Evaluation of MYBL1 as the master regulator for pachytene spermatocyte genes dysregulated in interspecific hybrid dzo.

Misregulation of spermatogenesis transcription factors (TF) in hybrids can lead to misexpression, which is a mechanism for hybrid male sterility (HMS). We used dzo (male offspring of Bos taurus ♂ × Bos grunniens ♀) in bovines to investigate the relationship of the key TF with HMS via RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses. RNA sequencing showed that the widespread misexpression in dzo was associated with spermatogenesis-related genes and somatic or progenitor genes. The transition from leptotene or zygotene spermatocytes to pachytene spermatocytes may be the key stage for meiosis arrest in dzo. The analysis of TF-binding motif enrichment revealed that the male meiosis-specific master TF MYB proto-oncogene like 1 (MYBL1, known as A-MYB) motif was enriched on the promoters of downregulated pachytene spermatocyte genes in dzo. Assay for transposase-accessible chromatin with high-throughput sequencing revealed that TF-binding sites for MYBL1, nuclear transcription factor Y, and regulatory factor X were enriched in the low-chromatin accessibility region of dzo. The target genes of the MYBL1-binding motif were associated with meiosis-specific genes and significantly downregulated in dzo testis. The transcription factor MYBL1 may be the candidate master regulator for pachytene spermatocyte genes dysregulated in interspecific HMS dzo. This study reported that a few upstream TF regulation changes might exert a cascading effect downstream in a regulatory network as a mechanism for HMS.

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 (m6A) 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 m6A-associated genes are linked with dzo male sterility. The mRNA expression pattern of m6A-associated genes and spermatogenesis-related genes modified by m6A was characterized in cattle, yak, and dzo. Compared with fertile cattle and yak, m6A 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 m6A 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 m6A-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 m6A-associated genes in dzo male sterility and the interaction of DNA methylation and miRNA with m6A-associated protein expression need to be further explored.