conversely, small interfering RNA (siRNA) silencing C4orf22 in GC-1 cells could cause an increase in the number of apoptosis cells and the cell cycle was arrested at G2/M phase. Western blot analysis and qRT-PCR results showed that C4orf22 over-expression significantly increased the expressions of anti-apoptotic bcl-2 and decreased the expression of caspase-3, caspase-8 and Bax. Our results suggest that C4orf22 may be involved in spermatogenesis, and for the first time, unravels its potential role in regulating cell apoptosis through bcl-2 regulatory pathway in GC-1 cells.