Department of Animal Reproduction, INIA, Madrid, Spain.
Physiology of Reproduction Group, Department of Physiology, Universidad de Murcia, Campus Mare Nostrum, Murcia, Spain.
Clin Epigenetics. 2021 Feb 3;13(1):27. doi: 10.1186/s13148-021-01003-x.
Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming.
Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage.
Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.
先前在小鼠中的研究表明，在生殖细胞的 DNA 甲基化重编程过程中，一些反转录元件仍保持大量甲基化。然而，关于这一过程在猪中的信息却很少。本研究旨在检测猪生殖细胞在表观遗传重编程过程中的甲基化谱。
对母猪进行人工授精，然后在 28、32、36、39 和 42 天后收集其胎儿。在每个时间点，从中肾解剖生殖嵴，并使用抗 SSEA-1 抗体通过磁激活细胞分选分离生殖细胞，并回收生殖细胞进行全基因组亚硫酸氢盐测序。使用 SeqMonk 软件通过进行无偏分析来量化甲基化水平，并确定每个性别中的持久甲基化区域（PMRs），以提取那些显示 50%或更高甲基化的区域。从第 28 天到第 36 天，大多数基因组元件经历了剧烈的去甲基化，此时达到最低水平。到第 42 天，有证据表明基因组重新甲基化的开始。我们分别在雄性和雌性生殖细胞中鉴定出总共 1456 和 1122 个 PMR，并且大量转座元件（SINEs、LINEs 和 LTRs）被发现位于这些 PMR 内。位于这些 PMR 中的大多数内含子被发现是基因的第一内含子，表明它们在这些基因的表达中具有调节作用。有趣的是，大多数鉴定出的 PMR 在囊胚阶段被去甲基化。
我们的研究结果表明，猪生殖细胞中的甲基化重编程遵循在小鼠和人类中显示的一般动态，揭示了雄性和雌性生殖细胞之间行为不同的基因组元件。
