Kyushu University [Nobuhiko Yamauchi (Associate Professor) Faculty of Agriculture, Department of Bioresource Sciences]

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Nobuhiko Yamauchi
Associate Professor
Department of Animal and Marine Bioresource Sciences
Department of Bioresource Sciences
Faculty of Agriculture
Last modified date：2023.11.27

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Department of Animal and Marine Bioresource Science
Graduate School of Bioresource and Bioenvironmental Sciences

Undergraduate School

Department of Bioresource and Bioenvironment
School of Agriculture

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Research

Research Interests

Analysis of the function of implantation specific genes using RNAi
keyword : RNAi, implantation, endometrium, gene, implantation window
2008.04.
Development of in vitro implantation model using the endometrial spheroid
keyword : rat, spheroid, embryo, implantation, endometrium
2006.04.
Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2
keyword : serine protease; implantation; rat; uterus; pregnancy
2005.04～2007.03 Implantation serine protease (ISP) was first identified in pregnant mouse uterus. It is expressed in the blastocysts and the uterus during implantation, suggested to be critical for successful implantation. However, the expression status and detailed functions of ISP remain unclear. The present study was performed to determine the expression patterns of ISP gene in the rat uterus at various stages and also the fetus during pregnancy. The analyses of two rat genes registered in GenBank, Tyrptase delta 1 (accession no. XM_220240) and Similar to ISP (accession no. XM_577076), respectively exhibited 90% and 86% of identity to mouse ISP gene at mRNA level. We herein considered them as rISP-1 and rISP-2 repectively. Then, primers and probes were designed based on these sequences for the following RT-PCR and in situ hybridization. Using RT-PCR, we found that the expression of both rISP-1 and rISP-2 were specific to the uterus. Second, we observed different patterns of rISP-1 and -2 expression during different stages of estrus cycle and the pregnancy period of the rat uterus. Specifically, rISP-1 mRNA was detected in the uterus throughout the pregnancy and the diestrus stage of estrus cycle, while rISP-2 was only expressed in the uterus from day 5 of the pregnancy until the end of gestation, but not at any stage of estrus cycle. Furthermore and interestingly, rISP-1 was also observed to be expressed in the fetus and placenta, but only in the fetus for rISP-2. Lastly, we found that both rISP mRNAs were mainly localized in the endometrial glands by in situ hybridization detection. These findings implicated that the present examined genes with high identity to mouse ISP may play significant roles not only during the implantation phase, but the development of embryo, estrus stage transition, and yet to be characterized reproduction-related processes. .
Development of endometrial spheroid using tissue engineering technique
keyword : endometrium, tissue engineering, spheroid, stromal cells, rat
2004.04～2006.03.

Academic Activities

Papers

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1.	Nishino D, Kotake A, Yun CS, Rahman AMI, El-Sharawy M, Yamanaka KI, Khandoker MAMY, Yamauchi N., Gene expression of bovine endometrial epithelial cells cultured in matrigel. , Cell Tissue Res. 2021 Apr 10. doi: 10.1007/s00441-021-03418-7. Online ahead of print., 2021.04, Glandular epithelial cells (GE) in the endometrium are thought to support the elongation and survival of ruminant embryos by secreting histotrophs. In the present study, the gene expression of bovine endometrial epithelial cells cultured in matrigel was analyzed and examined whether it could be an in vitro model of GE. Bovine endometrial epithelial cells (BEE) and stromal cells (BES) were isolated from the slaughterhouse uteri and cultured in DMEM/F12 + 10% FBS. BEE showed the gland-like structure morphological changes when cultured in 15% matrigel but could not be identified in higher concentrations of the matrigel (30% or 60%). The expression of typical genes expressed in GE, SERPINA14 and GRP, was substantially high in matrigel-cultured BEE than in monolayer (P
2.	Yun CS, Masaka H, Nishino D, Horaku S, Rahman AMI, Khandoker MAMY, Yamauchi N., Analysis of novel embryonic factors of cattle and effects on endometrial cells in vitro., Anim Reprod Sci. 2021 Mar;226:106696. doi: 10.1016/j.anireprosci.2021.106696., 226, 2021.03, Interferon tau (IFNT) is thought to have essential functions in maternal recognition and establishment of pregnancy in ruminants. There, however, is a lack of research on embryonic factors that affect pregnancy other than IFNT. The present study was conducted to determine what are other embryo-derived factors involved in pregnancy recognition and to identify effects on endometrial cells using an in vitro culture system. With use of LC-MS/MS procedures to evaluate the supernatant of elongating embryos of cattle in culture, there was detection of 78 secretary proteins including five cytokines and two growth factors. Then there was analysis for up-regulated genes using ingenuity pathway procedure, IFNT and MIF were identified as upstream regulators of 37 and five genes, respectively. The mRNA transcript of MIF receptors was identified in endometrial cells, however, not in embryos. Among genes induced by MIF, CCL2, IL7 and IL23A transcripts were identified in endometrial cells. When endometrial cells were treated with interferon alpha (IFNA) and MIF, the CCL2 transcript was in a larger abundance of endometrial epithelial and polymorphonuclear neutrophil cells, and there was a larger abundance of there mRNA transcripts as a result of MIF treatment of peripheral blood mononuclear cells. In conclusion, MIF secreted by elongating embryos of cattle synergistically regulates relative abundances of specific mRNA transcripts of endometrial cells when there is treatment with IFNA, indicating further there are several factors other than IFNT that have effects on gene expression in the endometrium during early stages of gestation in cattle..
3.	Rahman ANMI, Yamashita S, Fujihara T, Islam MR, Fujihara T, Yamaguchi H, Kawahara M, Takahashi M, Takahashi H, Gotoh T, Yamauchi N., Type-I interferon regulates matrix metalloproteinases clearance of the bovine endometrial spheroid, Anim Sci J. Jan-Dec 2020;91(1):e13350. doi: 10.1111/asj.13350., 89, 1609-1621, 2020.01, This study investigated the effect of type-I interferon (IFN) on the expression of matrix metalloproteinases (MMPs) of the bovine endometrial stromal cells (BES) and epithelial cells (BEE). The cells were separated and purified from the caruncles and cultured in DMEM/F-12 containing 10% fetal bovine serum. Spheroids were generated by using ascorbate. Zymograms of the supernatant showed that BEE predominantly expressed MMP-9, whereas MMP-2 was expressed in BES and homospheroids. While MMPs expression was not detected in hetero-spheroids. Real-time quantitative PCR revealed that type-I IFN and P4 suppressed the gene expression of MMP-2 and MMP-9 in hetero-spheroids, respectively. On the other hand, gelatin zymography analysis of the supernatant showed that type-I IFN strongly promote the clearance of MMPs. While zymograms of the MMPs stocked in the hetero-spheroids were significantly reduced by type-I IFN. Phenylmethanesulfonyl fluoride and leupeptin (both are serine proteinase inhibitors) significantly repressed the clearance of MMP-2 and MMP-9 induced by type-I IFN. Moreover, collagen fibers in heterospheroids significantly decreased after the treatment with type-I IFN. In conclusion, it was suggested that type-I IFN participate in the tissue remodeling by regulation the clearance of MMPs..
4.	Musavi SAA, Yamashita S, Fujihara T, Masaka H, Islam MR, Kim S, Gotoh T, Kawahara M, Tashiro K, Yamauchi N., Analysis of differentially expressed genes and the promoters in bovine endometrium throughout estrus cycle and early pregnancy., Anim Sci J. 2018 Nov;89(11):1609-1621. doi: 10.1111/asj.13091. Epub 2018 Sep 5., 2018 Nov;89(11):1609-1621. doi: 10.1111/asj.13091. Epub 2018 Sep 5., 89, 1609-1621, 2018.11, Endometrial gene expression is primarily regulated by the ovarian steroids and pregnancy recognition factors. The present study was aimed to characterize differential expression genes (DEGs) in bovine endometrium together with the analysis of their promoter region. Bovine uteri at follicular stage (FS), luteal stage (LS) and implantation stage (IS) at day18 of pregnancy were collected. Total RNA extracted and prepared cDNA were then subjected to high-throughput sequencing. For promoter analysis, 1kb upstream promoter region of each DEG was analyzed. The number of highly expressed DEGs were 496 and 597 at FS and LS, respectively. When compared the gene expression of IS with LS, 383 and 346 DEGs showed higher and lower expression at IS, respectively. It was also observed that 20-30 transcription factors (TFs) were included in each DEGs. Additionally, promoter analyses estimated 150-160 TFs for each stage. DLX4 and IRF4 at FS, and IRF5, IRF9, STAT1 and STAT2 at IS were in common to DEGs and estimated TFs, respectively. The present study highlighted potential molecular mechanisms controlling endometrial function during estrus cycle and implantation stage, which will further guide to better understand the endometrial functions in future studies..
5.	Islam MR, Ikeguchi Y, Yamagami K, El-Sharawy M, Yamauchi N., Development of an in vitro model to study uterine functions and early implantation using rat uterine explants., Cell Tissue Res., 370, 501-512, 2017.06, The study was conducted to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2 mm) were isolated, cultured and further characterized. Cultured explants after steroid hormone treatment exposed that both Muc1 and Pr are up-regulated significantly (P
6.	El-sharawy M, Eid E, Darwish S, El-razek IABD, Islam MR, Kubota K, Nobuhiko Yamauchi, El-shamaa I, Effect of Organic and Inorganic Selenium Supplementation on Semen Quality and Blood Enzymes in Buffalo Bulls., Anim Sci J., 21, 2016.05.
7.	Md. Rashedul Islam, Yamagami K, Yashii Y, Nobuhiko Yamauchi, Growth factor induced proliferation, migration, and lumen formation of rat endometrial epithelial cells in vitro. , J Reprod Dev, 2016.03.
8.	Egashira A, Nobuhiko Yamauchi, Md. Rashedul Islam, Yamagami K, Tanaka A, Suyama H, El-Sayed EM, Shoji Tabata, Kuramoto T, Kid depletion in mouse oocytes associated with multinucleated blastomere formation and inferior embryo development., Anim Sci J., 2016.02.
9.	Yamagami K, Md. Rashedul Islam, Yoshii Y, Mori K, KOSUKE TASHIRO, Nobuhiko Yamauchi, Preimplantation embryo-secreted factors modulate maternal gene expression in rat uterus., Cell Tissue Res. , 364, 453-463, 2015.12.
10.	Shirozu T, Sasaki K, Kawahara M, Yanagawa Y, Nagano M, Nobuhiko Yamauchi, Takahashi M, Expression dynamics of bovine MX genes in the endometrium and placenta during early to mid pregnancy., J Reprod Dev, 62, 1, 29-35, 2015.10.
11.	Egashira A, Nobuhiko Yamauchi, Tanaka K, Mine C, Otsubo H, Murakami M, Md. Rashedul Islam, Ohtsuka M, Yoshioka N, Kuramoto T, Developmental capacity and implantation potential of the embryos with multinucleated blastomeres., J Reprod Dev, 61, 6, 595-600, 2015.09.
12.	Yamauchi K, Nobuhiko Yamauchi, Yamagami K, Nakamura N, Yamashita S, Islam MR, Shoji Tabata, Yahiro K, Tamura T, Hashizume K, Masa-aki Hattori, Development of an in vitro model for the analysis of bovine endometrium using simple techniques., Anim Sci J. 2015 May;86(5):523-31., 2015.05.
13.	Yamagami K, Nobuhiko Yamauchi, Kubota K, Nishimura S, Vishwajit Sur Chowdhury, Yamanaka K, Takahashi M, Shoji Tabata, Masa-aki Hattori, Expression and Regulation of Foxa2 in the Rat Uterus during Early Pregnancy., J Reprod Dev, 2014.09.
14.	Nishimura K, Nakano N, Vishwajit Sur Chowdhury, Kaneto M, Torii M, Nobuhiko Yamauchi, Masa-aki Hattori, Kawai M, Effect of PPARβ/δ Agonist on the Placentation and Embryo-Fetal Development in Rats., Birth Defects Res B Dev Reprod Toxicol. 2013 Apr;98(2):164-9., 2013.04.
15.	Nishimura K, Yamauchi N, Chowdhury VS, Torii M, Hattori MA, Kaneto M., Expression of peroxisome proliferator-activated receptor isoforms in the rat uterus during early pregnancy., Cell Tissue Res. 2011 Aug;345(2):275-84., 2011.07.
16.	Kubota K, Yamauchi N, Yamagami K, Nishimura S, Gobaru T, Yamanaka K, Wood C, Soh T, Takahashi M, Hattori MA., Steroidal regulation of Ihh and Gli1 expression in the rat uterus., Cell Tissue Res. 2010 May;340(2):389-95. Epub 2010 Mar 16., 2010.03.
17.	Matsumoto K, Yamauchi N, Watanabe R, Oozono S, Kubota K, Nishimura K, Wood C, Soh T, Kizaki K, Hattori MA., In vitro decidualization of rat endometrial stromal cells., Cell Tissue Res. 2009 Mar;335(3):575-83. Epub 2008 Dec 17., 2008.12.
18.	Oozono S, Yamauchi N, Nishimura K, Matsumoto K, Watanabe R, Kubota K, Aramaki S, Sato F, Wood C, Soh T, Kizaki K, Hattori MA., Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2., J Exp Zoolog B Mol Dev Evol. 2008 Dec 15;310(8):642-9., 2008.12.
19.	Kubota K, Yamauchi N, Matsumoto K, Watanabe R, Oozono S, Aramaki S, Wood C, Soh T, Hattori MA., Expression of hedgehog family genes in the rat uterus during early pregnancy., J Reprod Dev. 2008 Oct;54(5):340-5. Epub 2008 Jul 8., 2008.07.
20.	Gamo T, Yamauchi N, Nishimura K, Watanabe R, Matsumoto K, Oozono S, Kubota K, He PJ, Soh T, Hattori MA., Effects of tumor necrosis factor-alpha on cell proliferation, prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro, J Exp Zool Part A Ecol Genet Physiol, 2007.12.
21.	N Yamauchi, T Takezawa, K Kizaki, CB Herath, K Hashizume, Proliferative potential of endometrial stromal cells, and endometrial and placental expression of cyclin in bovine., J Reprod Dev, 10.1262/jrd.49.553, 49, 6, 553-560, Vol.49(2003), 553-560., 2003.10.
22.	N Yamauchi, K Kizaki, O Yamada, T Takahashi, CB Herath, K Hashizume, Expression of integrin subunits depend on bovine endometrial stromal cells cultured in vitro., Connective Tissue, Vol.35(2003), 1-7., 2003.02.
23.	N Yamauchi, O Yamada, T Takahashi, K Imai, T Sato, A Ito, K Hashizume, A three-dimensional cell culture model for bovine endometrium; regeneration of a multicellular spheroid using ascoebate., Placenta, Vol.24(2002), 258-269., 2002.10.
24.	K Kizaki, O Yamada, H Nakano, T Takahashi, N Yamauchi, K Imai, K Hashizume, Cloning and localization of heparanase in bovine placenta., Placenta, Vol.24(2002), 424-430., 2002.10.
25.	N Yamauchi, O Yamada, T Takahashi, K Hashizume, Spheroid formation of bovine endometrial stromal cells using non- adherent culture plate., Journal of Reproduction and Development, Vol.47 (2001), 165-171., 2001.04.

Presentations

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1.	Tao Pan、CHISUN YUN、Nobuhiko Yamauchi, Regulation mechanism of β-catenin in Bovine Endometrium, 第1１４回日本繁殖生物学会, 2021.09, [Introduction] The elevation of β-catenin in the endometrium is reported in several mammalian species, including cattle. It has been considered that accumulated β-catenin acts as a transcription factor to regulate endometrial remodeling and implantation. It is generally known that the WNT induces the accumulation of β-catenin, but in some cases, other factors such as IGF1 also have a similar function. Each ligand involves phosphorylation of different proteins in the process of its signal transduction, LRP6 (WNT pathway) or GSK3β (IGF1 pathway), respectively. The present study aimed to clarify the regulation mechanism of β-catenin in the bovine endometrium. [Methods] Bovine endometrial epithelial cells (BEE) were treated without or with LiCl (positive control), WNT agonist AMBMP, and recombinant IGF1, respectively. The levels of β-catenin, phosphorylated LRP6 (pLRP6), and phosphorylated GSK3β (pGSK3β) were measured by western blotting. The potential downstream genes of the β-catenin (SLC2A1, MYC, and CCND1) were analyzed by qPCR. [Results] The treatment of LiCl, AMBMP, and IGF1 increased the levels of β-catenin and downstream genes in BEE. The levels of pLRP6 and pGSK3β in endometrial tissues were significantly high at luteal and implantation stages compared to the follicular stage. [Conclusions] The elevations of pGSK3β and pLRP6 suggested that both WNT and IGF1 pathways might exist in the bovine endometrium. Analysis of downstream genes in BEE showed that its expression pattern could be similar regardless of the ligand and its pathway. Collectively, these results imply that in the bovine endometrium, WNT and IGF1 pathways may contribute together to the regulation of β-catenin..
2.	CHISUN YUN、Tao Pan、Nobuhiko Yamauchi, CCL2 regulates prostaglandin circulation and embryo attachment of the bovine endometrium during implantation , 第1１４回日本繁殖生物学会, 2021.09, [Introduction] CCL2 (C-C motif chemokine ligand 2), which is known to recruit immune cells to the sites of inflammation, has been reported to be expressed in the bovine endometrium. We have reported that CCL2 induced proliferation and stimulated the expression of prostaglandin (PG) synthases in cultured endometrial cells. However, the details of how CCL2 involved in the implantation mechanism are still unclear. The purpose of present study is to analyze the functional properties of CCL2 in the bovine endometrium and embryo. [Materials and Methods] Bovine endometrial tissue, epithelial (BEE) and stromal (BES) cells, and bovine blastocyst-derived trophoblastic (BT) cells were used for the analysis. Gene expressions were analyzed by RT-PCR or qPCR. [Results] Since the previous results showed that both expressions of PGESs and PGFSs were high, we focused on the analysis of PG transporter genes (MRP4 and PGT). The amount of MRP4 and PGT were significantly high in CCL2 treated BEE and BES in vitro. The mRNA of chemokine receptors (CCR1, CCR2 and CXCR3) were detected in BT. The amount of PCNA and IFNt were significantly high in BT treated with CCL2. CCL2 significantly increased the attachment rate of BT vesicles to BEE in in vitro co-culture system. The amount of OPN increased in BEE, and ICAM-1 increased both in BEE and BT by CCL2 treatment. These results indicated that CCL2 has the potential to regulate the circulation of PGs in bovine endometrium and embryo growth. In addition, CCL2 has a possibility of regulating the process of bovine embryo attachment to endometrium by modulating expression of binding molecules..
3.	CHISUN YUN、Daichi Nishino、Shutaro Horaku、Nobuhiko Yamauchi, Expression and Function of Chemokines in Bovine Endometrium and Embryos , 第128回日本畜産学会, 2021.03, Chemokines, which mainly regulate immune cells, also involve the cellular remodeling in endometrium of human and rodents during pregnancy. However, the expression and function of chemokines in the bovine uterus remains still unclear. The purpose of this study is to analyze the expression pattern and functional properties of chemokines in the bovine endometrium and embryos. The qPCR analysis of the tissue showed that the amount of CCL2, CCL8 and CXCL10 transcripts were high at the implantation stage. The amount of CCL2 transcripts was significantly high in IFNa treated bovine endometrial stromal (BES) cells in vitro. In bovine endometrial epithelial (BEE) cells, however, the amount of CCL8 and CXCL10 were significantly high in the treatment group, but not for CCL2. The mRNA of each chemokine receptors was detected in the endometrial tissues and embryos by RT-PCR. Cellular proliferation of BEE and BES significantly increased by the CCL2 treatment. These results indicate that the expression of chemokines increased in endometrium during implantation and possible to regulate the proliferation of both endometrium and embryos..
4.	潘韜、尹治善、西野大地、寶楽修太郎、山内伸彦, Analysis of WNT/β-catenin pathway in Bovine Endometrial Cells In Vitro, 第128回日本畜産学会, 2021.03, It is reported that the WNT/β-catenin pathway is important for endometrial remodeling and embryo implantation. The present study aimed to analyze the WNT/β-catenin pathway in the bovine endometrial cells. Gene expression of potential WNT/β-catenin pathway components in bovine endometrial epithelial cells (BEE) and stromal cells (BES) were analyzed by RT-PCR. Then, BEE was treated with WNT/β-catenin pathway agonist AMBMP in vitro. The β-catenin protein level was measured by western blotting and the amounts of downstream genes were analyzed by qPCR. WNT/β-catenin pathway ligands Wnt2 was detected in both BEE and BES, Wnt3 was not detected in both types of cells, and Wnt7A was only detected in BEE. WNT/β-catenin pathway receptors LRP5, LRP6, FZD1, FZD4, and FZD7 were detected in both cells. The mRNA for the potential WNT7A-specific receptor FZD5 was expressed only in BEE. Treatment of AMBMP significantly increased the amount of β-catenin protein in BEE. In conclusion, it was demonstrated that the BEE process a functional WNT/β-catenin pathway in vitro. The amounts of downstream genes in the WNT/β-catenin pathway are currently being analyzed..
5.	Al-Nur Md. Iftekhar Rahman, Toru Takahashi, Nobuhiko Yamauchi, MMP3 mediates E2-induced endometrial cell proliferation by releasing , 第113回日本繁殖生物学会, 2020.09, 【Introduction】Cyclic cell proliferation and endometrial remodeling during the estrous cycle is important to maintain normal endometrial function. It has been reported that endometrial cells are actively proliferating at the follicular stage in cattle. However, the control mechanism of this cell proliferation has not yet been clarified. Recently it has been reported the possibility that protease enzyme matrix metalloproteinase-3 (MMP3) can regulate the cell proliferation. In the present study, the function of MMP3 on the proliferation of endometrial cells was analyzed.【Materials and Methods】Bovine endometrial stroma (BES) and epithelial (BEE) cells were cultured in DMEM/F12 containing 10%FBS. Gene expression was analyzed by qPCR. Protein expression of MMP3 and HB-EGF were analyzed by casein zymography and western bottling, respectively. Cell proliferation was analyzed by automated cell counter.【Results】 MMP3 highly expressed at follicle stage compared to luteal and implantation stage. E2 increased the gene expression and clearance of MMP3 in BES in vitro, but P4 and IFNα decreased the expression. E2 also increased the cell proliferation of BES, but the inhibitor of MMP3 and EGFR inhibited the proliferation induced by E2. Further, E2 increased the HB-EGF release in BES but MMP3 inhibitor suppressed this release. There was no direct effect of E2 on BEE cell proliferation. However, the condition medium of BES treated with E2 increased the BEE proliferation, but was inhibited by EGFR inhibitor. It was shown that E2 induces MMP3 expression and promotes HB-EGF release from BES. These results suggested that MMP3 is involved in the proliferation mechanism of endometrial cells in the follicular stage..
6.	Al-Nur Md. Iftekhar Rahman, Hayato Yamaguchi, Shutaro Horaku, Daichi Nishino, Qudratullah Qani, Toru Takahashi, Nobuhiko Yamauchi, Expression and regulation of MMP3 in bovine endometrial cells in vitro, 第126回日本畜産学会, 2019.09, Matrix Metalloproteinase 3 (MMP3) plays an important role in endometrial remodeling as well as for successful implantation in bovine. There are few information for MMP3 production and its regulation in bovine endometrium till now. Therefore, this study aimed to characterize MMP3 expression in bovine endometrial cells in vitro. Bovine endometrial stroma (BES) and epithelial (BEE) cells were cultured without or with E2, P4 and IFNα (type-I interferon) for gene and zymographic analysis, respectively. The results of qPCR showed that gene expression of MMP3 was significantly high for E2 treated BES cells compared with the other groups. BEE cells had no effects in either of these treatments for MMP3 gene expression. Casein zymography showed that BES cells secrete MMP3 after 48 h of culture, where as BEE did not show any zymographic activity. Furthermore, MMP3 clearance was significantly high when BES was treated with E2. Secretion of MMP3 was significantly decreased when treated with P4 or IFNα. These results indicate that BES release MMP3 and the expression was regulated by E2, P4 and type-I interferon..
7.	Qudratullah Qani, Al-Nur Md. Iftekhar Rahman, Hayato Yamaguchi, Daichi Nishino, Shutaro Horaku, Nobuhiko Yamauchi, Gene expression of IRFs in bovine endometrial epithelial and stromal cells in vitro, 第126回日本畜産学会, 2019.09, Interferon regulatory factors (IRFs) have a key role in IFN signaling for ISGs expression in the endometrium of ruminants including bovine. However, bovine endometrial cell types are not characterized for the expression of IRFs. The aim of this study was to characterize bovine endometrial epithelial (BEE) and stromal (BES) cells for the expression of the IRFs in vitro. Both types of cells were cultured without or with E2, P4 and IFNα for 24 h. Gene expression of IRF1, 4, 6 and 9 was analyzed. The result of RT-PCR showed that BEE expressed IRF1 and 6, and BES expressed only IRF1. IRF4 and 9 was not detected in both cell types. However, IRF9 was expressed in both cell types treated with IFNα. RT-qPCR results showed that IFNα significantly increased the expression of IRF9 for BEE, and IRF1 and 9 for BES (P
8.	Nobuhiko Yamauchi, Analysis of Bovine Endometrial Functions Using Three-dimensional Cultured Cells, The 18th International Symposium on Developmental Biology, 2018.11, Importance of the in vitro model of tissues or organs is now evident in tissue engineering and cell biology research. Till now, two-dimensional culture systems have been using for in vitro cell culture, and have contributed to cell function studies despite their limitations. Three-dimensional (3D) culture has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, unlike conventional monolayer cell culture. In our laboratory, we are developing 3D culture systems of bovine endometrial cells as a tool for the analysis of uterine endometrial functions. Among them, this lecture introduces spheroid culture and Matrigel culture. 1. Spheroid culture; Spheroids are a spherical mass composed of cells and extracellular matrices (ECMs). We have regenerated multicellular spheroids composed of bovine endometrial stromal and epithelial cells using ascorbate (1). Expression of MMPs, which are key enzymes for the tissue remodeling of the endometrium, were analyzed using the spheroid. E2, P4 and type-I IFN did not affect the gene expression of MMPs in the spheroid. However, treatment of type-I IFN increased the clearance of MMPs in the supernatant. These results suggest that IFN indirectly regulates endometrial tissue remodeling through clearance of MMPs. 2. Matrigel culture; It is reported that cells form lumens automatically by culturing cells in Matrigel (2). Matrigel is a solubilized basement membrane extracted derived from EHS mouse sarcoma cells. The bovine endometrial epithelial cells cultured in 15% Matrigel formed a circular or elliptical gland-like structure. Gene expressions of glandular epithelial specific factors (FOXA2, SERPINA14 and GRP) were significantly high in the Matrigel, compared to the monolayer cultured cells, except FOXA2. Further, SERPINA14 expression was affected by neither P4 nor IFN. However, when epithelial cells in Matrigel were co-culture with stromal cells, SERPINA14 expression increased significantly in the treatment of both P4 and IFN. These results suggest that bovine endometrial epithelial cells cultured in Matrigel show properties similar to the glandular epithelial cells in vivo, and regulated by the factors produced by the stromal cells. Finally, by using these 3D culture systems, it becomes possible to clarify not only factors regulating embryo elongation and implantation but also regulation of their expression. It will be able to reveal the mechanism of the embryo elongation and implantation to contribute to the improvement of the embryo transplantation technique. .
9.	Taisuke Fujihara, Seiya Yamashita, Kaname Nishino, Md. Rashedul Islam, Nobuhiko Yamauchi, Regulation of MMPs via type I interferon in the bovine endometrium during implantation stage, 4th World Congress of Reproductive Biology, 2017.09, Introduction Evidence suggests that matrix metalloproteinases (MMPs) play important role in bovine endometrium tissue remodeling. Previous studies in our laboratory reported that type I interferon (IFN) encourages the release of accumulated MMPs in the endometrium. However, the detail of its regulatory mechanism is still unknown. Recent findings revealed that MMPs bind to glycosaminoglycan (GAG) and accumulate in tissues. Therefore, in our study we focused on GAG degrading enzymes whose expression is fostered in the endometrium by type I IFN. Materials and Methods GAG degrading enzymes with significantly higher gene expression at implantation stage were analyzed using RNA-sequence data obtained from bovine endometrium. The effect of GAG degrading enzymes (heparitinase and hyalronidase) on release of MMPs was analyzed by zymography. Further, gene expression in stromal cells, epithelial cells and hetero spheroids was analyzed by real time quantitative polymerase chain reaction. Results and Discussion page1image11344 We found that galactosamine (N-acetyl)-6-sulfatase (GALNS) gene significantly increased at implantation stage. Likewise IFN alpha, both the GAG degrading enzymes upheld the MMPs release from the hetero spheroids. IFN alphaα promoted the GALNS gene expression in hetero spheroids and stromal cells. These results suggest that MMPs binds to GAGs and accumulated in tissues are released by GALNS whose expression is induced by type I IFN and resulted in tissue remodeling of the bovine endometrium during implantation stage..
10.	Mohamed El-Sharawy, Asami Tanaka, Hikaru Suyama, M. R. Islam, 山内伸彦, Effect of Cdc42 inhibitor on maturation rate of mouse cumulus and denuded oocytes during in vitro maturation, The 17th AAAP Animal Science Congress, 2016.08, Recent studies demonstrated that Cdc42 participated in asymmetric spindle orientation before polar body emission in mouse oocytes. Therefore, the study was aimed to investigate the effect of Cdc42 inhibitor (ML141) on maturation rate (MR) of mouse oocytes during in vitro maturation (IVM). Female mice (8–10 weeks old) were sacrificed by cervical dislocation after 46 hours of equine Chorionic Gonadotrophin (eCG; 5 IU) administration and ovaries were placed in M2 medium supplemented with 5 mg/ml bovine serum albumin. Cumulus and denuded oocytes (COC and DO) were collected and placed in M2 medium. Germinal vesicle (GV) oocytes were cultured in M16 media with 5μl/ml DMSO as control and 100μM from ML141 as treatment under mineral oil at 37°C with 5% CO2. ML141 was dissolved in 0.5% v/v DMSO and added to maturation medium to give a final concentration of 100 μM. For the analysis of MR, oocytes were observed to determine different stages of oocytes as GV, germinal vesicle breakdown, metaphase I, metaphase II, oocytes with big polar body (BPB) and degenerated oocytes. Results of this study showed a significant difference (P.
11.	S.A.A. Musavi, H. Masaka, M. R. Islam, 山内伸彦, KOSUKE TASHIRO, Differential gene expression analysis in bovine uterus at follicular, luteal and implantation stage , The 17th AAAP Animal Science Congress, 2016.08, In mammalian reproduction, uterine modulation is important for embryonic development, implantation and further placentation. This uterine modulation triggered by genes is not only steroids dependent but also the embryonic factors are critical during early pregnancy. Although the gene expression in uterus differs according to the reproductive cycles, the profiles of the functional genes yet to be identified. Therefore, the study was aimed to compare gene expressions of three different reproductive stages namely follicular, luteal and implantation in the bovine uterus. Bovine uteri were collected from the slaughterhouse and the follicular (n=5) and luteal stages (n=5) were defined by the morphology of the ovaries. For the uterus of implantation stages (n=5), cows were slaughtered on day 18 of pregnancy representing the conceptus elongation. Then the total RNA was extracted from endometrium tissues by using standard protocols of our laboratory and the quality was assessed by spectrophotometric UV absorbance at 260/280 nm. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using HiSeq 2500 sequencer (Illumina). Genes were selected by the criteria of fold change, considering >2 and
12.	M. R. Islam, Yuka Yoshii, Yuko Ikeguchi, 山内伸彦, Development of an in vitro model to study uterine functions using in vitro cultured rat uterine explants, The 17th AAAP Animal Science Congress, 2016.08, Uterine functions reflect on implantation and further placentation is mediated by a variety of factors produced by the endometrium and the blastocyst. Hence, it is important to investigate the uterine characters to understand the complex process of uterine functions. In this regard, an in vitro model would be a promising alternative. Considering the above perspectives, the present study was aimed to develop an in vitro model using rat uterine explants to explore complex uterine functions. Rat uterine explants (1-2 mm) were isolated, cultured and further characterized by phase contrast microscopy, histological analysis and indirect immunofluorescence staining. Then explants were treated with steroid hormones and the regulation of MUC1, PR, AREG and IGFBP1 were investigated. RT-qPCR data revealed that MUC1 and PR was upregulated significantly (PThe study revealed that the cultured uterine explants exhibited comparable characters of the in vivo uterine conditions, which suppose to mimic the morphology and physiology of the uterus. In conclusion, despite the necessity of comprehensive investigation, still the cultured rat uterine explants can be a useful in vitro model to explore endometrial functions. .
13.	H. Masaka, S. A. A. Musavi, M. R. Islam, 山内伸彦, Screening of embryo secreted factors using bovine elongated embryos, The 17th AAAP Animal Science Congress, 2016.08, It is suggested that some embryo-derived factors modulate the endometrial functions and play pivotal role in acquiring uterine receptivity. Thus the present was aimed to analyze proteins secreted from bovine elongated embryos to identify embryo-derived factors, those regulate endometrial function at implantation stage. Bovine elongated embryos at day 18 of pregnancy were cultured in DMEM/Ham’s F12 containing 1μM P4 and 1% PVA for 24 hours. Then, harvested supernatants were analyzed by LC-MS/MS. A total of 1091 proteins were identified and out of those 1069 proteins were converted to genes by protein database in NCBI. Annotated 904 genes were analyzed by GO analysis and observed that proteins coded by these genes contain the factors; those were not secreted outside the cells, such as cytoskeleton. Therefore, the factors with signal peptide were searched again for the genes, and 159 factors were identified as secretory factors. In reference to gene database in NCBI, secretory factors were categorized for enzyme, cell adhesion molecules, proteinase, protein regulators, proteinase inhibitors, molecule transporters, receptors, cytokines, growth factors, transcriptional factors and others. Identified cytokines (IFNT, FAM3C) and growth factors (MANF, GRN, MYDGF) were focused, as they have the possibility to become functional in signaling between embryo and endometrium. The gene expression of cytokines and growth factors in embryos and endometrium at the day 18 of pregnancy was analyzed by RT-PCR. The result showed that IFNT was expressed only in embryos (as observed in previous studies), while other genes were expressed both in embryos and endometrium. However, no remarkable number of embryo specific factors could screen except IFNT,　but the remaining genes might be a promising pool to screen the embryo specific factors. Thus, further study is necessary to analyze the factors by using IPA to sum up the embryo specific factors..
14.	Md. Rashedul Islam, 山上一樹, 吉井裕香, 山内伸彦, In Vitro Culture of Rat Uterine Explants: Characterization, Hormonal Regulation and In Vitro Decidualization, 第108回日本繁殖生物学会, 2015.09.
15.	山上一樹, Md. Rashedul Islam, 吉井裕香, 山内伸彦, Preimplantation embryo-secreted factors modulate maternal gene expression in rat uterus., Society for the Study of Reproduction, 48th Annual Meeting, 2015.06.
16.	山下聖世, 藤原泰佑, 田中綾, Md. Rashedul Islam, 山内伸彦, Type I interferon regulates Matrix Metalloproteinases (MMPs) expression of the bovine endometrial cells in vitro., Society for the Study of Reproduction, 48th Annual Meeting, 2015.06.
17.	M. R. Islam, K. Yamagami, Y. Yoshii, 山内伸彦, 服部眞彰, Effects of Epidermal Growth Factor and Hepatocyte Growth Factor on Rat Endometrial Epithelial Cells Proliferation, Migration and Lumen Formation In Vitro, The 16th AAAP Animal Science Congress, 2014.11, ABSTRACT: Several growth factors such as epidermal growth factor (EGF) and hepatocyte growth factor (HGF) modulate the function of the rat endometrium and are reportedly acts on various epithelial cells. The present study examined the expression of each receptors, EGFR and c-Met mRNA in cultured rat endometrial epithelial cells (REE) as well as investigated the biological effects of EGF and HGF on REE. Furthermore the effects of EGF and HGF on cell cycle regulation (emphasizing on p53 and cyclin D) were also investigated. The isolated and cultured REE were characterized by immunocytochemistry using endometrial epithelial cell specific antibodies. The expression of EGFR and c-Met mRNA in cultured REE were observed by isolation and purification of total mRNA followed by RT-PCR. Furthermore, the biological role of EGF and HGF on the regeneration of the endometrium was also studied in REE in their proliferative stage by using MTT assay. In the proliferation assay, the addition of EGF and HGF showed mitogenic effect in a dose dependent manner. Additionally, in vitro cell migration assay revealed that cell migration stimulated with the doses of EGF and HGF. The REE formed cell clusters followed by epithelial lumen formation were also prompted by EGF and HGF which were further investigated using a three-dimensional cell culture system. Furthermore the effect of EGF and HGF on cell cycle regulation (emphasizing on p53 and cyclin D) investigated by using real time quantitative PCR revealed that the cell cycle regulation was influenced by the EGF and HGF as did earlier. Thus the current study suggest that the EGF and HGF deserve the stimulatory function as well as trigger the cell cycle regulation and thus both have the role in proliferation, migration and lumen formation of rat endometrial epithelial cells in primary culture in vitro. .
18.	K. Yamauchi, 山内伸彦, K. Yamagami, S. Yamashita, M. R. Islam, Shoji Tabata, K. Yahiro, T. Tamura, 服部眞彰, Development of an in vitro model for the analysis of bovine endometrium using Micro Sphere Array, The 16th AAAP Animal Science Congress, 2014.11, ABSTRACT: The present study was conducted to develop an in vitro model for the analysis of bovine endometrial functions in a simple and easy method. We developed the hetero-spheroid as a model, consisting of bovine endometrial epithelial cells (BEE) and stromal cells (BES) using Micro Sphere Array® (MSA). MSA (STEM Biomethod Corporation, Kitakyushu, Japan) is a three-dimensional cell culture device, which has non-adherent cell culture wells. BEE and BES were mixed in an appropriate density and were seeded immediately on a MSA. Followed by seeding cells were cultured for 3 days and it was noticed that the cells in each well of the MSA gradually aggregated and finally formed a multicellular mass. After 4 days of the culture, transparent cells covered the outer layer of the cell mass and finally formed a hetero-spheroid. Immunocytochemical analysis showed that the outer layers of the spheroids were covered with epithelial cells and stromal cells were positioned in the inner part of the spheroids. The hetero-spheroids expressed ERα, PR, IFNR-1 and IFNR-2 mRNA, detected by RT-PCR. Reactivity to steroid hormones was investigated by the addition of E2 and P4 to each culture medium. Results of real-time RT-PCR showed that PR and Oxytocin R mRNA were increased by the addition of E2, and HGF mRNA was increased by P4. Gelatin zymography showed that MMP production was suppressed in the hetero-spheroids. In conclusion, we developed an in vitro model of the bovine endometrium, which could easily make in a short period. Since hormonal reaction and MMP production were similar to the character of endometrium in vivo, it was suggested that both models are effective for analysis of the endometrial functions in vitro. Thus, the hetero-spheroids developed in the present study provide a new platform for the bovine endometrial function research. .
19.	K. Yamagami, N. Nakamura, K. Yamauchi, S. Yamashita, K. Kubota, 山内伸彦, 服部眞彰, Expression and Regulation of Foxa2 in the Rat Uterus, The 15th AAAP Animal Science Congress, 2012.11.
20.	N. Nakamura, K. Isayama, K. Yamauchi, S. Yamashita, K. Yamagami, K. Kubota, 山内伸彦, 服部眞彰, Expression and Regulation of Indian Hedgehog in the Bovine Endometrium, The 15th AAAP Animal Science Congress, 2012.11.

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