Single-cell RNA sequencing and UPHLC-MS/MS targeted metabolomics offer new insights into the etiological basis for male cattle-yak sterility

The variety of species can be efficiently increased by interspecific hybridization. However, because the males in the hybrid progeny are usually sterile, this heterosis cannot be employed when other cattle and yaks are hybridized. While some system-level studies have sought to explore the etiological basis for male cattle-yak sterility, no systematic cellular analyses of this phenomenon have yet been performed. Here, single-cell RNA sequencing and UPHLC-MS/MS targeted metabolomics methods were used to study the differences in testicular tissue between 4-year-old male yak and 4-year-old male cattle-yak, providing new and comprehensive insights into the causes of male cattle-yak sterility. Cattle-yak testes samples detected 6 somatic cell types and one mixed germ cell type. Comparisons of these cell types revealed the more significant differences in Sertoli cells (SCs) and [Leydig cells and myoid cells (LCs_MCs)] between yak and cattle-yak samples compared to other somatic cell clusters. Even though the LCs and MCs from yaks and cattle-yaks were derived from the differentiation of the same progenitor cells, a high degree of overlap between LCs and MCs was observed in yak samples. Still, only a small overlap between LCs and MCs was observed in cattle-yak samples. Functional enrichment analyses revealed that genes down-regulated in cattle-yak SCs were primarily enriched in biological activity, whereas up-regulated genes in these cells were enriched for apoptotic activity. Furthermore, the genes of up-regulated in LCs_MCs of cattle-yak were significantly enriched in enzyme inhibitor and molecular function inhibitor activity. On the other hand, the genes of down-regulated in these cells were enriched for signal receptor binding, molecular function regulation, positive regulation of biological processes, and regulation of cell communication activity. The most significant annotated differences between yak and cattle-yak LCs_MCs were associated with cell-to-cell communication. While yak LCs_MCs regulated spermatogenic cells at spermatogonia, spermatocyte, and spermatid levels, no such relationships were found between cattle-yak LCs_MCs and germ cells. This may suggest that the somatic niche in male cattle-yak testes is a microenvironment that is ultimately not favorable for spermatogenesis.