Conceptus-derived prostaglandins regulate gene expression in the endometrium prior to pregnancy recognition in ruminants.
ABSTRACT: In cattle, the blastocyst hatches from the zona pellucida on days 8-9 and then forms a conceptus that grows and elongates into an ovoid and then filamentous shape between days 9 and 16. The growing conceptus synthesizes and secretes prostaglandins (PGs) and interferon ? (IFNT). Our hypothesis was that the ovoid conceptus exerts a local effect on the endometrium prior to maternal recognition of pregnancy on day 16 in cattle. In study one, synchronized cyclic heifers received no blastocysts or 20 in vitro-produced blastocysts on day 7 and their uteri were collected on day 13. IFNT was not detected by RIA in the uterine flushing samples of pregnant heifers containing multiple ovoid conceptuses; however, total PG levels were higher in the uterine lumen of pregnant heifers than in that of cyclic heifers. Microarray analysis revealed that the expression of 44 genes was increased in the endometria of day 13 pregnant heifers when compared with that in the endometria of cyclic heifers, and many of these genes were classical Type I IFN-stimulated genes (ISGs). In studies two and three, the effects of infusing PGs at the levels produced by the elongating day 14 conceptus into the uterine lumen of cyclic ewes on ISG expression in the endometrium were determined. Results indicated that the infusion of PGs increased the abundance of several ISGs in the endometrium. These studies support the hypothesis that the day 13 conceptus secretes PGs that act locally in a paracrine manner to alter gene expression in the endometrium prior to pregnancy recognition in cattle.
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 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:The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16-18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.
Project description:Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo-maternal communication during pregnancy recognition in cattle.
Project description:Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.
Project description:This study investigated bovine conceptus-induced modifications to the endometrial transcriptome related to effects of interferon tau (IFNT), conceptus origin (in vivo vs. in vitro), and conceptus sex. In vitro (IVF) or in vivo (superovulation and artificial insemination, AI) produced blastocysts were transferred into recipient heifers on day 7 of the estrous cycle. On day 15, IVF- or AI-derived conceptuses were obtained by uterine flushing and individually placed on endometrial explants in media for 6 h. Explants were also cultured with media alone as a control or media containing 100 ng/mL IFNT. Total explant RNA was analyzed by RNA-Seq. Incubation of endometrium with IFNT or IVF- or AI-derived conceptuses changed (P ? 0.001) expression of 491, 498, and 576 transcripts, respectively, compared to the control. Further, 369 differentially expressed genes (DEGs) were common between explants exposed to IFNT or a conceptus. A total of 240 DEGs were uniquely altered by conceptuses (IVF- and AI-derived) but not IFNT. Of these transcripts, 46 were shared between the IVF and AI groups, while 61 and 133 were specific to IVF and AI conceptuses, respectively. Five genes [melanophilin (MLPH), prominin-2 (PROM2), myeloid associated differentiation marker (MYADM), vomeronasal 1 receptor 4 like (VN1R4L) and 5-hydroxytryptamine receptor 1A (HTR1A)] were more abundant in endometrium exposed to female compared to male conceptuses (P < 0.001). A single gene [ADP-ribosylation factor like GTPase 4C (ARL4C)] was more abundant in response to male conceptuses (P < 0.001) than female conceptuses. These data support the hypothesis that conceptus regulation of gene expression in the endometrium is complex and involves factors other than IFNT that may have a biological role in pregnancy establishment.
Project description:Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.
Project description:Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to cell interactions for conceptus implantation to the maternal endometrium.
Project description:In cattle, conceptus-maternal interactions are critical for the establishment and maintenance of pregnancy. A major component of this early interaction involves the transport of nutrients and secretion of key molecules by uterine epithelial cells to help support conceptus development during the peri-implantation period of pregnancy. Objectives were to: 1) analyze temporal changes in the amino acid (AA) content of uterine luminal fluid (ULF) during the bovine estrous cycle; 2) understand conceptus-induced alterations in AA content; 3) determine expression of AA transporters in the endometrium and conceptus; and 4) determine how these transporters are modulated by (Progesterone) P4. Concentrations of aspartic acid, arginine, glutamine, histidine, lysine, isoleucine, leucine, phenylalanine and tyrosine decreased on Day 16 of the estrous cycle but increased on Day 19 in pregnant heifers (P<0.05). Glutamic acid only increased in pregnant heifers on Day 19 (P<0.001). Asparagine concentrations were greater in ULF of cyclic compared to pregnant heifers on Day 7 (P<0.05) while valine concentrations were higher in pregnant heifers on Day 16 (P<0.05). Temporal changes in expression of the cationic AA transporters SLC7A1 SLC7A4 and SLC7A6 occurred in the endometrium during the estrous cycle/early pregnancy coordinate with changes in conceptus expression of SLC7A4, SLC7A2 and SLC7A1 (P<0.05). Only one acidic AA transporter (SLC1A5) increased in the endometrium while conceptus expression of SLC1A4 increased (P<0.05). The neutral AA transporters SLC38A2 and SLC7A5 increased in the endometrium in a temporal manner while conceptus expression of SLC38A7, SLC43A2, SLC38A11 and SLC7A8 also increased (P<0.05). P4 modified the expression of SLC1A1, -1A4, -1A5, -38A2, -38A4, -38A7, -43A2, -6A14, -7A1, -7A5 and -7A7 in the endometrium. Results demonstrate that temporal changes in AA in the ULF reflect changes in transporter expression in the endometrium and conceptus during early pregnancy in cattle, some of which are modified by P4.
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.