Analysis Of Igf2, H19, Snrpn And Dazl Expression Level And Their DNA Methylation Modification Between Cattle Yaks And Their Parents

The yak, known as the "almighty livestock", is indispensable in the region of the Qinghai-Tibet Plateau and nearby areas, and is well adapted to high altitude environments. However, it is primitive and grows slowly, resulting in low production performance. To improve its productivity, hybridization between cattle and yaks has been carried out since the1950s. Cattle yaks, the F1hybrid between cattle and yaks, exhibit significant hybrid vigor. However, the males are sterile, which greatly restricts the utilization of this hybrid vigor, and is a "stumbling block" in the varietal improvement of the yak. To investigate the mechanism of male sterility of cattle yaks, in present study, the differentially methylated region （DMR） sequences of imprinting genes and spermato genesis-related gene in yak were obtained using homogenetic amplification, the expression of genes in cattle yaks and their parents was investigated by Real-time PCR, and the methylation patterns were examined using bisulfite sequencing. This would provide evidence for the epigenetic mechanism of sterility of cattle yaks.1. Molecular Cloning and Sequence Analysis of H19/IGF2Imprinting Control Region （ICR） in Yaks （Bos grunniens）Primers were designed from the sequence of the cattle H19DNA （NW₀01494548）, sequencing and assembly led to the establishment of the complete nucleotide sequence （6.5kb） of the H19ICR, promoter, and partial exon1. The sequence was submitted to GenBank under accession number EU502716. Sequence analysis revealed that the homology between yak and cattle, sheep, pig, human and mouse, was97%,83.13%,48%,47%,42%, respectively; six CpG islands were identified, and their lengths were1182,1186,707,277,281, and1311bp, respectively;6CTCF-binding sites were identified in HI9ICR, and they were all present in the CpG islands and located at approximately5.5kb （site1）,5.1kb （site2）,4.1kb （site3）,3.7kb （site4）,2.7kb （site5）, and1.3kb （site6） upstream of the transcription initiation site, respectively; computer assisted analysis revealed that the 5’-flanking region consisted of a putative core promoter region,2GC boxes, and several binding sites for Sp1, Sp2, AP2, NF-κB, STAT5, P53, WT1, GFI1, MTBP, CPBP, ZF9, E2F, and Myc-Max.2. Analysis of H19mRNA expression and its methylation status in testes between cattle yaks and their parentsBy Real-time PCR the expression of testes H19mRNA in cattle yaks and their parents was investigated. The result showed that the H19expression in cattle yaks was the lowest; and there was statistically significant difference between cattle yaks and their parents （P<0.01）, but no significant difference between cattle and yak （P>0.05）.The methylation patterns of H19ICR which contained CTCF-binding site III was examined in testis using bisulfite sequencing. The result showed that the methylation level of H19ICR in cattle yaks （48.82%） was significantly lower than that in cattle （70%）（P<0.01） and yaks （59.10%）（P<0.05）, and there was significant difference between cattle and yaks （P<0.05）; the methylation level of CTCF-binding site III in cattle yaks （48.33%） was significantly lower than that in cattle （75%） and yaks （68%）（P<0.01）. This indicated that the methylation status of H19ICR and CTCF-binding sites played an important role in transcriptional regulation.3. Analysis of IGF2mRNA expression and its methylation status in testes between cattle yaks and their parentsBased on the cattle IGF2gene （DQ298740）, primers were designed, and the partial sequence of IGF2exon10in yaks was obtained （GenBank No. FJ152104）. the IGF2mRNA expression of testes in cattle yaks and their parents was investigated by Real-time PCR and the methylation patterns of IGF2DMR in exon10was examined in testes using bisulfite sequencing. The results showed that IGF2expression in cattle yaks’testes was lower than their parents （significant, P<0.01）. The IGF2DMR was highly methylated （cattle, yak and cattle yak was90%,90.7%and92.0%respectively） in testes, with the highest level in cattle yaks （not significant, P>0.05）. Our study showed that IGF2played an important role in bovine spermatogenesis and might be involved in cattle yak male sterility. The methylation level of the IGF2DMR was irrelevant to the lower expression of IGF2in cattle yaks; other factors perhaps play roles in its expression.4. Analysis of SNRPN mRNA expression and its methylation status in testes between cattle yaks and their parentsPrimers were designed from the sequence of the cattle SNRPN DNA （NW₉35049）, the5’-flanking sequence （1137bp） in yaks was obtained, the homology between yaks and cattle was98.2%, and computer assisted analysis suggested that the region consisted of the putative methylation-sensitive binding sites, including YY1and SP1sites. SNRPN mRNA expression in cattle yaks and their parents and the methylation patterns of the SNRPN DMR were examined in testes. The results showed that there was no significant difference between cattle yaks and their parents in SNRPN expression, but the expression level in cattle yaks was lower than that in their parents; the methylation level of SNRPN gene in cattle yaks （38.53%） was very significantly greater than that in cattle （21.08%） and yaks （20.81%）（P<0.01）. This indicated that the methylation status of SNRPN5’-flanking in cattle yaks couldn’t lead to apparent different expression alone, other factors maybe play an important role in the SNRPN expression,.5. Relationship between Methylation Status of DAZL5’CpG Island and Male Sterility of Cattle YakIn our prophase research, we have found that DAZL gene was a candidate gene for male infertility in cattle yaks. To study the mechanism of DAZL gene expression regulation in cattle yaks, the yaks DAZL5’region sequence was firstly cloned and DAZL gene methylation patterns in cattle, yaks and cattle yaks were examined using bisulfite sequencing. The results showed that within the5’region of DAZL there was a CpG island which contained the promoter region, exon1and intron1; the methylation level of DAZL gene in cattle yaks （85.6%） was very significantly greater than that in cattle （69.8%） and yaks （71.4%）（P<0.01）. These results demonstrated that the hypermethylation of DAZL gene was in accordance with the phenotype of DAZL deficient expression and male infertility in cattle yaks, and indicated that the methylation status of DAZL gene played an important role in DAZL transcriptional regulation and maybe have a severe effect on meiotic process of spermatogenesis and male sterility in cattle yaks.