Cheng Yu, Vechtova Pavlina, Fussy Zoltan, Sterba Jan, Linhartová Zuzana, Rodina Marek, Tučková Vladimíra, Gela David, Samarin Azin Mohagheghi, Lebeda Ievgen, Xin Miaomiao, Zhang Songpei, Rahi Deepali, Linhart Otomar
South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Zátiší 728/II, 389 25 Vodňany, Czech Republic.
Faculty of Science, Institute of Chemistry and Biochemistry, University of South Bohemia in Ceske Budejovice, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic.
Int J Mol Sci. 2021 May 31;22(11):5925. doi: 10.3390/ijms22115925.
The purpose of the current study was to analyze phenotypic and functional characteristics of common carp () spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the genome in the public databases.
本研究的目的是分析鲤鱼精子在体外老化过程中的表型和功能特征，并研究精子老化是否会影响全基因组DNA甲基化。从五条雄性个体采集精液，采精后在冰箱中于冰上体外保存长达96小时，每隔1、24和96小时用于使卵子受精。采用计算机辅助精子分析和S3e细胞分选仪来确定精子的表型特征（活力、速度、浓度和存活率）。此外，在不同老化时间检测精液的pH值和渗透压以及精子的受精能力、孵化率和所产胚胎的健康状况。使用全基因组亚硫酸氢盐测序来比较新鲜精子和老化精子中的全基因组和基因特异性DNA甲基化。结果表明，鲤鱼精子老化显著影响其性能，从而影响人工受精的成功率。与采精后1小时的新鲜组相比，采精后24小时的精子中胞嘧啶-磷酸-鸟嘌呤（CpG）位点的甲基化水平显著升高，然后在采精后96小时显著下降。公共数据库中鲤鱼基因组注释不完整，阻碍了对DNA甲基化基因特异性差异的更详细研究。
