Kazuhiko Imakawa

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‐γ inducible protein 10 kDa (IP‐10, CXCL10), the chemokine receptor, CXCR3, also recognizes two other chemokines; monokine induced by IFN‐γ (MIG, CXCL9) and IFN‐inducible T cell α 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‐τ or IFN‐γ. 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. Mol. Reprod. Dev.

Expression of ovine interferon‐tau (oIFNτ), 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 oIFNτ expression is restricted to the trophectoderm have not been fully elucidated. The objective of this study was to determine whether oIFNτ 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 oIFNτ (−654 base pair, bp)‐luciferase reporter (−654‐oIFNτ‐Luc) construct and several transcription factor expression plasmids. Compared to −654‐oIFNτ‐Luc alone, transcription of the −654‐oIFNτ‐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 IFNτ gene transcription, the oIFNτ‐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 oIFNτ 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 oIFNτ gene transcription is regulated by Cdx2, and suggest that Cdx2 could be a key molecule in determining oIFNτ gene transcription by the trophectoderm. Mol. Reprod. Dev.

Conceptus implantation to the uterine endometrium represents a complex series of events, including synchronized development of conceptus and uterus through up‐ and/or down‐regulation of numerous gene products. In a previous study using the DNA microarray technique, we had discovered evidence that increase in a transcript for mesenchymal morphogen, epimorphin, was noted as the conceptus attached to the matrix in vitro (Qin et al., 2003 ). In the present study, the expression and potential function of epimorphin in developing conceptuses was investigated through the use of reverse transcription‐polymerase chain reaction (RT‐PCR), whole mount in situ hybridization/immunohistochemistry, and in vitro blastocyst culture. RT‐PCR and in situ hybridization analysis revealed that epimorphin mRNA was expressed weakly in murine conceptuses during early developmental stages (1 cell to post‐adhesion blastocyst stages) and higher levels of epimorphin transcripts were observed in both inner cell mass (ICM) and trophectoderm of outgrowing blastocysts. Immunohistochemical analysis confirmed that epimorphin was localized in outgrowing trophoblast cells and ICM. Treating blastocysts in culture with a 115 kDa form of recombinant epimorphin promoted trophoblast outgrowth (P < 0.05), but a 34 kDa form of recombinant epimorphin had no effect. Treatment with a function inhibitor, rat anti‐mouse epimorphin IgM, reduced the number of embryos progressing to blastocyst outgrowth to the levels similar to those observed with plain culture medium. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis also revealed that epimorphin increased the expression of a trophoblast cell differentiation marker, placental lactogen‐1 (PL‐1), mRNA (P < 0.01). These results suggest that epimorphin is involved in trophoblast outgrowth, a process required for conceptus implantation into the endometrium. Mol. Reprod. Dev. 70: 455–463, 2005.

Studies of ovine interferon‐tau (oIFNτ) 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 oIFNτ gene were identified; however, a molecular mechanism by which these factors regulate oIFNτ 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 oIFNτ gene transcription in a transient transfection system using human choriocarcinoma JEG3 cells. The oIFNτ 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 oIFNτ‐reporter construct; however, this coactivator had no effect on the oIFNτ‐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 oIFNτ 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 (Δ2‐36), suppressed oIFNτ gene transcription. These observations suggest that c‐jun and Ets‐2 are the most probable binding partners for CBP in the potentiation of oIFNτ gene transcription. Mol. Reprod. Dev. 65: 23–29, 2003.