Deep sequencing of the porcine endometrial transcriptome on day 12 of pregnancy. Sus scrofa

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal increases. Conceptus attachment to the uterine surface epithelium starts after Day 13 initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy (MRP), deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n=4) and corresponding non-pregnant controls (n=4) was performed using Illumina RNA-Seq. Between 30 and 35 million sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2,593 differentially expressed genes (DEG). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, of the DEG, and at the level of functional categories. This revealed specific gene expression patterns, reflecting the different functions of the endometrium during these stages, i.e. recognition of pregnancy and preparation for conceptus attachment. Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. In conclusion, this study identified comprehensive transcriptome changes in porcine endometrium associated with MRP and initiation of implantation and a number of genes and pathways potentially involved in regulation of these processes. Overall design: In total, 8 samples were analyzed, 4 biological replicates for pregnant animals (samples from 4 different animals) and 4 biological replicates for cyclic controls (samples from 4 different animals)

猪孕体（porcine conceptus）在妊娠第11至12天期间，会经历滋养层膜的快速分化与扩张。伴随滋养层伸长过程，作为猪胚胎妊娠识别信号的孕体雌激素分泌量显著升高。孕体在妊娠第13天后开始附着于子宫表面上皮，由此启动上皮绒毛膜型胎盘形成（epitheliochorial placentation）。
为解析母体妊娠识别（maternal recognition of pregnancy, MRP）进程中子宫内膜的转录组变化，本研究采用Illumina RNA-Seq技术，对妊娠第12天母猪的4个生物学重复样本及其对应的4个非妊娠对照样本的子宫内膜RNA进行了深度测序。每个样本可获得3000万至3500万条测序读段，这些读段被比对至猪参考基因组Sscrofa10.2。通过对读段计数的分析，共鉴定出2593个差异表达基因（differentially expressed genes, DEG）。本研究通过定量实时逆转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR）对部分筛选基因的表达水平进行了验证。生物信息学分析发现，上调基因与下调基因分别富集了若干特异性功能条目。
本研究还从所有表达基因、差异表达基因以及功能分类三个维度，对比了妊娠第12天与第14天的RNA-Seq数据，由此揭示了对应阶段子宫内膜的特异性基因表达模式，反映出该阶段子宫内膜在妊娠识别及孕体附着准备过程中的不同功能。本研究对与有丝分裂、免疫应答、上皮细胞分化发育、蛋白水解以及前列腺素信号通路与代谢相关的基因进行了详细探讨。
综上，本研究明确了猪子宫内膜在母体妊娠识别与着床起始过程中发生的全面转录组变化，以及若干参与调控这些过程的潜在基因与通路。
整体实验设计：本研究共分析8个样本，包括4例妊娠母猪的生物学重复样本，以及4例发情周期对照母猪的生物学重复样本。