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Nobuhiko Yamauchi Last modified date:2019.06.13

Associate Professor / Department of Animal and Marine Bioresource Sciences
Department of Bioresource Sciences
Faculty of Agriculture


Graduate School
Undergraduate School


E-Mail
Homepage
http://www.agr.kyushu-u.ac.jp/asweb/chiku1/yamauchi/top.html
Phone
092-802-4590
Academic Degree
Ph. D. (Agriculture)
Country of degree conferring institution (Overseas)
No
Field of Specialization
Animal Reproductive Physiology
Total Priod of education and research career in the foreign country
00years00months
Outline Activities
Research: I have been studied about animal reproductive physiology covered from oocyte maturation, fertilization, early development through implantation and placental development. Physiological characters of domestic animal oocytes in in vitro culture were investigated by using molecular and biochemical techniques. At present, functions of genes expressed during oocyte maturation and early development are being analyzed using RNA interference method. Furthermore, to clarify the complex implantation and placental development process, an in vitro model for analyzing the functions are being developed from endometrial and placental derived cells using 3-D tissue engineering techniques.
Education: Besides teaching in classes (Laboratory Exercise in Animal Reproduction and Basic Zoology), I am giving instructions for faculty and graduate students in the laboratory meeting and seminar. Also based on the above theme for the research, basic experimental protocols are taught practically for student遮s own thesis with a lot of discussions.
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
1. 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., 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..
2. 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<0.05) by E2. Areg is up-regulated significantly (P<0.05) by P4. Surprisingly, Igfbp1 is up-regulated significantly (P<0.05) by E2 and P4, although in rat Igfbp1 is E2 dependent. Furthermore, in vitro decidualization of cultured explants was induced and two potential markers of decidualization namely Prl8a2 and Bmp2 were analysed. Real time quantitative PCR data revealed that both Prl8a2 and Bmp2 are significantly (P<0.05) up-regulated in MPA and db-cAMP treated explants compared to the control group of explants. Then, individual hatched blastocyst and cultured explant was placed in a 96U (U shaped round bottom) well plate. Results showed that stable attachments are observed after 48 hours of co-culture, where embryos were stably attached to the explants and could not be dislodged after mild shaking and/or pipetting. Steroid hormone treatment revealed that the rate of attachment of embryo to the explant is significantly increased in P4 treated group (63.6%) compared to the control group (35.5%). On the other hand, rate of attachment is significantly reduced in E2 treated group compared to the control group, where no stable attachments are observed in E2 treated group (0.0%). Despite the necessity of comprehensive investigation, our results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation..
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
20. 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.
21. 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.
22. 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
1. 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.
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2. 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

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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..
3. 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<0.05) in MR and GV % of the COC oocytes (MR; 65.22 and 46.82%, GV; 7.47 and 16.94 %) in CNT and ML141 group, respectively. MR for DO oocyte was decreased in CNT and ML141 group (54.13 and 40.64%), respectively, but no significant differences were observed between COC and DO oocytes. In addition, the ratio of COC and DO oocytes, which had BPB was higher in ML141 group (3.92 and 9.36 %) than in CNT group (1.56 and 1.78%), respectively. From these results, it was supposed that Cdc42 was required for achievement of normal oocyte maturation. Also, Cdc42 deficient oocytes causing loss of polar body emission and contractile ring formation and resulted in inhibition of oocyte maturation.
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4. 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 <0.37. Genes according to their biological functions were clustered using gene ontology (GO). Then the gene clusters were compared as follicle vs luteal and luteal vs implantation stage. The results showed that 565 genes of 70 categories were highly expressed in implantation stages compared to luteal stages, whereas 188 genes of 30 categories showed reverse expression. On the other hand, in follicular stages 435 genes of 56 categories showed higher expression than luteal stages and 350 genes of 55 categories showed reverse expression pattern. The results showed that the implantation stages compared to follicular and luteal stages expressed higher number of genes reflects on biological functions that can be direct to utilize the genes to better understand the implantation mechanism..
5. 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 (P<0.05) by E2. On the other hand, AREG was upregulated significantly (P<0.05) by P4. Surprisingly, IGFBP1 was upregulated significantly (P<0.05) by E2 and P4, although in rat IGFBP1 is E2 dependent. Furthermore, the remodeling ability of the uterine explants in terms of in vitro decidualization was also investigated emphasizing on PRL8a2 and BMP2. RT-qPCR data revealed that the PRL8a2 and BMP2 were significantly (P<0.05) up regulated in treated explants compared to non-treated explants. Then, the cultured explants were co-cultured with the hatched blastocyst and the attachments of embryos to the explants were examined by phase contrast microscopy and histological analysis. Additionally, the steroid hormones reflects on attachment rates were also investigated, where progesterone induce the embryo attachment but estrogen prevent it compared to the control. .
The 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.
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6. 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..
7. 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.
8. 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.
9. 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.
10. 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.
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11. 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.
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12. 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.
13. 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.
Educational
Educational Activities
Laboratory Exercise in Animal Reproduction
Basic Zoology