N6-methyladenosine RNA demethylase ALKBH5 is testis-specifically downregulated in hybrid male sterile dzo and is a target gene of bta-miR-200a.

N6-methyladenosine (mA) is the most common RNA methylation modification in mammals, which is controlled in the male germline to ensure coordinated gene expression in the entire process of spermatogenesis. Dzo is the male offspring of a cross between the domestic cattle (Bos taurus) and yak (Bos grunniens), and is sterile. This study aimed to investigate whether mA-associated genes are linked with dzo male sterility. The mRNA expression pattern of mA-associated genes and spermatogenesis-related genes modified by mA was characterized in cattle, yak, and dzo. Compared with fertile cattle and yak, mA erasing (ALKBH5 and FTO), writing (METTL14, WTAP, and ZC3H13), and reading (YTHDC2, YTHDF1, and YTHDF2) were testis-specifically downregulated in infertile dzo. The expression of mA target genes in spermatogonial self-renewal and proliferation (BCL6B, FOXO1, TAF4B, and FGFR1) and differentiation genes (DNMT3B and SOHLH2) were dereguleted in dzo testis. Immunofluorescent staining showed that intense ALKBH5 immunoreactivity was present in spermatogonia, primary spermatocytes, and round spermatids of cattle and yak testis. However, the number of ALKBH5 immunoreactive-positive cells were significantly reduced in dzo testis, especially in primary spermatocytes and round spermatids. Whole genome bisulfite-seq data showed that the promoter regions of FTO and YTHDC2 genes were hypermethylated in dzo testis. Moreover, bta-miR-200a was significantly downregulated in dzo testis, and it targeted the mA-associated genes such as ALKBH5, FTO, WTAP, and YTHDF2. In conclusion, mRNA of ALKBH5 was testis-specifically downregulated in dzo, which may be because fewer specific spermatogenic cells express this gene. The role of mA-associated genes in dzo male sterility and the interaction of DNA methylation and miRNA with mA-associated protein expression need to be further explored.