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Bai H.,University of Tokyo | Sakurai T.,University of Tokyo | Konno T.,University of Tokyo | Ideta A.,Zen noh Center | And 3 more authors.
Molecular Reproduction and Development | Year: 2012

The transcription factor GATA1 is known to play an essential role in hematopoiesis, but its other roles have not been well characterized. The purpose of this study was to determine relationships between GATA1 and GATA2 and/or GATA3, and to identify their possible functions in ovine development. GATA1 mRNA was found in ovine conceptuses and endometrial epithelial regions of Day 15 (Day 0=day of estrus) cyclic and Days 15, 17, and 21 pregnant ovine uteri. GATA1 mRNA was strongly expressed in conceptuses on Day 21, when trophoblast attachment to the maternal endometrium progressed. Similarly, GATA1 protein expression was relatively high on Day 21. To localize GATA1 mRNA, ovine conceptuses and pregnant uteri were subjected to in situ hybridization on Days 15, 17, and 21, confirming that GATA1 mRNA was expressed in trophoblasts and uterine endometrial epithelial cells in these gestation days. The presence of GATA1 protein was further confirmed by immunohistochemistry. Because high GATA1 expression appeared to coincide with reduced GATA2/3 expression, a potential role of GATA1 was examined through transfection of a mouse Gata1 expression plasmid into bovine trophoblast F3 cells. This over-expression resulted in the down-regulation of endogenous GATA2 transcripts. These observations indicate that GATA1 exists in the ovine conceptus and uterus during the peri-attachment period, and suggest that GATA1 is integral to conceptus and endometrial development through the regulation of GATA2 and possibly other developmentally important genes. © 2011 Wiley Periodicals, Inc.

Bai H.,University of Tokyo | Sakurai T.,University of Tokyo | Fujiwara H.,Kyoto University | Ideta A.,Zen noh Center | And 3 more authors.
Reproductive Medicine and Biology | Year: 2012

The establishment of a successful pregnancy requires a "fine quality embryo", "maternal recognition of pregnancy", and a "receptive uterus" during the period of conceptus implantation to the uterine endometrium. In ruminants, a conceptus cytokine, interferon tau (IFNT), a major cytokine produced by the peri-implantation trophectoderm, is known as a key factor for maternal recognition of pregnancy. IFNT can be considered one of the main factors in conceptus-uterus cross-talk, resulting in the rescue of ovarian corpus luteum (CL), induction of endometrial gene expressions, activation of residual immune cells, and recruitment of immune cells. Much research on IFNT has focused on the CL life-span (pregnancy recognition) and uterine gene expression through IFNT and related genes; however, immunological acceptance of the conceptus by the mother has not been well characterized. In this review, we will discuss the progress in IFNT and implantation research made by us and others for over 10 years, and relate this progress to pregnancy in mammalian species other than ruminants. © Japan Society for Reproductive Medicine 2012.

Sakurai T.,University of Tokyo | Sakurai T.,Yokohama College of Pharmacy | Nakagawa S.,Tokai University | Kim M.-S.,University of Tokyo | And 7 more authors.
PLoS ONE | Year: 2013

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0 = day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1 , which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions 2 637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ. © 2013 Sakurai et al.

Bai H.,University of Tokyo | Sakurai T.,University of Tokyo | Someya Y.,University of Tokyo | Konno T.,University of Tokyo | And 3 more authors.
Journal of Reproduction and Development | Year: 2011

Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells. © 2011 by the Society for Reproduction and Development.

Yamakoshi S.,University of Tokyo | Bai R.,University of Tokyo | Chaen T.,University of Tokyo | Ideta A.,Zen noh Center | And 4 more authors.
Reproduction | Year: 2012

In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT. © 2012 Society for Reproduction and Fertility.

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