Elsevier BV Acta Medica Okayama 0016-6480 380 2026 Constitutive activation of MC1R in the large-billed crow (Corvus macrorhynchos) and its potential role in black plumage 114924 EN Saya Nakano Graduate School of Natural Science and Technology, Okayama University Yuichi Tashiro Graduate School of Natural Science and Technology, Okayama University Hibiki Fukuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sayaka Aizawa Graduate School of Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Natural Science and Technology, Okayama University Melanin-based plumage coloration in birds is largely regulated by the melanocortin 1 receptor (MC1R), a G protein–coupled receptor that promotes eumelanin synthesis via cAMP signaling. In domestic chickens, constitutively activating mutations such as the MC1R^E (E92K) allele cause melanistic phenotypes, demonstrating that persistent MC1R activation can drive generalized darkening. However, to our knowledge, no experimental study has directly demonstrated constitutive MC1R activation in wild birds exhibiting uniformly black plumage. We investigated the sequence and signaling properties of MC1R from the Large-billed Crow (Corvus macrorhynchos), a species with strongly eumelanin-dominant plumage. Crow MC1R exhibited elevated basal cAMP signaling and minimal responsiveness to α-melanocyte-stimulating hormone (α-MSH) in both stable Chinese hamster ovary (CHO-K1) cells and transient CRE-luciferase assays in HEK293T cells, demonstrating ligand-independent activation comparable to that observed in the melanizing chicken MC1R^E (E92K) allele. Comparative sequence analysis identified multiple substitutions conserved across Corvus species. Among these, E12K and E18K were functionally evaluated based on prior associations with melanism in other birds. Although E12K modestly increased basal signaling in chicken MC1R, E18K alone or in combination with E12K did not reproduce crow-level constitutive activity, and reciprocal substitutions in crow MC1R failed to abolish ligand-independent activation. These findings demonstrate that crow MC1R possesses constitutive activity and suggest that this phenotype reflects lineage-specific modifications rather than a single activating substitution. Our results provide experimental evidence that constitutive MC1R activation is a plausible molecular mechanism that may contribute to the black plumage in the Large-billed Crow, although a direct causal relationship remains to be established. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 1096-4959 281 2026 N-terminal domains and site-specific glycosylation regulate the secretion of avian melanocortin inverse agonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP) 111174 EN Hibiki Fukuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Ryoya Watanabe Graduate School of Natural Science and Technology, Okayama University Yuna Iida Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Saya Nakano Graduate School of Natural Science and Technology, Okayama University Aya Mizutani Graduate School of Natural Science and Technology, Okayama University Tatsuhiko Abo Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sayaka Aizawa Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Agouti signaling protein (ASIP) and agouti-related protein (AGRP) are paralogous inverse agonists of melanocortin receptors with distinct physiological roles, but their structural and biochemical properties in birds remain poorly understood. Here, we characterized chicken ASIP and AGRP proteins. Analysis of available sequences revealed that a motif resembling the mammalian proprotein convertase 1/3 (PC1/3, also known as PCSK1) cleavage site is conserved across a broad range of avian orders, but Western blot analysis of transfected Chinese hamster ovary (CHO-K1) cells and chicken hypothalamus detected no cleavage, suggesting that avian AGRP may not be post-translationally processed at this site. Chicken ASIP mRNA contains an in-frame upstream ATG (uATG) and a putative N-linked glycosylation site at Asn-42, both conserved across multiple avian orders. Overexpression in CHO-K1 cells showed that ASIP translated from either ATG produces a mature protein of the same size that is N-glycosylated at Asn-42 and exhibits markedly lower secretion efficiency than AGRP. Domain-swapping experiments revealed that the N-terminal domain reduces secretion, whereas a naturally occurring ASIP-b variant with an additional N-glycan at Asn-47 shows enhanced secretion. Proteasome inhibition increased intracellular ASIP, and endoglycosidase H (Endo H) sensitivity indicated endoplasmic reticulum (ER) retention, suggesting that the N-terminal domain limits secretion via ER-associated proteasomal degradation. These findings reveal species-specific post-translational regulation of avian melanocortin inverse agonists, in which N-terminal features and site-specific N-glycosylation determine secretion efficiency, likely contributing to their distinct roles in pigmentation and hypothalamic energy balance. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0016-6480 361 2025 Crosstalk between prolactin, insulin-like growth factors, and thyroid hormones in feather growth regulation in neonatal chick wings 114657 EN Yuri Nozawa Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Ayako Okamura Graduate School of Natural Science and Technology, Okayama University Hibiki Fukuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Masamichi Shinohara Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sayaka Aizawa Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University The elongation of primary feathers in neonatal chicks is delayed by the late-feathering K gene located on the Z chromosome. We recently found that the K gene slows feather growth by reducing the number of functional prolactin (PRL) receptor (PRLR) dimers. In this study, we investigated the molecular mechanisms by which PRL promotes feather elongation. RT-qPCR and immunohistochemistry analyses revealed that PRLRs are predominantly localized in the pulp rather than in the epidermal layer of the feather follicle. Treatment of primary cultured feather pulp cells with PRL increased the expression of mRNAs for insulin-like growth factors (IGFs; IGF-1 and IGF-2) and type 2 deiodinase (DIO2). Furthermore, treatments with IGF-1 and triiodothyronine (T3) reciprocally enhanced the expression of mRNAs for DIO2 and IGFs. Additionally, BrdU staining in neonatal chicks showed that T3 promoted cell proliferation in both the epidermal layer and pulp cells, while this effect was suppressed by an IGF-1 receptor (IGF1R) inhibitor. These findings suggest a novel model in which PRL upregulates IGFs and DIO2 in feather pulp cells, creating a positive feedback loop between IGFs and T3, ultimately leading to the promotion of cell proliferation in both the epidermal layer and the pulp cells by IGFs. This is the first report proposing crosstalk between PRL, thyroid hormone (TH), and IGFs in feather follicles. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0378-1119 941 2025 Identification of pennaceous barbule cell factor (PBCF), a novel gene with spatiotemporal expression in barbule cells during feather development 149244 EN Minori Nakaoka Graduate School of Natural Science and Technology, Okayama University Hibiki Fukuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Maho Ogoshi Graduate School of Natural Science and Technology, Okayama University Sayaka Aizawa Graduate School of Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Natural Science and Technology, Okayama University Bird contour feathers exhibit a complex hierarchical structure composed of a rachis, barbs, and barbules, with barbules playing a crucial role in maintaining feather structure and function. Understanding the molecular mechanisms underlying barbule formation is essential for advancing our knowledge of avian biology and evolution. In this study, we identified a novel gene, pennaceous barbule cell factor (PBCF), using microarray analysis, RT-PCR, and in situ hybridization. PBCF is expressed in barbule cells adjacent to the ramus during pennaceous barbule formation, where these cells fuse with the ramus to establish the feather’s branching structure. PBCF expression occurs transiently after melanin pigmentation of the barbule plates but before the expression of barbule-specific keratin 1 (BlSK1). Orthologues of PBCF, predicted to be secreted proteins, are conserved across avian species, with potential homologues detected in reptiles, suggesting an evolutionary lineage-specific adaptation. Additionally, PBCF is expressed in non-vacuolated notochord cells and the extra-embryonic ectoderm of the yolk sac, hinting at its broader developmental significance. The PBCF gene produces two mRNA isoforms via alternative splicing, encoding a secreted protein and a glycophosphatidylinositol (GPI)-anchored membrane-bound protein, indicating functional versatility. These findings suggest that PBCF may be involved as an avian-specific extracellular matrix component in cell adhesion and/or communication, potentially contributing to both feather development and embryogenesis. Further investigation of PBCF’s role in feather evolution and its potential functions in other vertebrates could provide new insights into the interplay between development and evolution. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0016-6480 357 2024 Revisiting the hormonal control of sexual dimorphism in chicken feathers 114601 EN Li You Graduate School of Natural Science and Technology, Okayama University Kaori Nishio Graduate School of Natural Science and Technology, Okayama University Kinue Kowata Graduate School of Natural Science and Technology, Okayama University Minaru Horikawa Department of Biology, Faculty of Science, Okayama University Hibiki Fukuchi Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Maho Ogoshi Graduate School of Natural Science and Technology, Okayama University Sayaka Aizawa Graduate School of Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Natural Science and Technology, Okayama University Sexual dimorphism in plumage is widespread among avian species. In chickens, adult females exhibit countershading, characterized by dull-colored round feathers lacking fringe on the saddle, while adult males display vibrant plumage with deeply fringed bright feathers. This dimorphism is estrogen-dependent, and administering estrogen to males transforms their showy plumage into cryptic female-like plumage. Extensive studies have shown that estrogen’s role in female plumage formation requires thyroid hormone; however, the precise mechanisms of their interaction remain unclear. In this study, we investigated the roles of estrogen and thyroid hormone in creating sexual dimorphism in the structure and coloration of saddle feathers by administering each hormone to adult males and observing the resulting changes in regenerated feathers induced by plucking. RT-PCR analysis revealed that the expression of type 3 deiodinase (DIO3), responsible for thyroid hormone inactivation, correlates with fringing. Estrogen suppressed DIO3 and agouti signaling protein (ASIP) expression while stimulating BlSK1, a marker of barbule cells, resulting in female-like feathers with mottled patterns and lacking fringes. Administration of thyroxine (T4) stimulated BlSK1 and proopiomelanocortin (POMC) expression, with no effect on ASIP, leading to the formation of solid black feathers lacking fringes. Triiodothyronine (T3) significantly increased POMC expression in pulp cells in culture. Taken together, these findings suggest that estrogen promotes the formation of solid vanes by suppressing DIO3 expression, while also inducing the formation of mottled patterns through inhibition of ASIP expression and indirect stimulation of melanocortin expression via changes in local T3 concentration. This is the first report describing molecular mechanism underlying hormonal crosstalk in creating sexual dimorphism in feathers. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 Neuromedin U-deficient rats do not lose body weight or food intake 17472 EN Kyoka Yokogi Department of Biology, Graduate School of Natural Science and Technology, Okayama University Yuki Goto Department of Biology, Graduate School of Natural Science and Technology, Okayama University Mai Otsuka Department of Biology, Graduate School of Natural Science and Technology, Okayama University Fumiya Ojima Department of Natural Sciences and Biology, Kawasaki Medical School Tomoe Kobayashi Division of Molecular Genetics, Shigei Medical Research Institute Yukina Tsuchiba Department of Biology, Graduate School of Natural Science and Technology, Okayama University Yu Takeuchi Department of Biology, Graduate School of Natural Science and Technology, Okayama University Masumi Namba Division of Molecular Genetics, Shigei Medical Research Institute Mayumi Kohno Division of Molecular Genetics, Shigei Medical Research Institute Minami Tetsuka Department of Biology, Graduate School of Natural Science and Technology, Okayama University Sakae Takeuchi Department of Biology, Graduate School of Natural Science and Technology, Okayama University Makoto Matsuyama Division of Molecular Genetics, Shigei Medical Research Institute Sayaka Aizawa Department of Biology, Graduate School of Natural Science and Technology, Okayama University Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species-specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats. No potential conflict of interest relevant to this article was reported.

Springer Acta Medica Okayama 0302-766X 375 3 2018 Runx3 regulates folliculogenesis and steroidogenesis in granulosa cells of immature mice 743 754 EN Fumiya Ojima Department of Biology, The Graduate School of Natural Science and Technology, Okayama University Yuka Saito Department of Biology, The Graduate School of Natural Science and Technology, Okayama University Yukiko Tsuchiya Department of Biology, The Graduate School of Natural Science and Technology, Okayama University Maho Ogoshi Department of Biology, The Graduate School of Natural Science and Technology, Okayama University Hiroshi Fukamachi The Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University Kenichi Inagaki The Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumio Otsuka The Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Sakae Takeuchi Department of Biology, The Graduate School of Natural Science and Technology, Okayama University Sumio Takahashi Department of Biology, The Graduate School of Natural Science and Technology, Okayama University We previously demonstrated that female Runx3 knockout (Runx3-/-) mice were anovulatory and their uteri were atrophic and that Runx3 mRNA was expressed in granulosa cells. To clarify how Runx3 regulates folliculogenesis and ovulation, we examine the effects of Runx3 knockout on the gene expression of growth factors associated with folliculogenesis and enzymes associated with steroidogenesis. In Runx3-/- mouse ovaries, the numbers of primary and antral follicles were lower than those in wild-type (wt) mice at 3 weeks of age, indicating that the loss of Runx3 affects folliculogenesis. The expression of genes encoding activin and inhibin subunits (Inha, Inhba and Inhbb) was also decreased in ovaries from the Runx3-/- mice compared with that in wt mice. Moreover, the expression of the genes Cyp11a1 and Cyp19a1 encoding steroidogenic enzymes was also decreased. In cultured granulosa cells from 3-week-old mouse ovaries, Cyp19a1 mRNA levels were lower in Runx3-/- mice than those in wt mice. Follicle-stimulating hormone (FSH) treatment increased Cyp19a1 mRNA levels in both wt and Runx3-/- granulosa cells in culture but the mRNA level in Runx3-/- granulosa cells was lower than that in wt ones, indicating that granulosa cells could not fully function in the absence of Runx3. At 3 weeks of age, gonadotropin α subunit, FSHβ subunit and luteinizing hormone (LH) β subunit mRNA levels were decreased in Runx3-/- mice. These findings suggest that Runx3 plays a key role in female reproduction by regulating folliculogenesis and steroidogenesis in granulosa cells. No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 33 2017 下垂体隆起部の発生期に特異的に発現する遺伝子Cytokine-like 1,Gap junction protein alpha 5の同定 31 34 EN Sayaka Aizawa Graduate School of Natural Science and Technology, Okayama University Yuriko Higaki Graduate School of Natural Science and Technology, Okayama University Maho Ogoshi Graduate School of Natural Science and Technology, Okayama University Sakae Takeuchi Graduate School of Natural Science and Technology, Okayama University Sumio Takahashi Graduate School of Natural Science and Technology, Okayama University Adenohypophysis delivered from oral ectoderm consists of pars distalis (PD), pars intermedia and pars tuberalis (PT). The mechanisms of development of PD has been well studied, and the cell differentiation of PD has been well understood. However, the morphogenesis and the differentiation of PT are still unclear, and the gene expression during the PT development remains largely unknown. In this study, we explored the specifically expressing genes in PT during development and analyzed its spatiotemporal expressions pattern. Microarray analysis on laser-captured PT and PD tissues obtained from chicken embryos on embryonic day 10 (E10.0) showed high expressing genes, Cytokine-like 1 (CYTL1) and Gap junction protein alpha 5 (GJA5) in PT. A detail analysis of spatiotemporal expressions pattern during chick embryo development by in situ hybridization revealed that CYTL1 mRNA was first detected in lateral head ectoderm and ventral head ectoderm in E1.5. The CYTL1 expressions moved into Rathke’s pouch at E2.5, then it was localized in PT primordium and continuously expressed in PT primordium until E12.0. On the other hand, GJA5 mRNA was transiently detected in PT primordium from E6 to E14.0, while the expression was not detected in PD during development. These results suggested that these genes may be involved in the regulation mechanism of PT development and could be a useful marker in the PT development. No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 32 2016 ウズラPBCF 遺伝子の構造と発現 19 21 EN Ayako Kugimoto Sayaka Aizawa Maho Ogoshi Sumio Takahashi Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 31 2015 マウス子宮内膜細胞におけるKallikrein の発現制御の解析 37 39 EN Megumi Tokumori Maho Ogoshi Sakae Takeuchi Sumio Takahashi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 30 2014 ニワトリにおける羽色調節の品種差 39 41 EN Toru Takahashi Kaori Nishio Maho Ogoshi Sumio Takahashi Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 28 2012 マイクロアレイによるニワトリ性的二色性の分子基盤の解析 40 43 EN Ayaka Fukao Minori Nakaoka Sumio Takahashi Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 22 9 2005 Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse 1011 1021 EN Takashi Ohtsuki Mariko Otsuki Yousuke Murakami Tetsuya Maekawa Takashi Yamamoto Koji Akasaka Sakae Takeuchi Sumio Takahashi Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17 beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 22 9 2005 Interleukin-18 (IL-18) mRNA Expression and Localization of IL-18 mRNA-Expressing Cells in the Mouse Uterus 1003 1010 EN Kenji Kusumoto Yousuke Murakami Mariko Otsuki Munetoshi Kanayama Sakae Takeuchi Sumio Takahashi Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and a subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17 beta (E-2) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E-2-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 24 3 2007 Alternative Leader-Exon Usage in Mouse IGF-I mRNA Variants: Class 1 and Class 2 IGF-I mRNAs 241 247 EN Takashi Ohtsuki Mariko Otsuki Yousuke Murakami Kensaku Hirata Sakae Takeuchi Sumio Takahashi The mouse IGF-I gene contains six exons, and exon 1 and exon 2 gene are considered to be leader exons. The regulatory mechanism of alternative usage of the leader exons is unclear in mice. The present study, was aimed at clarifying changes in class 1 (derived from exon 1) and class 2 (derived from exon 2) IGF-I mRNA expression in mice under various conditions. Both class 1 and class 2 IGF-I mRNAs were expressed in the mouse uterus, liver and kidney, and class 1 IGF-I mRNA was the major transcript in all organs studied. In the uterus, both class 1 and class 2 IGF-I mRNA expression changed markedly during the estrous cycle, with the highest level at proestrus, but in the liver and kidney there were no significant changes in IGF-I mRNA expression during the estrous cycle. Estrogen treatment increased both class 1 and class 2 IGF-I mRNA levels in the uterus of ovariectomized mice, but class 1 mRNA expression increased more in response to estrogen treatment than class 2 mRNA expression. These findings suggest that estrogen stimulates IGF-I gene expression in, uterine cells, and that a promoter involved in transcription of class 1 IGF-I mRNA is more responsive to estrogen. In conclusion, the present study revealed that two leader exons of mouse IGF-I gene are used in the uterus, liver and kidney. IGF-I mRNA levels of both classes changed during the estrous cycle in the uterus, but not in the liver or kidney. Estrogen increased IGF-I mRNA levels of both classes in the uterus. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 26 2 2009 Estradiol, Progesterone, and Transforming Growth Factor α Regulate Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Expression in Mouse Endometrial Cells 131 138 EN Tetsuya Maekawa Sakae Takeuchi Munetoshi Kanayama Sumio Takahashi Insulin-like growth factor 1 (IGF1) Is Involved in the proliferation of mouse and rat endometrial cells in a paracrine or autocrine manner. Insulin-like growth factor binding protein-3 (IGFBP3) modulates actions of IGFs directly or indirectly. The present study aimed to determine whether IGFBP3 is Involved In the regulation of proliferation of mouse endometrial cells. Mouse endometrial epithelial cells and stromal cells were isolated, and cultured In a serum free medium. IGF1 stimulated DNA synthesis by endometrial epithelial and stromal cells, and IGFBP3 Inhibited IGF1-induced DNA synthesis. Estradiol-17 beta (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas It Increased the Igf1 mRNA level. Transforming growth factor alpha (TGF alpha) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal cells. Progesterone (134) did not affect the E2-induced down-regulation of Igfbp3 mRNA expression in endometrial stromal cells, although P4 alone increased Igfbp3 mRNA levels. The present findings suggest that in mouse endometrial stromal cells E2 enhances IGF1 action through enhancement of IGF1 synthesis and reduction of IGFBP3 synthesis, and that TGF alpha affects IGF1 actions through modulation of IGFBP3 levels. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 15 4 1998 Insulin-Like Growth Factor-I and Its Receptor in Mouse Pituitary Glands 573 579 EN Junichi Honda Sakae Takeuchi Hiroshi Fukumachi Sumio Takahashi Insulin-like growth factor-I (IGF-I) is produced in the liver and other peripheral tissues in response to growth hormone (GH) stimuli. IGF-I regulates diverse physiological functions in an autocrine and/or paracrine manner. IGF-I and IGF-I receptor (type-I receptor) are expressed in human and rat pituitary glands. However, the cell types of IGF-I-expressing cells and target cells of IGF-I in the pituitary glands are not known. The present study was aimed to identify the cell types of IGF-I-expressing cells and of its type-I receptor-expressing cells in mouse pituitary glands. In the mouse pituitary glands, IGF-I mRNA and IGF-I receptor mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). IGF-I-expressing cells and its receptor-expressing cells were detected by non-radioisotopic in situ hybridization using mouse IGF-I cDNA and IGF-I receptor cDNA probes, and their cell types were immunocytochemically determined using antibodies raised against pituitary hormones. We found that somatotrophs expressed both IGF-I and IGF-I receptors, and some of corticotrophs expressed IGF-I receptors. Co-localization of IGF-I and GH in the same cultured pituitary cells was observed by dual-labelling immunocytochemistry. The present study demonstrated that pituitary IGF-I produced in somatotrophs regulated functions of somatotrophs and corticotrophs in an autocrine and/or paracrine manner. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 15 4 1998 Augmentation of Prolactin Release by α-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells 567 572 EN Yoshiaki Morooka Souichi Oomizu Sakae Takeuchi Sumio Takahashi Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by alpha-melanocyte stimulating hormone (a-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the alpha-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not alpha-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that alpha-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that alpha-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 17 5 2000 Epidermal Growth Factor Stimulates Proliferation of Mouse Uterine Epithelial Cells in Primary Culture 661 666 EN Masahiro Shiraga Noriko Komatsu Kiyoshi Teshigawara Akinobu Okada Sakae Takeuchi Hiroshi Fukamachi Sumio Takahashi Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/-8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/ or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-l-induced growth. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 19 7 2002 Induction of mammotroph development by a combination of epidermal growth factor, insulin, and estradiol-17β in rat pituitary tumor GH3 cells 789 795 EN Tomoshi Kakeya Sakae Takeuchi Sumio Takahashi Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E-2) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E-2 on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-Iabeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-Iabeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-Iabeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or VIS cells in GH3 cells by a combined treatment with EGF, insulin and E-2, while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells the transdifferentiation of GH cells or VIS cells, and a self-duplication of PRL cells. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 20 1 2003 Gene Expression and the Physiological Role of Transforming Growth Factor-α in the Mouse Pituitary 83 89 EN Seema Sharma Souichi Oomizu Tomoshi Kakeya Tohru Masui Sakae Takeuchi Sumio Takahashi Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-alpha-expressing cells and the physiological roles of TGF-a within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-alpha mRNA-expressing cells, and to clarify the involvement of TGF-alpha in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-alpha mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-alpha mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-alpha mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17beta (E2) and TGF-alpha treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-alpha treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-alpha with anti-TGF-alpha-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-alpha mRNA expression in ovariectomized female mice revealed that E2 increases TGF-alpha mRNA levels. These results indicate that the TGF-alpha produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 20 5 2003 Epidermal Growth Factor and Transforming Growth Factor-α Stimulate the Proliferation of Mouse Uterine Stromal Cells 639 645 EN Noriko Komatsu Tetsuya Maekawa Sakae Takeuchi Sumio Takahashi Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E-2) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-Iabeled cells in a dose-dependent manner. Administration of the combination of E-2 (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-Iabeled cells 2.3-fold. The stimulatory effect of EGF, TGF-a and the combination of E2 and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha, and EGF-mRNA was carried,out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors. No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 27 2011 転写因子Pit-1の下垂体外組織における発現と下垂体ホルモンの発現制御 31 35 EN Shusuke Taniuchi Sumio Takahashi Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 27 2011 雌マウス生殖器官におけるRunx3の役割 26 30 EN Yukiko Tsuchiya Atsuko Sakuma Sakae Takeuchi Sumio Takahashi Runx3(Runtdomaintranscriptionfactor3)はRunxファミリーに属する転写因子で雌マウスにおいてRunx3 mRNAは, 卵巣や子宮に発現していた。雌のRunx3(-/-)マウスは不妊であった。Runx3(-/-)マウスは卵胞形成異常を起こしており, 無排卵であった。一方で, 排卵能および黄体形成能は有していた。以上より, Runx3は卵胞形成および排卵制御に関与していることを明らかにした。Runx3(-/-)マウスの子宮は萎縮している。子宮内膜上皮細胞では, E2依存性の細胞増殖が起こらなかった。しかし,子宮内膜間質細胞では, E2, P4存在下で正常に細胞増殖が起きた。以上より, Runx3はE2による子宮の細胞増殖に関与していることを明らかにした｡ No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 25 2009 黒色鶏やカラスはなぜ黒い? 〜下垂体中葉を欠く鳥類に体色制御のα-MSH調節系は存在するか〜 3 6 EN Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 20 2003 下垂体インスリン様成長因子�Tの生理的意義について―下垂体内制御機構について― 36 39 EN Sumio Takahashi Junichi Honda Yoshie Manabe Ryusei Matsumura Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 21 2004 α-メラノサイト刺激ホルモンによるプロラクチン分泌の促進作用―下垂体中葉による前葉機能の制御機構― 26 30 EN Sumio Takahashi Ryusei Matsumura Sakae Takeuchi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 22 2005 マウス子宮におけるインターロイキン-18遺伝子の発現 20 23 EN Yousuke Murakami Kenji Kusumoto Mariko Otsuki Sakae Takeuchi Sumio Takahashi No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 24 2007 マウス下垂体におけるプロオピオメラノコルチン遺伝子の発現制御 16 18 EN Itsuo Murakami Sakae Takeuchi Sumio Takahashi マウス下垂体前葉のACTH産生細胞では､CRH-R1を発現していた。POMC遺伝子プロモーターのTpit/PitxRE配列は､POMC遺伝子の転写活性を維持するのに重要であることを明らかにした。 No potential conflict of interest relevant to this article was reported.