Project description: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. 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)

Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy. Endometrial biopsies were subjected to RNA sequencing from high fertile (HF; n=5), subfertile (SF; n=5) and infertile (IF; n=5) classified heifers on day 14 of pregnancy.

Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy.

Project description:The female reproductive tract provides a unique environment for a successful pregnancy. It is imperative that the non-pregnant uterus be transformed into a capable environment for the establishment and maintenance of pregnancy. To achieve this, intimate cross-communication between the endometrium and the embryo is necessary at an early stage of life (Almiñana et al., 2012) a dialog which further influences subsequent fetal developmental potential (Fleming et al., 2004) and even post-natal performance. Initially, the trophoblast-derived estrogen is one of the most important embryonic signals to activate the maternal uterus for attachment (Pope & First 1985). Once the conceptus has established its position in the uterus (>d15), its development and growth also require many other maternal-embryonic cellular and molecular interactions to ensure substantial vascular changes in the endometrium and the chorioallantois, mainly development of capillaries under the lamina propriae, essential to provide full function to the pig epiteliochorial placenta required for a successful pregnancy. It is during the peri-attachment window between days 12-30 of gestation when most (20-30%) of the embryos produced in pig natural or artificial breeding die (reviewed by Edwards et al., 2012); embryonic death with a substantial impact on pig production efficiency, especially because it significantly limits litter size. Different studies have focused on the elucidation of the complex embryo-maternal communication network to reduce pregnancy loss. Among these, several studies examined gene expression during the peri-implantation stage, when the majority of embryonic losses occur, or have compared the transcriptomic profiles of pregnant and non- pregnant animals. Interestingly, these studies identified changes in the expression of genes that can -directly or indirectly- contribute to reproductive success such genes related to cell proliferation, hormone synthesis and metabolism, cell adhesion or those related to cytokine production and immune local response (Almiñana et al., 2012, Lin et al, 2015; Smolinska et al., 2019; Yang et al., 2020, Martinez et al., 2020, 2022). However, we should not forget that changes in gene expression do not always lead to a corresponding alteration in the expression of proteins, which are crucial components in all biological processes. The knowledge of the proteome is therefore equally relevant to gene expression changes, being able to help detecting both normal and abnormal physiological conditions. Proteome characterization can led to a better understanding of physiological processes and to identify proteins that may serve as potential biomarkers. To date, only a limited number of studies have unfortunately explored the protein expression profiles of the pig endometrium during the crucial period of maternal recognition of pregnancy, which occurs between day 9 and 13 (Jalali et al., 2015; Kaiser et al., 2006) or during mid/last-gestation, from 40 to 93 days of pregnancy (Chae et al., 2011; Wang et al., 2019). These studies have identified several proteins associated with endometrial function, which could play a role in maternal recognition and progression of pregnancy. Therefore, in order to fully understand the molecular interaction between the conceptus and its chorion with the endometrium, we have aimed to characterize the proteome profile of both, the non-pregnant (control) endometrium compared with that of the “pregnant”endometrium with present extraembryonic membranes between the 3rd and the 4th week period, when the major conceptus loss occurs.

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Deep sequencing of the porcine endometrial transcriptome on day 12 of pregnancy

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