Identification of Protein Expression and Phosphorylation Associated with Spermatogenic Arrest in Cattle-yak Testes

The hybrid male offspring of cattle and yak constitute a classic model for investigating the genetic basis of hybrid incompatibility in domestic animals. Cattle-yak exhibit male-specific sterility due to azoospermia caused by meiotic arrest at the pachytene stage. Transcriptomic analysis of cattle-yak and their parental species has been extensively conducted to screen candidate genes associated with hybrid sterility. Phosphorylation is one of the main post-translational modifications that play important roles in directing germ cell differentiation; yet, the phosphoproteomic landscape associated with spermatogenic arrest in the cattle-yak remains unclear. In this study, we performed comparative proteomic and phosphoproteomic analyses of testicular tissues from yak, cattle-yak, and backcross offspring (BC1), which are progeny of female cattle-yak and demonstrate partial recovery of spermatogenesis. The results revealed that 779 and 536 proteins were differentially expressed (DEPs) and phosphorylated (DPPs) in cattle-yak testes, whereas 780 DEPs and 1025 DPPs were identified in BC1 testes. The DEPs in cattle-yak were enriched in mitochondrial double-strand break repair by homologous recombination, positive regulation of the DNA damage checkpoint, G2/M phase transition of the cell cycle, and stem cell fate decisions. In contrast, DEPs in BC1 offspring were enriched in DNA replication, regulation of heterochromatin assembly, double-strand break repair, and pyrimidine dimer repair by nucleotide-excision repair. Notably, DPPs were also enriched in key molecular events in meiosis, including positive regulation of the response to DNA damage stimulus, regulation of heterochromatin organization, and double-strand break repair. Integrated analysis revealed 26 proteins that were both differentially expressed and phosphorylated in cattle-yak testes, 8 of which were restored in the BC1 testes, including MRE11. Collectively, these findings provide crucial insights into the molecular mechanisms underlying sterility and fertility restoration in hybrid animals.