Zhang Shuai, Zhang Yan, Yang Chunhong, Zhang Wei, Ju Zhihua, Wang Xiuge, Jiang Qiang, Sun Yan, Huang Jinming, Zhong Jifeng, Wang Changfa
Dairy Cattle Research Center, Shandong Academy of Agricultural Science, Jinan, P.R. China College of Life Science, Shandong Normal University, Jinan, P.R. China.
Dairy Cattle Research Center, Shandong Academy of Agricultural Science, Jinan, P.R. China.
Biol Reprod. 2015 Jun;92(6):139. doi: 10.1095/biolreprod.114.126672. Epub 2015 Apr 22.
Transition nuclear proteins (TNPs), major proteins found in the chromatin of condensing spermatids, have been implicated in spermatogenesis and male fertility. In this study, DNA samples were collected from 404 Chinese Holstein bulls and sequenced to identify genetic variants in the 3'-untranslated region (UTR) of TNP1 and to investigate genetic variations in the TNP1 gene and their common haplotypes. This study was also conducted to determine whether these variations affect bovine semen quality traits and expression levels by PCR-restriction fragment length polymorphism, bioinformatics analyses, quantitative real-time PCR (qPCR), and fluorescence assay. Results showed that one new single-nucleotide polymorphism (SNP; g. 528 G>A, ss1388116558) and one reported SNP (g. 442 A>G, rs110469441) were found in the 3'-UTR of the TNP1 gene. Bioinformatics analysis results revealed that both loci were located in bta-miR-532-binding and bta-miR-204-binding regions, respectively. Association studies revealed that bulls with H1H1 (AGAG) and H1H3 (AGGG) haplotype combinations exhibited a lower deformity rate than those with other haplotype combinations (P < 0.05). The qPCR results showed that the relative mRNA expression of TNP1 in bulls with H1H1 haplotype combination was significantly higher than that in bulls with H4H4 haplotype combination (P < 0.05). MicroRNA qPCR results suggested that bta-miR-532 expression was downregulated by 5-fold in adult bull testicular tissues compared with that in fetal bull testicular tissues; by contrast, bta-miR-204 expression was downregulated by 1.6-fold. Luciferase assay results also indicated that TNP1 expression was directly targeted by bta-miR-532 and bta-miR-204 in murine Leydig tumor cell lines. These results provide the first indication of g. 442 A>G-mediated and g. 528 G>A-mediated translational suppression in which SNPs altered the binding of bta-miR-204 and bta-miR-532 to the 3'-UTR of TNP1; the mediated translational suppression could be involved in the regulation of TNP1 expression and may influence the morphological characteristics of Chinese Holstein bull sperm. We propose that SNPs on the TNP1 3'-UTR may help select semen quality trait in Chinese Holstein bulls in the dairy industry.
过渡核蛋白（TNPs）是浓缩精子细胞染色质中发现的主要蛋白质，与精子发生和雄性生育力有关。在本研究中，从404头中国荷斯坦公牛采集DNA样本并进行测序，以鉴定TNP1基因3'-非翻译区（UTR）中的遗传变异，并研究TNP1基因的遗传变异及其常见单倍型。本研究还通过聚合酶链反应-限制性片段长度多态性、生物信息学分析、定量实时聚合酶链反应（qPCR）和荧光测定，确定这些变异是否影响牛精液质量性状和表达水平。结果显示，在TNP1基因的3'-UTR中发现了一个新的单核苷酸多态性（SNP；g.528 G>A，ss1388116558）和一个已报道的SNP（g.442 A>G，rs110469441）。生物信息学分析结果表明，这两个位点分别位于bta-miR-532结合区和bta-miR-204结合区。关联研究显示，具有H1H1（AGAG）和H1H3（AGGG）单倍型组合的公牛畸形率低于其他单倍型组合的公牛（P<0.05）。qPCR结果显示，具有H1H1单倍型组合的公牛中TNP1的相对mRNA表达显著高于具有H4H4单倍型组合的公牛（P<0.05）。微小RNA qPCR结果表明，与胎牛睾丸组织相比，成年公牛睾丸组织中bta-miR-53表达式下调了5倍；相比之下，bta-miR-204表达下调了1.6倍。荧光素酶测定结果还表明，在小鼠睾丸间质细胞瘤细胞系中，TNP1表达直接受bta-miR-532和bta-miR-204靶向。这些结果首次表明g.442 A>G介导和g.528 G>A介导的翻译抑制，其中SNP改变了bta-miR-204和bta-miR-532与TNP1基因3'-UTR的结合；介导的翻译抑制可能参与TNP1表达的调控，并可能影响中国荷斯坦公牛精子的形态特征。我们提出，TNP1基因3'-UTR上的SNP可能有助于在奶牛业中选择中国荷斯坦公牛的精液质量性状。
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