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Exhaustive analysis of human endogenous retrovirus expression in cytotrophoblast differentiation into syncytiotrophobloast
Abstracts / Journal of Reproductive Immunology 112 (2015) 121–140
(TGF, IFN␥, TNF) were stained by Per-CP monoclonal antibodies. The stained cells were analyzed using flow cytometry performed with a FACS Caliber (Becton Dickinson). Results: The percentages of CD56+CD3− in decidual cells of PE were higher than those of NP. In those cells, CD16-cells of PE were higher than those of NP, but CD16+ cells of PE were lower than NP. The positive cells of CD314 in CD56+CD3− of PE were lower than those of NP in peripheral blood. The positive cells of CD337 in CD56+CD3− of PE were higher than those of NP in decidual cells. Cytokine production in CD56 positive cells of PE decidua was higher than PE peripheral blood. Conclusion: Changes of placenta decidual NK cells with NCR in preeclampsia may relate to roles in the pathology of this disorder. http://dx.doi.org/10.1016/j.jri.2015.09.032 28 Exhaustive analysis of human endogenous retrovirus expression in cytotrophoblast differentiation into syncytiotrophobloast Kazuki Morita ∗ , Takeshi Nagamatsu, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii Department of obstetrics and gynecology, Faculty of Medicine, University of Tokyo, Japan Introduction: Human endogenous retroviruses (HERVs) occupy about 8% of human genome. Though most of HERVs are nonfunctional due to mutagenic events, a few of them, such as syncytin1, syncytin2 and supressyn, have functional expression and are involved in cytotrophoblast (CT) fusion into syncytiotrophoblast (ST). We aimed to search novel functional HERVs expressed in human CTs using a new technology of exhaustive RNA expression analysis. Method: We collected placentas after caesarean delivery at 38 weeks gestational age. From the placentas, we isolated CTs and culture them. Spontaneous cell fusion into ST was confirmed in this culture model. We analyzed the purity of isolated CT with cell surface marker, HLA-ABC, integrin␣1, integrin␣6 and HLA-G using a flow cytometry. The secretion of hCG was examined using ELISA. Immunofluorescence staining targeting desmoplakin was performed to visualize the cell fusion progression. RNA in the cultured cells was isolated time-sequentially and was analyzed on RNA-seq. The obtained data sets were processed on a new endogenous retrovirus database. Result: The purity of isolated CTs was above 98%. The increase of hCG secretion and cell fusion detected by desmoplakin staining demonstrate the ST formation peaked at day 4 in culture. By referencing HERVs database, HERV-derived gene groups with solid expression was able to isolated in RNA-seq data, including known HERVs, syncytin1, syncytin2 and supressyn. Some HERVs showing remarkable change in gene expression level in fusion progression were identified as possible candidates of novel functional HERVs involved in CT differentiation into ST. Conclusion: By combining highly-purified CT culture model and a new database of HERVs, we successfully detected the candidates of novel functional HERVs. http://dx.doi.org/10.1016/j.jri.2015.09.033
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29 Identification and confirmation of carbohydrate structure recognized by an anti-sperm monoclonal antibody, Ts4, using lectin-binding and glycohydrolase-digestion assays Hiroshi Yoshitake 1,∗ , Noritaka Hashii 2 , Nana Kawasaki 3 , Risako Oda 1 , Yu Kawasaki 1 , Mayumi Sakuraba 1 , Shuichiro Endo 1 , Kenji Takamori 1 , Akiko Hasegawa 4 , Hiroshi Fujiwara 5 , Yoshihiko Araki 1 1
Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Japan 2 Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan 3 Laboratory of Proteomics, Yokohama City University Graduate school of Medical Life Science, Japan 4 Department of Obstetrics and Gynecology, Hyogo College of Medicine, Japan 5 Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, Japan Anti-sperm auto-antibodies (ASA-Abs) show various immunological activities such as sperm agglutination, reduction of sperm motility, and interference with gamete interaction. This diversity of effects induced by ASA-Abs suggests that the types of target antigens recognized by the Abs are abundant, and that these molecules have physiological significance in fertilization process. To identify ASA-antigen epitope, we previously established an ASA monoclonal Ab, Ts4, that reacts epididymal sperm head. Ts4 reacts N-linked oligosaccharide (OS) residue associated with some proteins. The aim of present study was to identify the molecular structure of the Ts4-epitope, to understand physiological significance of the OS residue. Using Ts4-immunoprecipitation combined with LC–MS analyses, the candidate OS structure of the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting GlcNAc or with GalNAc-GlcNAc motif. Further lectin-binding studies to the mouse testicular Ts4-immunoprecipitants showed positive staining with E-PHA, PSA, WGA, DSA and L-PHA, but not with DBA or SJA. In addition, Ts4-immunoreactivity was completely abrogated after digestion with -N-acetylglucosaminidase from Xanthomonas manihotis. These results indicate that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues. http://dx.doi.org/10.1016/j.jri.2015.09.034
(TGF, IFN␥, TNF) were stained by Per-CP monoclonal antibodies. The stained cells were analyzed using flow cytometry performed with a FACS Caliber (Becton Dickinson). Results: The percentages of CD56+CD3− in decidual cells of PE were higher than those of NP. In those cells, CD16-cells of PE were higher than those of NP, but CD16+ cells of PE were lower than NP. The positive cells of CD314 in CD56+CD3− of PE were lower than those of NP in peripheral blood. The positive cells of CD337 in CD56+CD3− of PE were higher than those of NP in decidual cells. Cytokine production in CD56 positive cells of PE decidua was higher than PE peripheral blood. Conclusion: Changes of placenta decidual NK cells with NCR in preeclampsia may relate to roles in the pathology of this disorder. http://dx.doi.org/10.1016/j.jri.2015.09.032 28 Exhaustive analysis of human endogenous retrovirus expression in cytotrophoblast differentiation into syncytiotrophobloast Kazuki Morita ∗ , Takeshi Nagamatsu, Kei Kawana, Yutaka Osuga, Tomoyuki Fujii Department of obstetrics and gynecology, Faculty of Medicine, University of Tokyo, Japan Introduction: Human endogenous retroviruses (HERVs) occupy about 8% of human genome. Though most of HERVs are nonfunctional due to mutagenic events, a few of them, such as syncytin1, syncytin2 and supressyn, have functional expression and are involved in cytotrophoblast (CT) fusion into syncytiotrophoblast (ST). We aimed to search novel functional HERVs expressed in human CTs using a new technology of exhaustive RNA expression analysis. Method: We collected placentas after caesarean delivery at 38 weeks gestational age. From the placentas, we isolated CTs and culture them. Spontaneous cell fusion into ST was confirmed in this culture model. We analyzed the purity of isolated CT with cell surface marker, HLA-ABC, integrin␣1, integrin␣6 and HLA-G using a flow cytometry. The secretion of hCG was examined using ELISA. Immunofluorescence staining targeting desmoplakin was performed to visualize the cell fusion progression. RNA in the cultured cells was isolated time-sequentially and was analyzed on RNA-seq. The obtained data sets were processed on a new endogenous retrovirus database. Result: The purity of isolated CTs was above 98%. The increase of hCG secretion and cell fusion detected by desmoplakin staining demonstrate the ST formation peaked at day 4 in culture. By referencing HERVs database, HERV-derived gene groups with solid expression was able to isolated in RNA-seq data, including known HERVs, syncytin1, syncytin2 and supressyn. Some HERVs showing remarkable change in gene expression level in fusion progression were identified as possible candidates of novel functional HERVs involved in CT differentiation into ST. Conclusion: By combining highly-purified CT culture model and a new database of HERVs, we successfully detected the candidates of novel functional HERVs. http://dx.doi.org/10.1016/j.jri.2015.09.033
131
29 Identification and confirmation of carbohydrate structure recognized by an anti-sperm monoclonal antibody, Ts4, using lectin-binding and glycohydrolase-digestion assays Hiroshi Yoshitake 1,∗ , Noritaka Hashii 2 , Nana Kawasaki 3 , Risako Oda 1 , Yu Kawasaki 1 , Mayumi Sakuraba 1 , Shuichiro Endo 1 , Kenji Takamori 1 , Akiko Hasegawa 4 , Hiroshi Fujiwara 5 , Yoshihiko Araki 1 1
Institute for Environmental and Gender-specific Medicine, Juntendo University Graduate School of Medicine, Japan 2 Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Japan 3 Laboratory of Proteomics, Yokohama City University Graduate school of Medical Life Science, Japan 4 Department of Obstetrics and Gynecology, Hyogo College of Medicine, Japan 5 Department of Obstetrics and Gynecology, Kanazawa University Graduate School of Medical Science, Japan Anti-sperm auto-antibodies (ASA-Abs) show various immunological activities such as sperm agglutination, reduction of sperm motility, and interference with gamete interaction. This diversity of effects induced by ASA-Abs suggests that the types of target antigens recognized by the Abs are abundant, and that these molecules have physiological significance in fertilization process. To identify ASA-antigen epitope, we previously established an ASA monoclonal Ab, Ts4, that reacts epididymal sperm head. Ts4 reacts N-linked oligosaccharide (OS) residue associated with some proteins. The aim of present study was to identify the molecular structure of the Ts4-epitope, to understand physiological significance of the OS residue. Using Ts4-immunoprecipitation combined with LC–MS analyses, the candidate OS structure of the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting GlcNAc or with GalNAc-GlcNAc motif. Further lectin-binding studies to the mouse testicular Ts4-immunoprecipitants showed positive staining with E-PHA, PSA, WGA, DSA and L-PHA, but not with DBA or SJA. In addition, Ts4-immunoreactivity was completely abrogated after digestion with -N-acetylglucosaminidase from Xanthomonas manihotis. These results indicate that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues. http://dx.doi.org/10.1016/j.jri.2015.09.034