Unknown,Transcriptomics,Genomics,Proteomics

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RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation

ABSTRACT: Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatchingembryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22?700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P < 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n = 236) and 6 (n = 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy. mRNA-seq study of bovine conceptuses at 5 stages of development (days 7, 10, 13, 16 and 19) post fertilization.

ORGANISM(S): Bos taurus

SUBMITTER: Paul McGettigan

PROVIDER: E-GEOD-56513 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation.

Mamo Solomon S Mehta Jai P JP McGettigan Paul P Fair Trudee T Spencer Thomas E TE Bazer Fuller W FW Lonergan Patrick P

Biology of reproduction 20110727 6

Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatching embryo and elongating conceptus. This period of development is argu ...[more]

PMID: 21795669

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Project description:The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endo- metria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status. mRNA profiles of pregnant vs not pregnant cross-bred beef heifers at days 13 and 16 (n=5 per group)

Project description:In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus gene expression and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes were differently expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid with male compared to female conceptuses, while female conceptuses had higher expression of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in gene expression and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different response in the endometrium. Following a synchronized estrous cycle, all heifers observed in standing estrus (=Day 0, n=30) were inseminated with semen from a proven sire. All samples were recovered at slaughter on Day 19 following estrus corresponding to the initiation of implantation in cattle, flushed with 10 ml of PBS and the presence of a conceptus was observed under a stereo-microscope (n=24). Each conceptus was dissected into 4 pieces, 3 containing only trophectoderm cells and one containing the embryonic disc along with associated trophectoderm cells, and immediately snap-frozen in liquid nitrogen along with the corresponding intercaruncular endometrium from the uterine horn ipsilateral to the corpus luteum. DNA was extracted from each conceptus with phenol/chloroform treatment and finally re-suspended in 200 Î¼L of milliQ water. Two microliters of each sample were used to perform embryo sexing by PCR amplification of sex-specific polymorphic fragments in the amelogenin gene. N=5 samples of intercaruncular endometirum and the corresponding trophectoderm only sample were anaylsed for gene expression.

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.

Dataset Information

RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation

Publications

RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation.

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