nd consists of three members, DAZ, DAZL, and Boule. Boule is the recently 1 / 14 Promoter Methylation Regulates bBoule identified ancestral “grandfather” gene in the DAZ family; it is expressed in prophase and metaphase spermatocyte meiosis in the testis, and highly expressed in meiotic pachytene spermatocytes. Boule is found in vertebrates and invertebrates. DAZL is regarded as the “father” gene in the DAZ family and evolved from ancestral Boule. It is expressed in spermatogonia and spermatocytes of the testis and ovary. DAZL is only detected in vertebrates. DAZ maps to the Y chromosome, is obtained by gene transposition, duplication, and exon splicing from autosomal DAZL, and is highly expressed in meiotic prophase germ cells in the testis. DAZ is only found in Old World monkeys and humans. The proteins encoded by DAZ family genes are all RNA-binding proteins with typical RNA-recognition motifs and DAZ repeats; they play an important role in spermatocyte meiosis and are associated with male infertility. Boule, a recently identified member of the DAZ family, was first detected in Drosophila and human testes. In Drosophila, the testes of boule mutants produce no sperm and have germ cells that are arrested before meiosis, resulting in azoospermia and male infertility. A fly boule transgene or a human BOULE transgene can rescue the reproductive defects of boule mutant flies. Testicular BOULE expression is decreased in some patients with abnormal spermatogenesis, and spermatogenesis is arrested before the primary spermatocyte stage; no BOULE expression is detected in testes of patients with complete meiotic arrest. Lin et al. also found that BOULE mRNA levels are significantly decreased in azoospermic male testes, and are progressively decreased with increasing severity of testicular failure; patients with successful sperm retrieval have significantly higher BOULE levels than patients with failed sperm retrieval. Boule-/- mice are male sterile and azoospermic, similar to boule mutant flies PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19699128 and some men with DAZ deletions. Li et al. demonstrated that over-expression of Boule promotes the expression of meiosis-related genes such as Stra8 in goat male germline stem cells. Thus, these results suggest that the expression of Boule is associated with mammalian spermatocyte meiosis and male infertility, and that it may be the key regulatory factor of spermatocyte meiosis. The transcriptional regulation of DAZ family genes has been extensively studied. However, little is known about the regulation of Boule, and particularly its epigenetic regulation. Our previous study suggested that bovine Boule may MedChemExpress R-7128 function in bovine spermatogenesis, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19697363 and that low bBoule expression might lead to male sterility in cattle-yak hybrids. In the present study, we examined the epigenetic mechanisms of low bBoule expression in testes of cattle-yak hybrids. Materials and Methods Bioinformatic analysis The genomic DNA sequence of the bBoule gene was obtained by a BLAST search of the genome database of cattle based on the cDNA sequence of bBoule that was previously cloned by our group. The putative promoter region of bBoule was predicted using Proscan software. CpG islands were searched by the online CpG Island Searcher program. We searched the transcription factor binding sites of the bBoule core promoter using the web tool TFSEARCH v1.3 using a threshold score of 85.0. PCR and sequencing Genomic DNA was isolated from testes using the phenol-chloroform method. Three primers