Fig 2: The localization and expression of TSSK1B in cattle–yak and yak testes. Testes from different stages (fetal, juvenile, and adult) of yak and cattle–yak development were embedded in paraffin, and sections of 4 μm thickness were prepared and stained with anti-TSSK1B antibody (brown) and counterstained with hematoxylin to visualize DNA (blue). The following pictures offer an enlarged view of the red box in the above figures. SSC, spermatogonial stem cells; SC, Sertoli cells; SP, spermatogonium; PS, primary spermatocyte; SS, secondary spermatocyte; IC, Leydig cells; RS, round spermatid; ES, elongating spermatid.

Fig 3: The phylogenetic tree of the TSSK1B gene established from the abovementioned alignment. The asterisk “★” represents the TSSK1B sequence obtained from yak testis.

Fig 4: The physicochemical properties of predicted protein coding by TSSK1B gene. (A) The predicted secondary structure of protein encoded by TSSK1B gene. Blue line, alpha helix; green line, beta-turn; red line, extended strand; orange line, random coil. (B) The predicted tertiary structure of protein. (C) The predicted hydrophobicity profile of protein encoded by TSSK1B gene. Hydrophobic: The mean hydrophobicity of each position is more than 0. Hydrophilic: The mean hydrophobicity of each position is less than 0. (D) The transmembrane structure of protein coding by TSSK1B gene. The square frame represents the position transmembrane site. (E) The phosphorylation sites analysis of TSSK1B. The prediction scores above 0.5 indicate positive predictions.

Fig 5: The methylation status of TSSK1B promoter region. (A,B) Ten BSP-amplified products of bisulfite-converted DNA sequence selected from TSSK1B promoter region, including the CpG island, in adult yak and cattle–yak testis, respectively. (C,D) The methylation status of CpG sites from adult yak and cattle–yak testes drew out with the sequencing results. The black circles represent methylated CpGs, and white circles represent unmethylated CpGs. (E) The methylation percentages of CpG sites in adult yak and cattle–yak testis calculated from (C,D). ** represents p < 0.01.