Regulation of conceptus adhesion by endometrial CXC chemokines during the implantation period in sheep.

To gain a better understanding of biochemical mechanisms of conceptus adhesion to the maternal endometrium in ruminant ungulates, the present study was performed to clarify roles of chemokines and extracellular matrix (ECM) components in the regulation of ovine blastocyst attachment to the endometrium. In addition to the chemokine, interferon-gamma inducible protein 10 kDa (IP-10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN-gamma (MIG, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11). Similar to CXCL10, CXCL9, and CXCL11 were expressed in the uterus during the peri-implantation period, and CXCL9 mRNA expression was stimulated in endometrial explants from day 14 cyclic ewes by the addition of IFN-tau or IFN-gamma. Without ECM components, conceptus cell adhesion was low on day 14 of gestation and exhibited a 2.5-fold increase on day 17; adhesiveness on day 20 was 1/10 of that on day 14. Among various ECM components examined, trophoblast adhesion was greatest when fibronectin was used. Although day 14 conceptuses did not show much adhesive activity to fibronectin, day 17 trophoblast, and day 20 chorionic membrane exhibited 2.3-fold and 50-fold increase, respectively, which was enhanced by treatment with CXCL9 or CXCL10. These results indicate that through endometrial fibronectin and chemokines, ovine conceptus cells gain the ability to attach to the endometrium during pre-implantation period; however, elucidation of molecular mechanisms by which the conceptus acquires the adhesive ability during this time period awaits further investigation.

Regulation of the ovine interferon-tau gene by a blastocyst-specific transcription factor, Cdx2.

Expression of ovine interferon-tau (oIFNtau), a factor essential for the process of maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast. However, the molecular mechanisms by which oIFNtau expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNtau gene transcription could be regulated through Cdx2 expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Human choriocarcinoma JEG3 cells were co-transfected with an oIFNtau (-654 base pair, bp)-luciferase reporter (-654-oIFNtau-Luc) construct and several transcription factor expression plasmids. Compared to -654-oIFNtau-Luc alone, transcription of the -654-oIFNtau-Luc increased more than 30 times when this construct was co-transfected with Cdx2, Ets-2, and c-jun. The degree of transcription decreased to 1/4 levels when the upstream region was reduced to -551 bp, and became minimal with further deletions; this was confirmed with the use of the reporter constructs with mutated c-jun, Ets-2, and/or Cdx2 sites. In trophoblast unrelated NIH3T3 cells, which do not support IFNtau gene transcription, the oIFNtau-Luc transcription was enhanced approximately eightfold when the cells were co-transfected with the Cdx2/Ets-2 or Cdx2/Ets-2/c-jun expression plasmids. These findings were confirmed by gel-shift assays examining Cdx binding site on the oIFNtau gene's upstream region, by immunohistochemical study identifying the presence of Cdx2 in day 15 and 17 ovine conceptuses, and by Western blot detecting Cdx2 in day 17 conceptuses. Our results indicate that oIFNtau gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNtau gene transcription by the trophectoderm.

Different levels of ovine interferon-tau gene expressions are regulated through the short promoter region including Ets-2 binding site.

Regulation of interferon-tau (IFNtau) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNtau (oIFNtau) gene sequences characterized, approximately 75% of oIFNtau transcripts expressed in utero is derived from oIFNtau-o10 gene and amounts of transcripts from other oIFNtau genes such as oIFNtau-o8 or oIFNtau-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNtau-o10 and oIFNtau-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNtau genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNtau genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNtau-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNtau-o10 gene inserted into the oIFNtau-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNtau-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNtau-o10 gene was required for oIFNtau-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNtau-o10 and oIFNtau-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNtau gene expressions seen in utero.

Changes in immune cell distribution and IL-10 production are regulated through endometrial IP-10 expression in the goat uterus.

PROBLEM: Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon-gamma (IFN-gamma)-inducible protein 10 kDa (IP-10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP-10 during the period of pregnancy establishment. METHOD OF STUDY: Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of IP-10, IFN-gamma, tumor necrosis factor-alpha, interleukin-10 (IL-10), CXCR3 mRNA and leukocyte cell surface markers, CD4, CD8, CD11b and CD45 mRNA during the caprine early pregnancy was investigated. The ability of IP-10 to stimulate peripheral blood mononuclear cells (PBMCs) migration was demonstrated using a chemotaxis assay. Changes in migration of PBMCs' immune cell population and cytokine expressions with IP-10 stimulation were investigated using flow cytometry and RT-PCR respectively. RESULTS: Levels of IP-10, IL-10, CD4 and CD11b mRNA, and the number of CD4 and CD11b positive cells in pregnant goat endometrium were higher than those of cyclic goat endometrium. Migration of PBMCs was stimulated by recombinant caprine IP-10, and the effect was significantly reduced by neutralization with the use of an anti-IP-10 antibody. In the flow cytometric and RT-PCR analyses, migrated cells stimulated by IP-10 increased the expression of IL-10 and CD11b mRNA. Furthermore, IP-10 could stimulate the expression of IL-10 mRNA from PBMCs. CONCLUSION: Endometrial chemokine IP-10 could regulate IL-10 production by resident and possibly migrated cells expressing CD11b, probably natural killer cells, and these changes may result in immune environments of the uterus suitable for conceptus implantation in ruminants.

Pre-implantation conceptus and maternal uterine communications: molecular events leading to successful implantation.

Implantation, a critical step for mammals in establishing pregnancy, requires successful completion of sequential events such as maternal uterine development, conceptus development and attachment, and placental formation. To reach the stage of placental formation, synchronized development of the conceptus and uterus throughout the implantation period is absolutely required. A number of factors expressed at the uterine endometrium and/or conceptus, which are associated with peri-implantation development, have been identified. In addition to a temporal and spatial expression of these factors, their roles in intra- and inter-cellular interactions make it difficult to fully understand physiological roles played during the critical period. This paper focuses on early conceptus development, maternal preparation for implantation and uterine-conceptus communication during the pre-implantation period, rather than the subsequent events such as conceptus attachment to the maternal endometrium. New aspects of pre-implantation processes are evaluated through simultaneous expressions of transcription factors as they possibly regulate the complex processes of implantation events in murine species and ruminant ungulates.

Increase in DNA methylation downregulates conceptus interferon-tau gene expression.

Expression of ovine interferon-tau (oIFNtau) genes, essential for the maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast and is not detected in any other cell types or tissues. Substantial secretion of oIFNtau starts on day 12-13 of pregnancy (day 0 = day of estrus), reaches the highest on day 16-17, and then declines rapidly. Ovine IFNtau mRNA, on the other hand, reaches the highest level on day 14 of pregnancy, 2-3 days before peak production of the protein. In this study, day 14 and 17 conceptuses treated with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, were cultured in vitro and only day 17, not day 14, conceptuses resulted in upregulation of oIFNtau gene expression. To gain insight into the molecular mechanism of oIFNtau gene downregulation, the methylation status within 1 kb of the 5'-flanking region of oIFNtau-o10 gene was investigated: CpG dinucleotides of this gene in day 14 ovine conceptuses were hypomethylated compared to day 20 conceptuses or other tissues. In vitro methylation of oIFNtau-o10-reporter constructs caused suppression of reporter activity in transient transfections. Cotransfection of methyl-CpG-binding protein (MeCP2) with the reporter construct elicited further suppression of the reporter activity. In electrophoretic mobility shift assay (EMSA), patterns of shifted bands did not show much difference between methylated and unmethylated probes in distal regions, but exhibited differences in the proximal region of upstream sequences of the oIFNtau gene. These results provide evidence that changes in the degree of DNA methylation could be one of the major mechanisms leading to downregulation of the oIFNtau-o10 gene during early gestation, and possibly its silencing in nonconceptus tissues.

Molecular cloning and endometrial expression of porcine amphiregulin.

The porcine amphiregulin gene was previously reported to be within the quantitative trait locus (QTL) for uterine capacity on chromosome 8. Because amphiregulin stimulates cell proliferation, the amphiregulin gene might be responsible for this QTL. The objectives of this study were to clone amphiregulin cDNA and compare endometrial expression of its mRNA in pregnant Meishan (M) and White composite (WC) pigs. We obtained two amphiregulin cDNAs, one with 1,221 bp and another with 1,109 bp. The 112 bp difference corresponded to exon 5 of the human amphiregulin gene, which codes for the cytoplasmic domain. Endometrial mRNA expression of amphiregulin was significantly lower in M pigs than in WC pigs during early pregnancy (day 15 - 40 of gestation). Amphiregulin mRNA expression in the endometrium of both M and WC pigs increased (P < 0.01) from days 15 to 20, decreased (P = 0.01) from days 20 to 30, and did not change between days 30 and 40. This may result in reduced amphiregulin protein production leading to the slower development of M conceptuses, contributing to greater uterine capacity and litter size in prolific Chinese M pigs. Porcine genomic sequences isolated from a bacterial artificial chromosome genomic library contained exon 5, suggesting that the deletion of exon 5 in the mRNA may be due to differential splicing. The amphiregulin gene consisted of six exons and five introns spanning 10.3 kb. Mol.

Regulation of blastocyst migration, apposition, and initial adhesion by a chemokine, interferon gamma-inducible protein 10 kDa (IP-10), during early gestation.

For a pregnancy to be established, initial apposition and adhesion of the blastocyst to maternal endometrium must occur in a coordinated manner; however, a key factor(s) that mediates the trophoblast cell migration and attachment to the apical surface of the endometrium has not been identified. In this study, we examined the effect of an endometrial chemokine, interferon-gamma-inducible protein 10 kDa (IP-10), on conceptus migration to the endometrial epithelium. We first studied endometrial IP-10 mRNA expression, which was localized in the subepithelial stromal region, and detected the protein in the uterine flushing media during early pregnancy. Expression of IP-10 mRNA by the endometrium of cyclic animals was stimulated by the addition of a conceptus factor interferon-tau (IFN-tau). Immunofluorescent analysis revealed that IP-10 receptor, CXCR3, was localized in the trophoblast cells, to which biotinylated-recombinant caprine IP-10 (rcIP-10) bound. Chemotaxis assay indicated that rcIP-10 stimulated the migration of trophoblast cells, and the effects of rcIP-10 were neutralized by the pretreatment with an anti-IP-10 antibody. Adhesive activity of trophoblast cells to fibronectin was promoted by rcIP-10, and the effect was inhibited by the use of anti-IP-10 antibody. Further adhesion experiments demonstrated that binding of trophoblast cells to fibronectin was completely inhibited by a peptide of the Arg-Gly-Asp (RGD) sequence, which binds to integrins alpha5beta1, alphaVbeta1, alphaVbeta3, and alphaVbeta5, whereas non-binding peptide containing Arg-Gly-Glu (RGE) had minimal effects. More importantly, rcIP-10 promoted the adhesion of trophoblast cells to primary cells isolated from endometrial epithelium. Furthermore, rcIP-10 stimulated the expression of integrin alpha5, alphaV, and beta3 subunit mRNA in trophoblast cells. These findings suggest that endometrial IP-10 regulates the establishment of apical interactions between trophoblast and epithelial cells during early gestation.

Coactivator CBP in the regulation of conceptus IFNtau gene transcription.

Studies of ovine interferon-tau (oIFNtau) gene regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, have been carried out, but a definitive mechanism for its spatial-temporal transcription has not been elucidated. Recently, specific binding regions for transcription factors AP-1 and Ets-2 on the oIFNtau gene were identified; however, a molecular mechanism by which these factors regulate oIFNtau gene transcription has not been characterized. In the present study, we investigated the potential relationship between AP-1 and Ets-2, and their association with a coactivator, cAMP-response element binding protein-binding protein (CBP), on oIFNtau gene transcription in a transient transfection system using human choriocarcinoma JEG3 cells. The oIFNtau gene promoter/enhancer (-654 to + 1 bases, wild type)-luciferase reporter construct (pGL3-654) or its mutant at the AP-1 or Ets-2 site was cotransfected with CBP (pRc/RSV-CBP) construct along with c-jun, c-fos, and/or Ets-2 expression plasmid. CBP enhanced transcription of the wild type oIFNtau-reporter construct; however, this coactivator had no effect on the oIFNtau-reporter construct with mutated AP-1 or Ets-2 site. Cotransfection of CBP with c-jun and/or Ets-2, but not with c-fos, further increased oIFNtau gene transactivation although amounts of c-jun and c-fos expression, resulting from expression vectors, were similar. In addition, CBP inhibitor adenovirus 12S E1A (E1A), but not the mutant of E1A without CBP binding domain (Delta2-36), suppressed oIFNtau gene transcription. These observations suggest that c-jun and Ets-2 are the most probable binding partners for CBP in the potentiation of oIFNtau gene transcription. Mol. Reprod. Dev. 65: 23-29, 2003.

Molecular characterization and expression of porcine bone morphogenetic protein receptor-IB in the uterus of cyclic and pregnant gilts.

Previous gene mapping analyses revealed a quantitative trait locus for uterine capacity on chromosome 8. Comparison of porcine and human genetic maps suggests that the bone morphogenetic protein receptor IB (BMPR-IB) gene may be located near this region. The objectives of this study were to 1) clone the full coding region for BMPR-IB, 2) examine BMPR-IB gene expression by the endometrium and its cellular localization in cyclic and pregnant gilts, and 3) map the BMPR-IB gene. By iterative screening of an expressed sequence tag library, we obtained a 3559-base pair cDNA clone including the full coding region of BMPR-IB. Endometrial BMPR-IB mRNA expression of White composite gilts was determined by Northern blotting in Days 10, 13, and 15 cyclic and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial BMPR-IB mRNA expression was elevated on Days 13 and 15 (P < 0.01) compared with Day 10. Expression of BMPR-IB mRNA was localized in both luminal and glandular epithelium on Day 15. However, in pregnant gilts, BMPR-IB mRNA expression was not significantly different in the endometrium from Day 10 to Day 20, and it was significantly decreased on Days 30 and 40 (P = 0.011). The BMPR-IB gene was mapped to 108 cM on chromosome 8. These findings show that BMPR-IB mRNA expression is regulated differently in cyclic and pregnant gilts; this pattern of gene expression may be important for endometrial function during the luteal phase of the estrous cycle as compared with early pregnancy.

Antibody repertoire development in fetal and neonatal piglets. VI. B cell lymphogenesis occurs at multiple sites with differences in the frequency of in-frame rearrangements.

B cell lymphogenesis in mammals occurs in various tissues during development but it is generally accepted that it operates by the same mechanism in all tissues. We show that in swine, the frequency of in-frame (IF) VDJ rearrangements differs among yolk sac, fetal liver, spleen, early thymus, bone marrow, and late thymus. All VDJ rearrangements recovered and analyzed on the 20th day of gestation (DG20) from the yolk sac were 100% IF. Those recovered at DG30 in the fetal liver were >90% IF, and this predominance of cells with apparently a single IF rearrangement continued in all organs until approximately DG45, which corresponds to the time when lymphopoiesis begins in the bone marrow. Thereafter, the proportion of IF rearrangements drops to approximately 71%, i.e., the value predicted whether VDJ rearrangement is random and both chromosomes were involved. Unlike other tissues, VDJs recovered from thymus after DG50 display a pattern suggesting no selection for IF rearrangements. Regardless of differences in the proportion of IF rearrangements, we observed no significant age- or tissue-dependent changes in CDR3 diversity, N region additions, or other characteristics of fetal VDJs during ontogeny. These findings indicate there are multiple sites of B cell lymphogenesis in fetal piglets and differences in the frequency of productive VDJ rearrangements at various sites. We propose the latter to result from differential selection or a developmentally dependent change in the intrinsic mechanism of VDJ rearrangement.

A chemokine, interferon (IFN)-gamma-inducible protein 10 kDa, is stimulated by IFN-tau and recruits immune cells in the ovine endometrium.

Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-gamma-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-tau on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-alpha, IFN-gamma, and IFN-tau in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-tau. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-tau stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-tau regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.

Chromosomal location, structure, and temporal expression of the platelet-activating factor receptor (PAFr) gene in porcine endometrium and embryos relative to estrogen receptor alpha gene expression.

Although platelet-activating factor receptor (PAFr) gene was well characterized in the human, little was known about it in domestic animals. Porcine PAFr gene was mapped using fluorescence in situ hybridization (FISH). The structure of this gene was investigated using a 5' rapid amplification of cDNA ends (RACE) technique. Temporal expression of PAFr and estrogen receptor alpha genes (ER), and distribution of the PAFr transcripts in porcine endometrial and embryonic tissues on days 0, 10, 12, 14, 16, and 18 were analyzed using DNA competitors and reverse transcription and polymerase chain reaction (RT-PCR). The porcine PAFr gene was mapped to SSC6q26-27. Alternative splicing of primary transcripts of the PAFr gene produced two different transcripts. Transcript 1 was expressed in all tissues and cells, and transcript 2 was detected in all tissues but white blood cells. The temporal expression of the PAFr gene in endometrial (P > 0.05) and embryonic (P < 0.05) tissues of pregnant sows increased from day 10 to 16. The temporal expression of ER genes in endometrial tissues of pregnant sows decreased from day 10 to 18 (P < 0.05). In addition, ER expression was detectable in 20-60% of embryonic tissue samples, which generally decreased. In combination with previously obtained data on PAF and estradiol-17beta (E(2)) concentrations in pregnant uterine luminal fluids (pULF), endometrial and embryonic tissues, the present results indicated that the increasing PAFr transcripts were positively associated with increasing levels of PAF. Both ER transcripts and E(2) found in pULF decreased correspondingly from day 13 to 16. These results indicate that via PAFr, PAF could play a dominant role in peri-implantation development in pigs as compared to E(2).

Temporal expression of type I interferon receptor in the peri-implantation ovine extra-embryonic membranes: demonstration that human IFNalpha can bind to this receptor.

Interferon-tau (IFNtau), produced by the trophectoderm of ruminant ungulates, binds to the type I IFN receptor (IFNAR) located at the uterine endometrium in a paracrine manner. Since IFNtau attenuates the secretory pattern of an endometrial luteolysin, prostaglandin F2alpha, IFNtau has been considered as a conceptus factor implicated in the process of maternal recognition of pregnancy. Here we report the presence of IFNAR subunit (IFNAR1) in ovine conceptuses during the period of peri-implantation development and demonstrate that 125I-human (h) IFNalpha binds to membrane preparations from ovine corpus luteum and conceptus. Using an antibody against hIFNAR1, immunohistochemical analysis revealed that IFNAR1 protein was present in day 14 and 16 conceptuses (day 0 = day of estrus) and luminal and glandular epithelia of the endometrium. Conceptus membrane proteins analyzed by western blot with the same antibody displayed immunoreactive bands at 95, 60 and 55 kDa while endometrial membrane proteins showed bands at 200, 95 and 55 kDa. Northern blot analysis revealed that IFNAR1 mRNA was present in days 15-19 conceptuses and day 18-19 allantoic membranes. Receptor binding studies indicated that 125I-hIFNalpha binding to day 16, but not earlier, conceptus membrane proteins could be displaced with hIFNalpha or ovine IFNtau. Based on Scatchard analysis, day 16 conceptus membranes contained 28 fmol IFNAR/mg protein with a dissociation constant of 300 pM. Cross-linking experiments demonstrated that 125I-hIFNalpha-receptor complex migrated at 120 kDa, indicating that the receptor component(s) was approximately 100 kDa. These data provide evidence that although the binding does not occur until day 16, ovine conceptuses possess IFNAR1 near or at the time of implantation, suggesting that IFNtau, a factor produced by the trophectoderm of ruminant ungulates, could act on the conceptus in an autocrine manner. In addition to functioning as an antiluteolytic factor, therefore, IFNtau may have a direct effect on conceptus development.