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Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation

ABSTRACT: Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern pre-implantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil (CO) vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many of the differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of pre-implantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and pre-implantation blastocyst growth and development in sheep.

ORGANISM(S): Bos taurus Ovis aries

PROVIDER: GSE17401 | GEO | 2009/08/01

SECONDARY ACCESSION(S): PRJNA118847

REPOSITORIES: GEO

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Project description:Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern pre-implantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil (CO) vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many of the differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of pre-implantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and pre-implantation blastocyst growth and development in sheep. Ewes were mated at estrus to intact Suffolk rams and then assigned randomly to receive daily intramuscular (i.m.) injections from days 1.5 to 9 of either corn oil vehicle (CO) or 25 mg progesterone (P4) (Sigma Chemical Co., St. Louis, MO) in CO vehicle, and hysterectomized on either day 9 or day 12 of pregnancy (n=5 per day and treatment). Microarray hybridization was conducted with a bovine whole genome spotted oligonucleotide array. This 24,000-element array was developed by the Bovine Oligo Microarray Consortium at the University of Missouri in collaboration with Texas A&M University and Iowa State University. The array includes 16,846 probes designed from expressed sequence tags (ESTs) that were aligned to homologous vertebrate proteins and to the 6X bovine genome assembly. The probe set was supplemented with oligos designed from 703 predicted RefSeq genes, and 5943 reproductive tissue ESTs. A link to probe sequences and annotations is available at http://www.bovineoligo.org. The annotation included assigning a human gene symbol based on homology using BLAST. The oligos were synthesized by Illumina and spotted onto aminosilane-coated glass slides by Dr. David Galbraith's laboratory at the University of Arizona (http://www.ag.arizona.edu/microarray/BOM.html). Hybridizations were performed with the Genisphere Array 350 kit (Genisphere Inc., Hatfield, PA) using dual channel design with dye-swapping. Preparation of cDNA was performed according to the manufacturer's instructions. Briefly, endometrial total RNA (9 Âµg) from each ewe (n=5 per day and treatment) was reverse-transcribed with Superscript II (Invitrogen, Carlsbad, CA) with the Genisphere oligo d(T) primer containing a capture sequence for the Cy3 or Cy5 labeling reagents. Each cDNA sample containing the capture sequence for the Cy3 or Cy5 label was co-hybridized with a cDNA sample from the opposite treatment for direct comparison of differences between treatments.

Project description:Although somatic cell nuclear transfer (SCNT) cloning is more efficient in bovine than in all other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the pre-implantation period. Therefore, we evaluated the transcriptome response of the endometrium to SCNT embryos (produced from five different donor cell cultures) as compared to embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (Day 8) and transferred to recipients. The recipients were slaughtered at day 18 of pregnancy and the uterus was recovered. Pregnancy was verified by the presence of at least one normally developed embryo. Transcriptome profiling of endometrium samples using a custom cDNA microarray covering transcripts expressed in the endometrium and/or oviduct epithelium revealed 58 transcripts that were differently abundant between endometrium samples from SCNT vs. IVF pregnancies. Prominent examples are NR2F2 (encoding the orphan nuclear receptor COUP-TFII) and GJA1 (encoding connexin 43). Both transcripts are known to play important roles in placentation and were significantly less abundant in endometrium from SCNT vs. IVF pregnancies. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period. Endometrium transcriptome profiles may serve as a novel readout to evaluate SCNT embryos for their ability to induce pregnancy with a functional placenta. Keywords: response to different embryos Nineteen German Fleckvieh (Simmental) heifers were slaughtered at day 18 of pregnancy. Cycle-synchronized recipient heifers received either IVP or SCNT embryos at day 7 of the estrous cycle. Animals were slaughtered at day 18. Endometrial (intercaruncular) tissue samples were obtained from 10 pregnant animals after transfer of IVP embryos and from 9 pregnant animals after transfer of SCNT embryos.

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:Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.

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Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation

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