Molecular characterization of the Bvh gene
The present study identified and characterized the bovine vasa homolog (Bvh) gene from cattle, yaks and their interspecific hybrid cattle-yaks. Sequence analysis indicated that the Bvh protein is a true DEAD-box family member and vasa family member. Like other members of the DEAD-box family, Bvh also contains two recombinase A (RecA)-like helicase domains, Domain 1 (DEADc domain) and Domain 2 (HELICc domain) [2, 23]. Within the helicase domains, there are at least eleven characteristic sequence motifs at conserved positions of Bvh, with seven conserved motifs (Q, I, Ia, Ib, GG, II and III) in Domain 1 and four motifs (IV, Iva, V and VI) in Domain 2, which is consistent with other mammals [2, 6, 19]. Previous investigations found that these conserved motifs are all involved with the function of Vasa, among which motifs Q, I and II are related to ATP binding, motif III related to hydrolysis, motif Ia, Ib, IV, and V related to RNA binding with RNA, and motif VI has a role in ATP activity and helicase activity [4, 23]. Thus, the amino acid sequence, constitution, arrangement and location of functional domains and motifs of Bvh are highly similar to the Vasa proteins from other mammals, which suggests that the Bvh protein is a member of DEAD-box protein family with ATP-dependent RNA helicase activity, and plays an important role in bovine spermatogenesis .
Alternative splicing of the Bvh gene
Alternative splicing (AS) is a major mechanism for the enhancement of transcriptome and proteome diversity, and plays important roles in development, physiology and in the pathology of various diseases, particularly in mammals . Previous studies showed that at least 74% of human multi-exon genes are alternatively spliced . Alternative splicing is a central tool of evolution that significantly increases the size of the transcriptome and generates functional specification. In the post-genomics era, AS has attracted the attention of researchers [34, 35]. In this study, two splice variants were identified within the coding regions of Bvh: Bvh-V4 and Bvh-V45. The alternative splice sites in Bvh are all located in the first five exons of the N-terminus (Bvh-V4 lacks exon 4, and Bvh-V45 lacks exon 4 and exon 5) and lead to amino acid deletions of the Bvh protein sequence.
Previous data showed that at least one expressed splice variant lacking an exon within the N-terminal region is present in other species, such as tammar and zebrafish [2, 28]. In tammar and zebrafish, the shorter-splice variants all lack exon 4. In addition, screening of the GenBank database using BLAST showed that three splice variants exist in the human Vasa gene, compared with the full-length human Vasa cDNA (GenBank ID: NM_024415.2). Splice variant 1 (GenBank ID: NM_001166533.1) lacks 60 bp from exon 7 and exon 8, splice variant 2 (GenBank ID: NM_001142549.1) lacks 102 bp from exon 7 and exon 9, and the shortest, splice variant 3 (GenBank ID: NM_001166534.1), lacks 447 bp from exons 2–6 and exon9. The alternative splicing patterns of Vasa in chimpanzee and marmoset were exactly consistent with the human gene. The mouse Mvh transcript variant (GenBank ID: NM_010029.2) lacks 78 bp from exon 4. The lack of sequence conservation suggests that if the N-terminal region plays a specific role in Vasa regulation, it appears to be species specific . The alternative splicing of Bvh occurred in the region encoding the N-terminal part of the protein, which does not contain functional domains and motifs; therefore, we speculated that protein isoforms Bvh-V4 and Bvh-V45 have similar functionality to Bvh.
Expression of the Bvh gene
The Vasa gene is particularly expressed in mammalian germplasm cells, and is closely related to spermatogenesis and meiosis [19, 20, 36]. Previous studies found that many RNA metabolism-related processes, such as transcription, ribosome biogenesis, RNA splicing, editing, transferring and translation were regulated by Vasa[37, 38]. Recently, studies observed that Vasa was involved in small RNA pathway, especially those closely related to mammalian spermatogenesis, such as the Piwi-interacting RNA (piRNA) [39, 40]. In this study, we found that Bvh and two splice variants, Bvh-V4 and Bvh-V45, are specifically expressed in the testes and ovary of adult cattle, which is consistent with the expression profile of Vasa in other mammals [19, 41, 42]. The results indicated that Bvh, Bvh-V4 and Bvh-V45 might, as in other mammals, make a significant contribution to the process of meiosis and Bvh might represent an important candidate gene that could influence bovine spermatogenesis. By real-time PCR, we found that the mRNA expression levels of Bvh in the testis of cattle and yaks with normal meiosis and spermatogenesis were significantly higher than that of cattle-yak hybrids with meiotic arrest (MA) and male sterility. The phenotype of MA and male sterility in cattle-yak hybrids  is consistent with the phenotype of Mvh gene knockout mice , suggesting that the mRNA levels of Bvh in the testicular tissue may be associated with the male sterility of cattle-yak hybrids. Ando et al.  found that transcription levels of Vasa in testicular tissue of successful testicular sperm extraction (TESE) patients with nonobstructive azoospermia (NOA) were higher than that of unsuccessful TESE groups, and suggested that measuring Vasa mRNA in testis could be a useful adjunct to conventional parameters for predicting sperm retrieval by micro-TESE in patients with NOA. The Vasa mRNA and protein levels were significantly decreased in patients with oligozoospermia: their mRNA level was only 1/5 of the normozoospermic men . Thus, the low expression of Vasa is related to the pathogenesis of some subtypes of male infertility, and Vasa could be used as a molecular marker for the diagnosis of male infertility .
In cattle testes, the relative ratio for Bvh-FL: Bvh-V4: Bvh-V45 was 2.2:1.6:1, and the differences in their expression levels were significant (P < 0.01 or P < 0.05). Bvh-FL and Bvh-V4 were the most abundantly expressed isoforms in the testes of cattle with complete spermatogenesis. In the testes of cattle-yak hybrids with MA of spermatogenesis, transcript levels of the two splice variants were significantly decreased (P < 0.01). Collectively, these data suggest a major physiological role for Bvh-V4 in bovine spermatogenesis between two splice variants.
Promoter methylation status of Bvh in testes
During transcription, the regulation of TF binding sites and TF interaction can be achieved by epigenetic modifications of the DNA, including DNA methylation, one of the main genome epigenetic modifications [45, 46]. To further study the mechanism of epigenetic regulation of Bvh expression in bovine testicular tissue, BSP was used to detect the methylation status of the Bvh promoter region in cattle, yaks and their interspecific hybrid cattle-yaks. The methylation level of the Bvh promoter region in the testicular tissue of cattle-yak hybrids (86.5%) was significantly higher than that of cattle (54.0%) and yaks (67.0%). These results indicated that the promoter region methylation of Bvh in testes is involved in transcriptional regulation, which was consistent with previous findings. The Vasa genes in humans and mice are regulated by the methylation state of tissue-specific differentially methylated regions (TDMRs). The methylation status of the CpG islands region in the promoter is related to the specific expression of Vasa and spermatogenesis, in which the Vasa promoter is hypomethylated in the testes but methylated in all other tissues that do not express Vasa. A clinical study showed that spermatogenesis defects, such as idiopathic azoospermia or severe oligospermia, were also associated with a hypermethylated Vasa promoter in some individuals . Lin et al.  reported that some germ cell-specific genes (e.g. Nanog, Pou5f1, and Zp1) in the marmoset and mouse testis showed different expression patterns and methylation patterns, but the expression patterns and methylation patterns of Vasa and some imprinted genes are conserved.
In addition, of the 20 CpG sites in the Bvh promoter, only CpG3, CpG4, CpG11 and CpG16 showed different methylation levels between cattle-yaks and their male parent (cattle). DNA methylation regulates gene transcription mainly through two mechanisms [50, 51]. Firstly, gene transcription may be inhibited by blocking the binding between a TF and its binding sites in the promoter region. Secondly, the recognition and specific binding to DNA methylation sites by methyl-CpG-binding proteins (MBPs) influences TF binding, and thus inhibits transcription initiation. To explore the probably mechanism by which differentially methylated (DM) CpG sites affect the expression level of Bvh, the putative transcription factor binding patterns associated with the differentially methylated (DM) CpG sites were determined using the web tools TFSEARCH (with a threshold score of 85.0), MatInspector and Proscan. The results showed that CpG site CpG3 is located in the binding site for transcription factor GATA-1, while CpG16 is located in the binding site for transcription factors Sp1 and T-Ag. The transcription factor Sp1 is a member of the Sp family, whose zinc finger domain near the C-terminus can specifically recognize a GC Box on the DNA sequence. Sp TFs regulate transcription in multiple tissues . Methylation of Sp1 binding sites in a promoter region tends to inhibit the transcription of the gene [53, 54]. Therefore, we speculate that the hypermethylation of the Sp1 binding site (CpG16) in the Bvh promoter in the testicular tissues of cattle-yak hybrids is probably responsible for the lower expression of Bvh. Hypermethylation of Sp1 binding sites probably prevents Sp1 from binding to its binding sites by recruiting MBPs, thus inhibiting Bvh expression [53, 55].