Possible Roles of CC- and CXC-Chemokines in Regulating Bovine Endometrial Function during Early Pregnancy.
ABSTRACT: The aim of the present study was to determine the possible roles of chemokines in regulating bovine endometrial function during early pregnancy. The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10, was higher in the endometrium at 15 and 18 days of pregnancy than at the same days in non-pregnant animals. Immunohistochemical staining showed that chemokine receptors (CCR1, CCR2, CCR3, and CXCR3) were expressed in the epithelial cells and glandular epithelial cells of the bovine endometrium as well as in the fetal trophoblast obtained from a cow on day 18 of pregnancy. The addition of interferon-? (IFNT) to an endometrial tissue culture system increased CCL8 and CXCL10 expression in the tissues, but did not affect CCL2, CCL11, and CCL16 expression. CCL14 expression by these tissues was inhibited by IFNT. CCL16, but not other chemokines, clearly stimulated interferon-stimulated gene 15 (ISG15) and myxovirus-resistance gene 1 (MX1) expression in these tissues. Cyclooxygenase 2 (COX2) expression decreased after stimulation with CCL8 and CCL14, and oxytocin receptor (OTR) expression was decreased by CCL2, CCL8, CCL14, and CXCL10. Collectively, the expression of chemokine genes is increased in the endometrium during early pregnancy. These genes may contribute to the regulation of endometrial function by inhibiting COX2 and OTR expression, subsequently decreasing prostaglandin production and preventing luteolysis in cows.
Project description:The aim of the present study was to evaluate CCL8 and CXCL10 expression and its regulatory mechanism in peripheral blood leukocytes (PBLs) at the time of maternal recognition in cows. Blood samples were collected on 14, 15, 16, 17 and 18 d after artificial insemination (AI). Based on the day of return of estrus, cows were divided into three groups, pregnant (n = 5), early embryonic mortality (EEM; n = 5) and late embryonic mortality (LEM; n = 5). The gene expression levels in PBLs were assessed with quantitative real-time reverse transcription PCR.The expression of CCL8 and CXCL10 mRNA in PBLs gradually increased from 14 to 18 d of pregnant cows and significant differences were observed on 18 d (P < 0.05), whereas no significant changes were observed both in EEM and LEM cows. Interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance gene (MX) 1 and MX2 mRNA expression in PBLs increased from 14 to 18 d which was significant on 18 d of pregnant cows as well as in LEM cows (P < 0.05), but no changes were observed in EEM cows. To determine whether the expression of CCL8 and CXCL10 in PBLs was regulated by pregnancy-related substances or not, expression level was assessed after exposure to interferon-τ (IFNT) and CCL16. Monocytes, granulocytes and lymphocytes were obtained using density-gradient centrifugation and flow cytometry. The addition of IFNT (100 ng/mL) and CCL16 (100 ng/mL) to cultured PBLs increased the expression of CCL8 and CXCL10 mRNA (P < 0.05). The expression of ISG15, MX1 and MX2 mRNA in PBLs was also stimulated by IFNT and CCL16 (P < 0.05).The expression of CCL8 and CXCL10 genes increased in PBLs during early pregnancy. Since IFNT stimulated CCL8 and CXCL10 expression in cultured PBLs, the increase of CCL8 and CXCL10 might be pregnancy-dependent events. The expression of both CCL8 and CXCL10 in PBLs was stimulated by CCL16 as well as IFNT, suggesting a chemokine interaction that at least includes CCL8, CXCL10 and CCL16, and may play a role in regulating maternal recognition in cows.
Project description:The aim of the present study is to determine possible roles of chemokines in regulating bovine endometrial function during early pregnancy. A 15 K bovine oligo DNA microarray detected 344 and 1336 differentially expressed genes (>2-fold; P<0.05) in the bovine endometrium of 15 and 18 days of pregnancy compared with these days of the estrous cycle (cyclic, n=4). The expression of six chemokines, including CCL2, CCL8, CCL11, CCL14, CCL16 and CXCL10 was higher in the endometrium of early pregnancy than that in the cyclic stage. Overall design: Bovine uteri were obtained from Japanese-Black cows in the institute ranch within 10-30 min of exsanguination. Tissue samples were collected from cows on 15 and 18 days of the estrous cycle (NP15 and NP18) and the gestation (P15 and P18)(n=4 animals/each day). The day of artificial insemination was designated as day 1.
Project description:Previous studies highlighted chemokines as potential factors regulating changes in the endometrium during early pregnancy. The current study aimed to screen the effects of a broad range of chemokines and indicate those that are involved in porcine luminal epithelial (LE) cell remodelling. Messenger RNA expression of chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL10 and CXCL12) and both the mRNA and protein expression of their receptors (CCR1, CCR2, CCR3, CCR5, CXCR2, CXCR3, CXCR4) were detected in LE cells. Exogenous CCL8 enhanced the proliferative and migration potential of LE cells and their motility in the environment with its stable concentration. The adhesive properties of LE cells were negatively affected by CCL8. However, CXCL12 positively affected the proliferation, motility and adhesion of LE cells as well as caused a decrease in MUC1 mRNA expression. To conclude, our studies determined that exogenous chemokines affected critical endometrial epithelial cell functions in the context of embryo implantation. We suggest that of all the examined factors, chemokine CCL8 participates in the establishment of a proper environment for embryo implantation, whereas CXCL12, apart from participation in endometrial receptivity, promotes embryo attachment.
Project description:This study combined in vitro production of bovine blastocysts, multiple embryo transfer techniques, and a conceptus-endometrial explant co-culture system to test the hypothesis that bovine endometrium exposed to long vs. short day 15 conceptuses would exhibit a different transcriptome profile reflective of potential for successful pregnancy establishment. Bovine endometrial explants collected at the late luteal stage of the estrous cycle were cultured in RPMI medium for 6 h with nothing (control), 100 ng/mL recombinant ovine interferon tau (IFNT), a long day 15 conceptus, or a short day 15 conceptus. Transcriptional profiling of the endometrial explants found that exposure of endometrium to IFNT, long conceptuses, or short conceptuses altered (P < 0.05) expression of 491, 498, and 230 transcripts, respectively, compared to the control. Further analysis revealed three categories of differentially expressed genes (DEG): (i) commonly responsive to exposure to IFNT and conceptuses, irrespective of size (n = 223); (ii) commonly responsive to IFNT and long conceptuses only (n = 168); and genes induced by the presence of a conceptus but independent of IFNT (n = 108). Of those 108 genes, 101 were exclusively induced by long conceptuses and functional analysis revealed that regulation of molecular function, magnesium-ion transmembrane transport, and clathrin coat assembly were the principal gene ontologies associated with these DEG. In conclusion, bovine endometrium responds differently to age-matched conceptuses of varying size in both an IFNT-dependent and -independent manner, which may be reflective of the likelihood of successful 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: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: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: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:Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".
Project description:Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased ?H2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.