Cytosine methylation of sperm DNA in horse semen after cryopreservation
Cytosine methylation of sperm DNA in horse semen after cryopreservation
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DOI：
10.1016/j.theriogenology.2016.04.077
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发表时间：
2016-09-15
期刊：
影响因子：
2.8
通讯作者：
Scarlet, Dragos
中科院分区：
文献类型：
--
作者：
Aurich, Christine;Schreiner, Bettina;Scarlet, Dragos
精液加工可能导致精子的表观遗传变化。因此，我们研究了由种马精液冷冻保存引起的精子DNA胞嘧啶甲基化的变化。用ELISA法分析5-甲基胞嘧啶相对量与精子DNA基因组胞嘧啶含量的关系。实验1：对6匹公马的生精液进行电击冷冻。解冻后精液活力和膜完整性完全缺失，而原始精液（0.4 +/- 0.2%）和休克冷冻精液（0.3 +/- 0.1%）的DNA甲基化相似（不显著）。试验2每匹马3次射精（n = 6）。在加入冷冻扩展剂前和冻融后分别用根特(G)或BotuCrio （BC）扩展剂评估精液质量和DNA甲基化。精子活力、形态和膜完整性显著降低，但不受扩展剂的影响（例如，总活力：G 69.5 +/- 2.0, BC 68.4 +/- 2.2%；与离心相比P < 0.001）。冷冻保存显著（P < 0.01）提高了DNA甲基化水平（冷冻前为0.6 +/- 0.1%，解冻后为6.4 +/- 3.7，BC为4.4 +/- 1.5%,P < 0.01），但不同冷冻延长剂间差异不显著（P < 0.01）。DNA甲基化水平与精液活力、形态或膜完整性无关。结果表明，低温保存的精液处理提高了种马精液DNA甲基化水平。我们的结论是，评估精子DNA甲基化允许评估一个额外的参数表征精液质量。用冻融精液授精后的母马生育率较低，至少部分原因可能是冷冻保存过程引起的精子DNA胞嘧啶甲基化。(C) 2016 Elsevier Inc.版权所有。
Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 +/- 0.2%) and shock-frozen (0.3 +/- 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 +/- 2.0, BC 68.4 +/- 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 +/- 0.1%, postthaw G 6.4 +/- 3.7, BC 4.4 +/- 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm DNA induced by the cryopreservation procedure. (C) 2016 Elsevier Inc. All rights reserved.