Comparative RNA-Seq analysis of differentially expressed genes in the sertoli cells of yak and cattle-yak

To study the problem of male sterility of cattle-yak and improve the yak crossbreeding, this study obtained the testicular Sertoli cells of yak and cattle-yak and compared the differences in transcriptome levels between the two bovine species. The testicular tissues of 3 healthy male cattle-yaks and 3 F₁ generation male yaks were collected at the age of 24 months. The Sertoli cells were isolated after enzymatic digestion, differential adhesion and starvation treatment. DATA-4 and SOX9 immunofluorescence staining were used to identify the cell type. Sertoli cells were subjected to transcriptome sequencing, GO analysis, KEGG analysis and differentially expressed gene were validated by RT-qPCR and Western blotting.

The study successfully isolated and purified Sertoli cells of yak and cattle-yak. The transcriptome sequencing data were compared, analyzed and annotated. Compared to yak Sertoli cells, 6592 differentially expressed genes were identified, with 3007 genes upregulated and 3585 genes downregulated in cattle-yak Sertoli cells. GO analysis suggested that the upregulated genes might be mainly involved in processes such as translation, peptide biosynthetic process, amide biosynthetic process, peptide metabolic process, ribosome, cytoplasmic part, structural constituent of ribosome, structural molecule activity, endomembrane system, protein kinase activity, and phosphotransferase activity. The downregulated genes appeared to be primarily involved in protein phosphorylation, phosphorylation, endomembrane system, protein kinase activity, and phosphotransferase activity. KEGG analysis compared differential genes across 316 pathways, with 8 pathways showing significant enrichment. The upregulated pathways were potentially enriched in cattle-yak Sertoli cells, including ribosome, thermogenesis, and oxidative phosphorylation, while the downregulated pathways seemed to be significantly enriched in adherens junction, mTOR signaling pathway, AMPK signaling pathway, FoxO signaling pathway, and focal adhesion. Compared with yak Sertoli cells, ISOC2, RPL27A and FISI were highly expressed in cattle-yak, as confirmed by RT-qPCR analysis. PDPN, SORBS2, TF, PLSCR1, TJP2, KIF2C, ITGA3, SMTNL2, DSP, ADGRG1, DDR1, GSK3A, RBBP6, ZC3H15 and Claudin 11 showed low expression levels in cattle-yak.

Compared with yak Sertoli cells, the expression of genes related to protein activation, cell function, and membranous organelle composition in cattle-yak Sertoli cells appeared to be abnormal. The potential defects in cattle-yak Sertoli cells may hinder the creation of a suitable environment for spermatogenesis, which could be one of the factors contributing to male cattle-yak sterility. Claudin-11 might be a potentially important gene for further research into cattle-yak male sterility.