Construction of Sows DNA Methylation Profiles and Pool of Early Pregnancy Factors

[Background] DNA methylation plays a crucial role in regulating cell proliferation, differentiation, gene expression, and embryonic development. However, the comprehensive changes in genome-wide DNA methylation during early pregnancy in sows remain unknown. This study aimed to investigate the DNA methylation profiles and to construct an early gene pool of sows during early gestation.[Results] In this investigation, we utilized blood samples from 20 healthy sows and applied Reduced Representation Bisulfite Sequencing (RBSS) technology to systematically examine alterations in DNA methylation in sow blood during early pregnancy. Screening and validating the Methylation differential genes by using bisulfite sequencing PCR (BSP) and Quantitative Real-time PCR (RT-qPCR). Our findings indicated a significant increase in methylation levels on day 28 of pregnancy (pre28) compared to non-pregnancy (ctrl) across various gene elements, including CGI, promoter, and genebody. A total of 64,908 differentially methylated sites (DMCs) and 12,119 differentially methylated regions (DMRs) were identified, predominantly exhibiting methylation upregulation. Randomly selected five differentially methylated genes—VBP1, UPRT (upregulated methylation), RPL10, RAB33A, and CD93 (downregulated methylation)—were validated. Between the pregnant (pre28) and non-pregnant (ctrl) groups the BSP results showed highly significant methylation differences (P<0.01) in VBP1, UPRT, RPL10, and RAB33A. DMC gene ontology (GO) analysis revealed significant enrichment in nuclear receptor activity, negative regulation of cysteine-type endopeptidase activity during apoptosis, heme binding, and hormonal activity. DMRs GO analysis highlighted significant enrichment in 1-acylglycerol-3-phosphate O-acyltransferase activity, regulation of transforming growth factor ß receptor signaling pathway, mitochondrial outer membrane and myotubular cell development, and cellular calcium homeostasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in the MAPK signaling pathway and the PI3K-Akt signaling pathway.[Conclusion] During sow early gestation, DNA methylation is significant changes and is mainly upregulated. The observed upregulation of DNA methylation during early gestation in sows may regulate the MAPK and PI3K-Akt signaling pathways, potentially influencing embryonic development. Methylated differential genes VBP1 and RPL10 can be used as potential markers for early sow pregnancy diagnosis. These findings offer novel insights into the epigenetic changes occurring in sows during early gestation. Overall design: To investigate the genome-wide DNA methylation changes in sows during early pregnancy. Experiments were conducted to systematically analyze the changes of DNA methylation in the blood of sows in early pregnancy using the RRBS sequencing method with blood samples from 28-day pregnant (pre28) and non-pregnant (ctrl) sows. Differential genes were screened and validated using BSP with the RT-qPCR method.

[背景] DNA甲基化（DNA methylation）在调控细胞增殖、分化、基因表达以及胚胎发育中发挥着至关重要的作用。然而，母猪妊娠早期全基因组DNA甲基化的全面变化仍未明确。本研究旨在探究母猪妊娠早期的DNA甲基化谱，并构建其早期基因库。
[结果] 本研究采集了20头健康母猪的血液样本，采用简化代表性亚硫酸氢盐测序（RBSS）技术，系统分析了母猪妊娠早期血液中DNA甲基化的变化情况。通过亚硫酸氢盐测序PCR（BSP）与实时定量PCR（RT-qPCR）筛选并验证了差异甲基化基因。研究发现，相较于未妊娠对照组（ctrl），妊娠第28天组（pre28）在CpG岛（CGI）、启动子、基因体等多种基因元件上的甲基化水平均显著升高。本研究共鉴定出64908个差异甲基化位点（DMCs）与12119个差异甲基化区域（DMRs），且二者主要呈现甲基化上调趋势。随机选取5个差异甲基化基因进行验证：VBP1、UPRT（甲基化上调）、RPL10、RAB33A与CD93（甲基化下调）。BSP结果显示，妊娠组（pre28）与未妊娠组（ctrl）间VBP1、UPRT、RPL10及RAB33A的甲基化差异极显著（P<0.01）。差异甲基化位点基因本体（GO）富集分析显示，其显著富集于核受体活性、凋亡过程中半胱氨酸型内肽酶活性的负调控、血红素结合以及激素活性等功能条目。差异甲基化区域GO富集分析则显著富集于1-酰基甘油-3-磷酸O-酰基转移酶活性、转化生长因子β受体信号通路调控、线粒体外膜、肌管细胞发育以及细胞钙稳态等功能。京都基因与基因组百科全书（KEGG）富集分析显示，其显著富集于丝裂原活化蛋白激酶（MAPK）信号通路与磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-Akt）信号通路。
[结论] 母猪妊娠早期的DNA甲基化水平发生显著变化，且主要呈现上调趋势。母猪妊娠早期DNA甲基化的上调可能通过调控MAPK与PI3K-Akt信号通路，进而影响胚胎发育。差异甲基化基因VBP1与RPL10可作为母猪早期妊娠诊断的潜在标志物。本研究结果为母猪妊娠早期的表观遗传变化提供了新的研究视角。
[整体实验设计] 为探究母猪妊娠早期的全基因组DNA甲基化变化，本研究以妊娠28天母猪（pre28）与未妊娠母猪（ctrl）的血液样本为材料，采用RRBS测序技术系统分析母猪妊娠早期血液中的DNA甲基化变化，并通过BSP结合RT-qPCR方法筛选验证差异基因。