The g.-165 T>C Rather than Methylation Is Associated with Semen Motility in Chinese Holstein Bulls by Regulating the Transcriptional Activity of the HIBADH Gene

• Published in: PloS one Vol. 10; no. 7; p. e0127670
• Main Authors: Zhang, Shuai, Zhang, Yan, Yang, Chunhong, Ju, Zhihua, Wang, Xiuge, Jiang, Qiang, Sun, Yan, Huang, Jinming, Zhong, Jifeng, Wang, Changfa
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.07.2015; Public Library of Science (PLoS)
• Subjects: 3-Hydroxyisobutyrate dehydrogenase; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Animals; Base Sequence; Biomarkers - metabolism; Bisulfite; Cattle; Cell Line, Tumor; Correlation analysis; Deoxyribonucleic acid; DNA; DNA Methylation; Enzymes; Epididymis; Epididymis - cytology; Epididymis - enzymology; Epigenetics; Epithelium; Gene expression; Gene Expression Regulation; Gene regulation; Genes, Reporter; Genomes; Genotype; Genotypes; Immunofluorescence; Immunohistochemistry; Infertility; Leydig Cells - cytology; Leydig Cells - enzymology; Luciferases - genetics; Luciferases - metabolism; Male; Mice; Molecular modelling; Molecular Sequence Data; Motility; Neck; Plasmids - chemistry; Plasmids - genetics; Polymerase chain reaction; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Proteins; Quality; Reproductive technologies; Reverse transcription; Science; Semen; Semen Analysis; Single-nucleotide polymorphism; Sperm; Sperm Motility - genetics; Spermatids; Spermatids - enzymology; Spermatids - ultrastructure; Spermatogenesis; Studies; Testis - cytology; Testis - enzymology; Transcription, Genetic; Transfection
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0127670

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) is regarded as a human sperm-motility marker. However, the molecular mechanisms involved in the regulation of expression of the HIBADH gene in bulls remain largely unknown. HIBADH was detected in the testis, epididymis, and sperm via reverse transcription polymerase chain reaction and Western blot analysis. It is also expressed in the seminiferous epithelium, spermatids, and the entire epididymis, as detected by immunohistochemistry. Furthermore, HIBADH was expressed in the neck-piece and mid-piece of bull spermatids, as shown in the immunofluorescence assay. Using serially truncated bovine HIBADH promoters and luciferase constructs, we discovered an 878 bp (-703 bp to +175 bp) fragment that constitutes the core promoter region. One SNP g.-165 T>C of HIBADH was identified and genotyped in 307 Chinese Holstein bulls. Correlation analysis revealed that bulls with the TT genotype had higher initial sperm motility than those with the CC genotype (P < 0.05). Furthermore, the T- or C-containing loci (designated as pGL3-T and pGL3-C) were transiently transfected into MLTC-1 to test the effect of SNP on HIBADH expression. The luciferase reporter assay showed that the pGL3-T genotype exhibited 58% higher transcriptional activity than the pGL3-C genotype (P < 0.05). The bisulfite sequencing analysis revealed that the methylation pattern of the core promoter presented hypomethylation in the ejaculated semen in high-motility and low-motility bulls. The results demonstrated for the first time that the g.-165 T>C rather than methylation in the 5'-flanking region could affect the bovine sperm motility through the regulation of HIBADH gene transcriptional activity.