Unknown,Transcriptomics,Genomics,Proteomics

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Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy

ABSTRACT: 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.

ORGANISM(S): Bos taurus

SUBMITTER: Thomas Spencer

PROVIDER: E-GEOD-81449 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Interferon tau (IFNT), a Type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal, produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In study one, endometrial tissue samples were obtained on days (D) 12, 15, and 18 post-mating from nonpregnant or pregnant heifers. In study two, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or placebo lipid extrudates or PBS only as controls. Endometrial biopsies were collected after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Study one revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In study two, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the datasets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. Vice versa, genes were found as differentially expressed during pregnancy but not after IFNA2 treatment. In study three, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. Study I: Early pregnancy; day 12 of pregnancy (n=5 heifers), day 15 of pregnancy (n=3), day 18 of pregnancy (n=4), day 12 cyclic controls (n=5), day 15 cyclic controls (n=3), day 18 cyclic controls (n=4). Study II: Treatment with human interferon alpha (IFNA); IFNA treatment group (IFNA, n=3 heifers), placebo group (PLAC, n=3 heifers), control group (CONT, n=3 heifers).

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: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:An increase in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our objective was to ascertain differential effects of elevated P4 concentrations following conception on endometrial gene expression in beef heifers on Days 5, 7, 13 and 16 of pregnancy, corresponding to the morula, blastocyst, elongation and maternal recognition of pregnancy stages, respectively. Estrus was synchronized in beef heifers (N=263). Two-thirds (N=140) were inseminated (Day 0), and all animals were randomly assigned to one of the following treatments: (i) pregnant, high P4; (ii) pregnant, normal P4; (iii) cycling, high P4; (iv) and cycling, normal P4. All high P4 groups received a P4 release intravaginal device (PRID) on Day 3 post-estrus/mating. Tissue was collected on Days 5, 7, 13 or 16 of the cycle or pregnancy, and pregnancy was confirmed by the presence of an appropriately developed embryo/conceptus. PRID insertion elevated (P<0.05) P4 concentrations from Day 3.5 to 8 compared with untreated animals and conceptus size was larger (P<0.05) in animals with elevated P4 on Days 13 and 16 compared with normal P4. Total RNA was extracted from predominantly intercaruncular endometria from the ipsilateral uterine horn. Samples from individual heifers were selected on the basis of their P4 profiles and gene expression was analyzed using bovine Affymetrix microarrays (N=5 per treatment per time point). Microarray data from analyses using Bioconductor GCRMA and Limma packages were subjected to a modified t-test and P-values were adjusted for multiple testing using the Benjamin and Hochberg false discovery rate method. Differentially expressed genes were selected on the basis of an adjusted P-value of <0.01. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7 and 13 post-estrus, but, the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). On Days 5 and 7, elevated P4 in pregnant heifers, altered the expression of 36 and 124 genes respectively but on Days 13 and 16 there were relatively few DEG between high and normal P4 heifers (15 and 25). Of the genes that were differentially regulated by P4, the majority were unique to a specific day of the estrous cycle/early pregnancy. In conclusion, gene expression in endometria did not differ between pregnant and cycling heifers until Day 16 of pregnancy (i.e. the time of maternal recognition of pregnancy and production of interferon tau by conceptus trophectoderm); however, elevating P4 in early pregnancy programmed changes in gene expression in endometria that are hypothesized to impact early conceptus growth and development. Thus, on Days 5, 7 and 13 differential gene expression was affected by P4, but on Day 16 the conceptus primarily influenced gene expression in uterine endometria of heifers. Endometiral samples were taken from estrous synchronized beef heifers that were either cyclic or confirmed pregnant with either normal or high concentrations of progesterone on Day 5, 7, 13 and 16 of the estrous cycle/early pregnancy.

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Identification of Beef Heifers with Superior Uterine Capacity for Pregnancy

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