Takeda Kumiko, Kobayashi Eiji, Akagi Satoshi, Nishino Kagetomo, Kaneda Masahiro, Watanabe Shinya
Animal Breeding and Reproduction Research Division, Institute of Livestock and Grassland Science, NARO, Ibaraki 305-0901, Japan.
Beef Cattle Institute, Ibaraki Prefectural Livestock Research Center, Ibaraki 319-2224, Japan.
J Reprod Dev. 2017 Jun 21;63(3):279-287. doi: 10.1262/jrd.2016-160. Epub 2017 Mar 19.
The methylation status of sperm DNA differs between individual bulls. However, the relationship between methylation status and bull sperm parameters is not well elucidated. The present study investigated genome-wide methylation profiles at 450,000 CpG sites in bull spermatozoa by using a human DNA methylation microarray. Semen samples from three adult Japanese Black bulls with different in vitro fertilization (IVF) results and from a young Holstein bull through sexual maturation (at ages 10, 10.5, 15, and 25 months) were used for the analysis. The heatmap displaying the results of microarray analysis shows inter- and intra-individual differences in methylation profiles. After setting a cut-off of 0.2 for differences between ages (10, 10.5 vs. 15, 25 months) or between IVF results (developed to the blastocyst-stage, > 20% vs. < 10%), different methylation levels were detected at approximately 100 CpGs. We confirmed the different DNA methylation levels of CpG sites by using combined bisulfite restriction analysis (COBRA); five of the CpG sites reflected methylation levels similar to those detected by the microarray. One of the CpG sites was thought to reflect an age-related increase in methylation levels, which was confirmed by COBRA and bisulfite sequencing. However, the relationship between methylation status and IVF results could not be shown here. In conclusion, methylation profiles of individual and age-related alterations in bull spermatozoa can be revealed using a human microarray, and methylation changes in some CpG sites can be easily visualized using COBRA. Combined analysis of DNA methylation levels and sperm parameters could be considered an effective approach for assessing bull fertility in the future.
不同公牛精子DNA的甲基化状态存在差异。然而，甲基化状态与公牛精子参数之间的关系尚未得到充分阐明。本研究使用人类DNA甲基化微阵列，对公牛精子中45万个CpG位点的全基因组甲基化谱进行了研究。分析使用了来自三头体外受精（IVF）结果不同的成年日本黑牛以及一头通过性成熟（10、10.5、15和25月龄）的年轻荷斯坦公牛的精液样本。展示微阵列分析结果的热图显示了甲基化谱在个体间和个体内的差异。在设定年龄（10、10.5与15、25月龄）或IVF结果（发育至囊胚阶段，>20%与<10%）之间差异的截止值为0.2后，在大约100个CpG位点检测到了不同的甲基化水平。我们通过使用联合亚硫酸氢盐限制分析（COBRA）证实了CpG位点不同的DNA甲基化水平；其中五个CpG位点反映的甲基化水平与微阵列检测到的相似。其中一个CpG位点被认为反映了与年龄相关的甲基化水平增加，这通过COBRA和亚硫酸氢盐测序得到了证实。然而，这里未能显示甲基化状态与IVF结果之间的关系。总之，使用人类微阵列可以揭示公牛精子中个体及与年龄相关的甲基化谱变化，并且使用COBRA可以轻松观察到一些CpG位点的甲基化变化。DNA甲基化水平与精子参数的联合分析可能被认为是未来评估公牛生育能力的一种有效方法。
