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Kamishihoro, Japan

Bai R.,University of Tokyo | Bai H.,University of Tokyo | Bai H.,Meiji Feed Co. | Kuse M.,Okayama University | And 7 more authors.
Reproduction | Year: 2014

Following bidirectional communication, the conceptus and the uterine epithelium must establish a proper cell-cell interaction, resulting in the progression of implantation processes. To clarify the mechanism of conceptus attachment to the uterine endometrium, we studied whether vascular cell adhesion molecule (VCAM1) was expressed in bovine conceptuses or endometrium during the peri-attachment period. Uterine VCAM1 expression was minimal in day 17 (day 0Zday of estrus) cyclic and pregnant animals, but increased between days 20 and 22 of pregnancy. In the intercaruncular regions, VCAM1 protein was localized to the luminal and glandular epithelia, whereas in the caruncular regions, VCAM1 protein was detected in the stroma and endothelia of the uterine endometrium. In cultured endometrial epithelial cells (EECs), VCAM1 expression was up-regulated when treated with uterine flushings or growth factor and further increased when EECs were cocultured with bovine trophoblast CT1 cells. VCAM1 expression in CT1 cells was also up-regulated with the use of uterine flushings, and further increased when these cells were cocultured with EECs. Expression of VCAM1 receptor, integrin a 4 (ITGA4) mRNA, increased significantly in day 22 conceptuses. In day 22 pregnant uteri, VCAM1 protein was found in both EECs and conceptuses, but ITGA4 was localized only to trophoblasts. These observations indicate that cell-cell interactions between conceptuses and uterine epithelial cells are required for sufficient VCAM1 and ITGA4 expression in the bovine species and suggest that uterine VCAM1 and conceptus ITGA4 play a role in the establishment of conceptus adhesion to the uterine endometrium.

Bai R.,University of Tokyo | Kusama K.,University of Tokyo | Sakurai T.,University of Tokyo | Sakurai T.,Tokyo University of Science | And 9 more authors.
Biology of Reproduction | Year: 2015

A successful pregnancy depends on the blastocyst's implantation to the maternal endometrium; however, the initial interaction between blastocyst and uterine epithelium has not been well characterized. The objectives of this study were to determine if selectins and their ligands were expressed in the bovine conceptus and/or uterus during the periattachment period and to study whether selectins were associated with conceptus attachment to the uterine epithelium. Through the RNA-sequence analysis of bovine conceptuses on Days 17, 20, and 22 (Day 0=day of estrus), only the SELL ligand, podocalyxin (PODXL), and P-selectin (SELP) ligand, SELPLG, were found. Quantitative PCR analysis confirmed the presence of PODXL and SELPLG in these conceptuses and revealed that SELL, mRNA and protein, detected in the uterine epithelium but not in conceptuses increased during the periattachment period. In the cultured endometrial epithelial cells (EECs), SELL transcript was up-regulated when uterine flushings from Day 20 pregnant animals were placed onto these cells. SELL was also up-regulated when cultured EECs were treated with progesterone, EGF, or bFGF, but not with IFNT. In the coculture system with EECs and bovine trophoblast CT-1 cells, SELL expression in EECs was effectively reduced by its small interfering RNA; however, IFNT, a marker for CT-1 cell attachment to EECs, was not reduced, nor was a transcription factor of IFNT, CDX2. These observations suggest that the conceptus could attach to the uterine epithelium through the use of endometrial SELL and embryonic selectin ligands, possibly initiating the conceptus attachment process in the bovine species. © 2015 by the Society for the Study of Reproduction, Inc.

Kusama K.,University of Tokyo | Bai R.,University of Tokyo | Sakurai T.,Tokyo University of Science | Bai H.,Hokkaido University | And 3 more authors.
Domestic Animal Endocrinology | Year: 2016

Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNT gene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down-regulates IFNT gene transcription after conceptus attachment to the uterine endometrium. © 2016 Elsevier Inc.

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