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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.

DOI： 10.1007/s00441-018-2947-2

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Chicken early (EF) and late feathering (LF) are sex-linked phenotypes conferred by wild-type k + and dominant K alleles on chromosome Z, respectively. Besides prolactin (PRL) receptor (PRLR) and sperm flagellar 2 (SPEF2) genes, the K allele contains a fusion gene in which partially duplicated PRLR (dPRLR) and SPEF2 (dSPEF2) genes are linked in a tail-to-tail manner. The causative dPRLR gene encodes a C-terminal truncated receptor. LF chickens have short or no primaries at hatching; however, their feather growth rate is higher than that of EF chickens. This study aimed to elucidate the molecular basis of the K allele's biphasic effect on feather development. By 3′RACE and RT-PCR analyses, we demonstrated that dSPEF2 gene transcription occurred beyond all coding exons of the dPRLR gene on the opposite strand and that dPRLR mRNA was less abundant than PRLR mRNA. In addition, a 5′UTR splice variant (SPV) of PRL receptor mRNAs was increased in LF chickens. In vitro expression analysis of 5′UTR linked to the luciferase reporter gene revealed higher translation efficiency of SPV. RT-qPCR showed that the dPRLR mRNA level was higher in embryos; conversely, SPV was higher in hatched chickens, as was dSPEF2 mRNA. These findings suggest that the K allele inhibits feather development at the fetal stage by expressing dPRLR to attenuate PRLR function and promotes feather growth after hatching by increasing PRLR through dSPEF2 mRNA expression. Increased SPV may cause greater feather growth than that in EF chickens by increasing the availability of PRLR homodimers and enhancing PRL signaling.

DOI： 10.1016/j.ygcen.2018.12.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

We previously demonstrated that the Runx3 transcription factor is expressed in the hypothalami, pituitaries, and ovaries of mice, and that Runx3 knockout (Runx3(-/-)) mice are anovulatory and their uteri are atrophic. Runx3 mRNA expression was detected in the granulosa cells of ovarian follicles, and in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). In the present study, we examined the effects of Runx3 knockout on the gene expression of enzymes associated with steroidogenesis. We found decreased Cyplla1 mRNA expression in Runx3(-/-) mouse ovaries compared with that in wild-type (wt) mouse ovaries at the age of 8 weeks. In situ hybridization analysis showed that the percentages of Cyplla1 mRNA-expressing theca cells in follicles of Runx3(-/-) mice were decreased compared with those of wt mice. In accord with the alterations in Runx3(-/-) mouse ovaries, Kiss1 mRNA levels in ARC were increased, whereas mRNA levels of kisspeptin in AVPV were decreased, and gonadotropin-releasing hormone in the preoptic area and follicle-stimulating hormone beta subunit gene were increased in Runx3(-/-) mice. Following an ovarian transplantation experiment between Runx3(-/-) mice and wt mice, corpora lutea were observed when ovaries from Runx3(-/-) mice were transplanted into wt mice, but not when those from wt mice were transplanted into Runx3(-/-) mice, suggesting that Runx3 in the hypothalamo-pituitary system may drive gonadotropin release to induce ovulation in the ovary. These findings indicate that Runx3 plays a crucial role in the hypothalamo-pituitary-gonadal axis.

DOI： 10.1262/jrd.2016-005

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Melanocortin receptor 3 (MC3R) is expressed in the hypothalamus and pituitary in humans and rodents, and is involved in the control of feeding, energy metabolism, and pituitary function. In the mouse pituitary, MC3R is detected in mammotrophs. This study aimed to clarify the regulatory mechanism for Mc3r expression in the mouse pituitary. The promoter activities of reporter constructs for the MC3R gene 5'-flanking region up to -4000 bp (transcription initiation site designated as +1) were analyzed. The promoter activity significantly increased in the -86/+109 construct, but decreased in the -38/+109 construct, indicating that the minimal promoter required for basal expression of Mc3r is located in the -86/+109 region. Putative binding sites for transcription factors AP-1 and ATF4 were found in the 5'-flanking region of Mc3r. Site-directed mutation or deletion of these sites affected the promoter activities. In gel-shift assays with a nuclear extract of mouse anterior pituitary cells, band-shifts were detected for both sites after the addition of the nuclear extract, and were decreased in the presence of excess unlabeled probe competitors. These results indicated that both sites were involved in the regulation of Mc3r expression in anterior pituitary cells. Estradiol-17β treatment increased the Mc3r promoter activity, indicating that the gene is regulated by estradiol-17β. In conclusion, we have demonstrated the minimum promoter region required for Mc3r expression, and identified two binding sites for AP-1 and ATF4 and in the 5' upstream-flanking region of Mc3r that are essential for Mc3r expression.

DOI： 10.1016/j.gene.2015.02.043

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Insulin-like growth factor 1 (IGF-1) is involved in regulations of reproductive functions in rats and mice. IGF-1 expression is regulated by estrogen in several reproductive organs including the uterus and ovary. Two types of estrogen receptor (ER alpha and ER beta) are expressed in mouse uteri and ovaries, and it is unclear whether they differently mediate IGF-1 gene transcription. To clarify the roles of ER alpha and ER beta, mouse endometrial stromal cells and ovarian granulosa cells were treated with ligands specific for individual estrogen receptors. In endometrial stromal cells, propyl-pyrazole-triol (PPT; ER alpha-selective agonist) increased Igf1 mRNA expression, which was suppressed by methyl-piperidino-pyrazole (MPP, ER alpha-selective antagonist), while diarylpropionitrile (DPN, ER beta-potency selective agonist) increased Igf1 mRNA expression, which was inhibited by MPP but not by 4[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-alpha]pyrimidin-3-yl]phenol (PHTPP; ER beta antagonist). PHTPP enhanced the DPN-induced increase in Igf1 mRNA expression. In ovarian granulosa cells, E2 and DPN decreased Igfl mRNA expression, whereas PPT did not affect Igfl mRNA levels. In these cells, PHTPP inhibited the DPN-induced decrease in Igf1 mRNA expression. These results suggest that ERa facilitates le transcription, whereas ER beta appears to inhibit Igfl gene transcription in mouse endometrial stromal cells and ovarian granulosa cells.

DOI： 10.1262/jrd.2013-085

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of beta-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BISK1). The BISK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BISK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BISK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2014.03.027

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Reproductive functions decline with the onset of diabetes in female mice. Diabetic mice have smaller uteri with an underdeveloped endometrium, suggesting diminished estrogen-induced growth. We aimed to clarify the changes in the estrous cycle and in insulin-like growth factor 1 (IGF1) expression in the uteri of streptozotocin (STZ)-treated diabetic mice, because IGF1 is one of the main growth factors involved in estrogen-induced uterine growth. ICR female mice were intraperitoneally administered STZ (10 mg/100 g BW), and blood glucose levels were determined. Mice with blood glucose levels > 200 mg/dl were classified as diabetic mice. The onset of diabetes was associated with acyclic estrous cycles. Diabetes was also induced with STZ in ovariectomized mice. Uterine Igf1 mRNA levels were reduced in ovariectomized STZ-treated diabetic mice. Estrogen is known to stimulate Igf1 mRNA expression in the uterus, but estrogen action was abolished in the uteri of STZ-treated diabetic mice. mRNA expressions of estrogen receptor α (ERα) and steroid hormone receptor coactivators (SRC-1/Ncoa1, SRC-2/Ncoa2, SRC-3/Ncoa3 and CBP/p300/Crebbp) were reduced in the uteri of ovariectomized STZ-treated diabetic mice. The present study demonstrates that diabetes induces a decline in female reproductive functions in mice. Igf1 expression in ovariectomized diabetic female mice was decreased, and decreased responsiveness to estrogen in the uteri of diabetic mice is probably associated with a reduction in ERα and steroid receptor coactivator mRNA expression. © 2013 by the Society for Reproduction and Development.

DOI： 10.1262/jrd.2012-169

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Brilliant plumage is typical of male birds, thus sexual plumage dichromatism is seen in many avian species; however, the molecular mechanism underlying this remains unclear. The agouti signaling protein (ASIP) is a paracrine factor that stimulates yellow/red pigment (pheomelanin) synthesis and inhibits black/brown pigment (eumelanin) synthesis in follicular melanocytes. In mammals, the distal promoter of the ASIP gene acts exclusively on the ventral side of the body to create a countershading pigmentation pattern by stimulating pheomelanin synthesis in the ventrum. Here, we examined the role of the distal ASIP promoter in controlling estrogen-dependent sexual dichromatism in chickens. Reverse-transcription polymerase chain reaction analyses revealed that ASIP class 1 mRNAs transcribed by the distal promoter were expressed exclusively on the ventral side of chicks and adult females displaying countershading. In showy adult males, the ASIP class 1 mRNAs were expressed in gold-colored ornamental feathers grown on the back. In the presence of estrogen, males molted into female-like plumage and ASIP class 1 mRNAs expression was altered to female patterns. These results suggest that the distal ASIP promoter produces countershading in chicks and adult females, similar to the ventral-specific ASIP promoter in mammals. In addition, the class 1 promoter plays an important role for creating sexual plumage dichromatism controlled by estrogen. This is the first evidence for a pigmentation gene having been modified in its expression during evolution to develop phenotypic diversity between individuals of different sexes. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2012.04.016

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Hair and feather pigmentation is mainly determined by the distribution of two kinds of melanin, eumelanin and pheomelanin, which produce brown to black and yellow to red colorations, respectively. The agouti signaling protein (ASIP) acts as an antagonist or an inverse agonist of the melanocortin 1 receptor (MC1R), a G protein-coupled receptor for alpha-melanocyte-stimulating hormone (alpha-MSH). This antagonism of the MC1R by ASIP on melanocytes initiates a switch of melanin synthesis from eumelanogenesis to pheomelanogenesis in mammals. In the present study, we isolated multiple ASIP mRNA variants generated by alternative splicing and promoters in chicken feather follicles. The mRNA variants showed a discrete tissue distribution. However, mRNAs were expressed predominantly in the feather pulp of follicles. Paralleling mRNA distribution, ASIP immunoreactivity was observed in feather pulp. Interestingly, ASIP was stained with pheomelanin but not eumelanin in pulp areas that face developing barbs. We suggest that the elaborate color pattern of individual feathers is formed in part by the antagonistic action of ASIP that is produced by multiple mRNA variants in chicken feather follicles. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2011.12.009

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Pit-1 is a POU-domain transcription factor that promotes growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone β subunit (TSHβ) gene expression in the pituitary gland. Alternative splicing of Pit-1 gene transcripts has been shown to give rise to several variants with discrete transactivation properties. Recently, we identified a mouse Pit-1w that is generated by alternative promoter usage and is expressed in a variety of tissues including the testis. Using a combination of reverse-transcription polymerase chain reaction analyses and luciferase reporter gene assays, we investigated the possible role of Pit-1w in the mouse testis. In postnatal testicular development, the expression of Pit-1w mRNA was significantly up-regulated between 18 and 20. days after birth when the numbers of secondary spermatocytes and spermatids have been reported to increase in mice. The PRL mRNA, but not the mRNAs for GH or TSHβ, showed intratesticular expression patterns that were similar to those of the Pit-1w mRNA. In experimental unilaterally cryptorchid testes of adult mice, spermatid numbers were extremely low and the expression levels of both the Pit-1w and PRL mRNAs dropped dramatically. Furthermore, in the luciferase reporter gene assays, we found that Pit-1w specifically transactivated the PRL promoter but had no effect on the promoters of GH or TSHβ. These results suggested that Pit-1w could be involved in the paracrine/autocrine system in mice and may be necessary for normal testicular function via its possible role in regulating PRL expression in testicular germ cells. This is the first report demonstrating the possible role of Pit-1w in mammals. © 2012 Elsevier Inc.

DOI： 10.1016/j.ygcen.2012.05.004

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Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGF on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17β (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription. © 2012 Zoological Society of Japan.

DOI： 10.2108/zsj.29.377

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Runx3 is a transcription factor that belongs to the Runx family. We studied the localization of Runx3 mRNA in the mouse uterus, and its function in the mouse endometrium using Runx3 knockout (Runx3-/-) mice. Runx3 mRNA was detected in the endometrial luminal epithelial cells, glandular epithelial cells and stromal cells below the epithelial cell layer on the luminal side. The uteri of Runx3-/- mice were smaller than those of wt mice. The endometrial layer and uterine glands of Runx3-/- mice were less developed than those of wild-type mice, and the endometrial stromal layer was thinner. Transforming growth factor β1 and β3 (TGFβ1 and β3) mRNA levels in endometrial stromal cells of Runx3-/- mice were low compared with those of wild-type mice. Estradiol-17β (E2) increased Tgfb2 mRNA levels in endometrial stromal cells of Runx3-/- mice, but not in those of wild-type mice. E2 increased epidermal growth factor (EGF) mRNA levels in endometrial stromal cells of wild-type mice, but did not increase those of Runx3-/- mice. The diminished Tgfb1 and Tgfb3 mRNA expressions may lead to the reduced proliferation of endometrial stromal cells. Alterations of E2-associated expressions of Tgfb2 and Egf mRNA in endometrial stromal cells of Runx3-/- mice may be associated with suppression of E2-dependent endometrial epithelial cell proliferation in Runx3-/- mice. Thus, Runx3 is likely to be a regulatory factor responsible for endometrial growth. © 2012 by the Society for Reproduction and Development.

DOI： 10.1262/jrd.2012-066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

In our laboratory, a single autosomal recessive mutation in a phenotype similar to ruby-eye (ru/Hps6(ru)) or ruby-eye 2 (ru2/Hps5(ru2)) spontaneously occurred in siblings of C57BL/10JHir (+/+, black) mice in 2006. RT-PCR analysis revealed that this novel mutation, named ru2(d)/Hps5(ru2-d), exhibited frameshift by 997G deletion in the Hps5 gene. To clarify the mechanism of the hypopigmentation, the characteristics of proliferation and differentiation of ru2(d)/ru2(d) epidermal melanoblasts and melanocytes cultured in a serum-free medium were investigated. The proliferation of ru2(d)/ru2(d) melanoblasts and melanocytes did not differ from that of +/+ melanoblasts and melanocytes. However, the differentiation of ru2(d)/ru2(d) melanocytes was greatly inhibited. Tyrosinase (TYR) activity, expression of TYR, TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT), eumelanin synthesis, and the number of stage IV melanosomes markedly decreased in ru2(d)/ru2(d) melanocytes. However, excess L-tyrosine (Tyr) added to culture media from initiation of the primary culture rescued the reduced differentiation through increase in TYR activity, expression of TYR, TRP1, TRP2 and Kit, eumelanin synthesis, and stage IV melanosomes. L-Tyr injected into ru2(d)/ru2(d) mice also stimulated melanocyte differentiation. These results suggest that the ru2(d) allele inhibits melanocyte differentiation, and that its impaired differentiation is rescued by excess Tyr.

DOI： 10.2108/zsj.28.790

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Alternative splicing of Pit-1 gene transcripts has been shown to give rise to several variants with discrete transactivation properties: however, those arising from alternative promoters such as avian Pit-1w have not yet been identified in mammals. Here, comparative genomics analysis followed by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of 5' cDNA ends (5'RACE) were used in identifying Pit-1w mRNA in the mouse pituitary. The mouse Pit-1w mRNA is generated by using an alternative promoter located in the first intron, as with chicken Pit-1w, and is expressed in a wide variety of tissues besides the pituitary. In the testis, Pit-1w is expressed as the predominant variant and a protein of 33 kDa. During the first wave of spermatogenesis, expression of Pit-1w mRNA at substantial levels was observed from 3 weeks, but not at 1 or 2 weeks after birth. A combination of immunohistochemistry and in situ hybridization detected Pit-1 mRNA and Pit-1 immunoreactivity in the spermatogonia, spermatocytes, and spermatids in the testis of adult mice. Because secondary spermatocytes and haploid spermatids increase in number between 18 and 20 days after birth in mice, it is possible that mouse Pit-1w plays a role in spermatogenesis. This is the first report demonstrating the expression of Pit-1 variants arising from alternative promoters in mammals. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2011.06.016

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In the present study, we expressed chicken (ch) Pit-1 alpha (chPit-1 alpha) and chPit-1 gamma in vitro to compare the roles of chPit-1s in the transcription of the chicken growth hormone (chGH) gene. Both green fluorescence protein (GFP)-fused chPit-1 gamma and GFP-fused chPit-1 alpha were localized in the nuclei of COS-7 cells. In a luciferase reporter gene assay, both chPit-1 alpha and chPit-1 gamma transactivated the chGH promoter, and chPit-1 alpha showed a more potent effect than chPit-1 gamma. On the other hand, an increase of cellular cAMP induced by forskolin promoted transactivation of the chGH gene with chPit-1 alpha and chPit-1 gamma to similar extents. These results suggest that chPit-1 gamma may modulate the basal promoter activity of the chGH gene to the same degree as chPit-1 alpha; however, a structural difference observed at the N-terminus transactivation domains in chPit-1 alpha and chPit-1 gamma could be associated with the efficiency of basal activation of the chGH promoter. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2011.06.007

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Feather coloration in chickens mainly depends on melanin produced by melanocytes located in the feather follicles. The melanocortin 1 receptor (MC1R) on follicular melanocytes regulates melanin synthesis; however, the source of the melanocortins that interact with the receptors remains unclear. In this study, we examine the potential expression of melanocortins and characterize the mRNAs for the precursor pro-opiomelanocortin (POMC) in chicken feather follicles. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of mRNAs for POMC, prohormone convertase 1 (PC1) and PC2, and western blotting detected adrenocorticotropic hormone (ACTH)-related products of POMC processing in feather follicles, suggesting that melanocortins are produced locally in the tissues of chickens. A combination of 5'RACE (rapid amplification of cDNA 5' end), 3'RACE and RT-PCR analyzes identified two classes of POMC mRNA, class a and class b, which encode the same full-length POMC protein but have different non-coding leader exons. Class a mRNAs were expressed specifically in feather follicles, whereas class b mRNAs were expressed in the pituitary, hypothalamus, and various peripheral tissues that we examined. Within the feather follicles, the class a mRNAs were distributed in epidermal layers from middle to distal locations, whereas the class b mRNAs were mainly expressed in pulp at proximal locations. Our findings suggest that feather pigmentation is regulated by locally produced melanocortins, and indicate that the melanocortins encoded by the different classes of POMC mRNAs may play different intra-follicular roles in chickens. This is the first report that demonstrates alternative promoter usage generating different full-length POMC mRNAs in vertebrates. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2010.12.018

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Prolactin (PRL)-releasing peptides (PrRP) have been identified in mammals, amphibians and fishes, and these animals have several PrRPs that consist of different numbers of amino acids such as 20, 31 and 37. In the present study, we identified the cDNA encoding chicken prepro-PrRP, which can generate putative PrRPs, and cloned and sequenced it. Sequences for the coding region suggested the occurrence of putative PrRPs of 20, 31 and 32 amino acid residues. The amino acid sequence of chicken PrRP20 showed 100%, 95% and 70% identity with those of PrRP20s from teleosts, Xenopus laevis and mammals, respectively. On the other hand, chicken PrRP31 showed approximately 90% and 52-55% homology to PrRP31s of X. laevis and mammals, respectively. Native chicken PrRPs were purified from an acid extract of chick brain by a Sep-Pak C18 cartridge (Waters Corp., Milford, MA, USA), affinity chromatography using anti-salmon PrRP serum, and reverse phase high-performance liquid chromatography (HPLC) on an ODS-120T column (TOSOH, Tokyo, Japan). The existence of chicken PrRP20 and PrRP31 in the brain was demonstrated by comparing them with the synthetic peptides using HPLC and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Chicken PrRP31 increased plasma PRL concentration when administered peripherally, whereas central administration decreased the concentration, suggesting that chicken PrRP31 has a distinct effect on PRL secretion between tissues in chicks. On the other hand, plasma growth hormone concentration decreased with both peripheral and central administrations of chicken PrRP31. Furthermore, central administration of chicken PrRP31 increased food intake in chicks compared to those observed in mammals and fishes. Taken together with the results indicating that chicken PrRP20 did not show endocrine and behavioural effects, we showed that chicken PrRP has a similar amino acid sequence to teleosts, Xenopus laevis and mammals, although the actions were variable among vertebrates. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2826.2010.02078.x

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The agouti signaling protein (ASIP)/melanocortin signaling system regulates lipid metabolism in humans but not in mice. To examine whether the system is present in adipose tissues in chickens, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using mRNAs extracted from subcutaneous and visceral adipose tissues. This revealed that mRNAs for melanocortin 1receptor (MC1R), MC5R, ASIP, pro-opiomelanocortin (POMC), prohormone convertase 1 (PC1) and PC2 were expressed in both adipose tissues, suggesting the presence of a local ASIP/melanocortin signaling system in these tissues in chickens. To investigate the potential biological role of this system, the adipose tissue mRNA expression of ASIP and POMC was examined under food-deprived conditions. Forty-eight-hour fasting significantly decreased body weight and reduced the cell size in adipose tissues. In addition, the plasma levels of free fatty acids were increased while those of glucose, triacylglycerols and insulin were decreased, suggesting the activation of lipolysis and gluconeogenesis in the fasted chickens. Under this condition, the mRNA expression of ASIP and POMC was decreased and increased, respectively, in subcutaneous and visceral adipose tissues. ASIP and melanocortins are lipogenic and lipolytic hormones, respectively. Therefore, these findings suggest that lipid metabolism in the adipose tissues is regulated by a local ASIP/melanocortin signaling system in chickens. This is the first report demonstrating changes in the expression of ASIP mRNA in adipose tissues in the negative-energy state in vertebrates. © 2010, Japan Poultry Science Association.

DOI： 10.2141/jpsa.009110

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DOI： 10.3330/hikakuseiriseika.26.3

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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-17p (E2) decreased the Igfbp3 mRNA level in endometrial stromal cells, whereas it increased the Igf1 mRNA level. Transforming growth factor a (TGFa) significantly decreased IGFBP3 expression at both the mRNA and secreted protein levels in endometrial stromal ceils. Progesterone (P4) 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 TGFα affects IGF1 actions through modulation of IGFBP3 levels. © 2009 Zoological Society of Japan.

DOI： 10.2108/zsj.26.131

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Runx3 is a transcription factor that belongs to the Runx family. We studied the function of Runx3 in the mouse ovary and uterus using the Runx3 knockout (Runx3-/-) mouse. Ovaries were collected from 8-week-old wild type (wt) and Runx3-/- mice. Histological studies showed that follicles were present at various developmental stages in the Runx3-/- and wt mouse ovaries. The numbers of primary, preantral and antral follicles in the Runx3-/- mice were significantly less than those in the wt mice while the number of primordial follicles in the Runx3-/- mice was not significantly different from that in the wt mice. Corpora lutea were not detected in the Runx3-/- mouse ovary. Gonadotropin treatment in immature female mice induced ovulation in Runx3-/- mice as well as in wt mice, indicating that ovaries of Runx3-/- mice respond to gonadotropin treatment as those in wt mouse ovaries. This suggests that failure of ovulation is due to dysfunction of regulatory mechanism of gonadotropin secretion. In addition, the uteri of Runx3-/- mice were atrophic, showed thin epithelial layers compared with those of the wt mice, and did not respond to estrogen in terms of DNA replication in endometrial epithelial cells. These results suggest that Runx3 takes part in the regulation of reproductive functions. © 2008 Wiley-Liss, Inc.

DOI： 10.1002/mrd.20904

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We previously described the cloning of the full-length chicken thyrotropin receptor (TSHRa) and two splice variants, lacking exon 3 (TSHRb) or both exons 2 and 3 (TSHRc). Here we report the identification of three novel splice variants of the chicken TSHR, named TSHRd, -e and -f, differing in their C-terminal region and containing unique exonic sequences that are not present in the other TSHR variants. This finding suggests a TSHR gene structure with 13 rather than the previously assumed 10 exons. The three novel exons appear to be chicken-specific, as no equivalents of these exons were found in other vertebrate genomes. Like the full-length receptor, the five TSHR splice variants are most abundantly expressed in thyroid gland. TSHRb, -d, -e and -f mRNA was also present in virtually all extra-thyroidal tissues expressing TSHRa, whereas TSHRc shows a more restricted tissue distribution. Whether these receptor transcripts are translated to functional proteins needs to be verified, but if so, they could be attributed various physiological roles. © 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2008.03.003

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Tpit/Pitx-responsive element (Tpit/PitxR-E), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/ PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxR-E markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmoduhn-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRH-induced activation of POMC gene expression in a calcium-dependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/ PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression. © 2007 Society for Endocrinology.

DOI： 10.1677/JOE-06-0143

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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. © 2007 Zoological Society of Japan.

DOI： 10.2108/zsj.24.241

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Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) α subunit was analyzed. IL-18Rα mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Rα riboprobe demonstrated that IL-18Rα mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Rα mRNA expression level changed with the estrous cycle. The uterine IL-18Rα mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Rα mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17 β or progesterone. These findings suggest that IL-18Rα gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.

DOI： 10.1262/jrd.18036

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TSH and the interaction with its receptor (TSHR) in the thyroid gland play a crucial role in the pituitary-thyroid axis of all vertebrates. Released upon stimulation by TSH, thyroid hormones influence numerous processes in the body and are extremely important during the last week of chicken embryonic development. In this study, we have cloned and functionally characterized the chicken TSHR (cTSHR), which was found to be a G protein-coupled receptor consisting of 10 exons. Besides the full-length cDNA, we detected two splice variants lacking either exon 3, or exons 2 and 3, both part of the extracellular domain of the receptor. Bovine TSH increased intracellular cAMP levels in HEK-239 cells transiently expressing the full-length cTSHR (EC50 = 1.43 nM). In situ hybridization showed the expression of cTSHR mRNA in the thyroidal follicular cells. cTSHR mRNA expression, as determined by real-time PCR, was also found in several other tissues such as brain, pituitary, pineal gland, and retina, suggesting that the TSH-TSHR interaction is not only important in regulating thyroid function. TSHR mRNA expression in the thyroid gland did not change significantly during the last week of embryonic development, which suggests that an increased thyroidal sensitivity is not part of the cause of the concomitant increasing T4 levels. Copyright © 2006 by The Endocrine Society.

DOI： 10.1210/en.2005-1223

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.3312/jyio.38.40

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The mammalian melanocortin system has been established as a crucial regulatory component in an extraordinarily diverse number of physiological functions. In contrast, comparatively little is known about the avian melanocortin system: interest in the physiological role of alpha-MSH in birds has been limited by the fact that birds lack the intermediate lobe of the pituitary, the main source of circulating alpha-MSH in most vertebrates. Recently, however, the main avian melanocortin system genes, including POMC, AGRP, and all the melanocortin receptors, have been cloned and their physiological roles are the beginning to be elucidated. This review outlines our improved understanding of the avian melanocortin system, particularly in relation to two of the most widely studied physiological functions of the melanocortin system in mammals, the regulation of pigmentation and energy homeostasis. The data reviewed here indicate that the melanocortin system has been strongly conserved during vertebrate evolution and that alpha-MSH is produced locally in birds to act as an autocrine/paracrine hormone. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2004.11.039

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type 1) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10(-7) M of the MAP kinase inhibitor PD098059 and 10(-5) M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10(-5) M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial TGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.

DOI： 10.1262/jrd.16076

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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-17β (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. © 2005 Zoological Society of Japan.

DOI： 10.2108/zsj.22.1011

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Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-γ inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and α 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-17β (E2) 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 E2-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. © 2005 Zoological Society of Japan.

DOI： 10.2108/zsj.22.1003

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Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17β(E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/ mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Rα mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.

DOI： 10.1262/jrd.17029

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Alpha-melanocyte stimulating hormone (alpha-MSH) added to serum-free primary culture of melanoblasts derived from epidermal cell suspensions of 0.5 d old C57BL/10JHir mice induced their differentiation. Analysis using the reverse transcription-polymerase chain reaction showed that the expression of the melanocyte-specific alpha-MSH receptor gene, melanocortin receptor-1 (MC1-R), had already been initiated before addition of alpha-MSH, and, in addition, no up-regulation of the MC1-R gene was observed after addition of alpha-MSH. However, no expression of the proopiomelanocortin (POMC) gene was observed before or after the addition of alpha-MSH. The expression of the MC1-R and POMC genes in the epidermis and dermis of the dorsal skin was surveyed from 13 d old embryos to 5.5 d old neonates. The expression of the MC1-R gene was first observed in the epidermis of 13 d old embryos, and gradually increased up to 0.5 d after birth, and thereafter remained constant. By contrast, the expression of the MC1-R gene in the dermis was first observed in 16 d old embryos, and gradually increased up to 3.5 d after birth, and thereafter remained constant. However, no expression of the POMC gene was observed in the epidermis or dermis of the dorsal skin at any age of mice tested. These results suggest that the expression of the MC1-R gene, but not of the POMC gene, plays an important role in the regulation of melanocyte differentiation in mouse skin.

DOI： 10.1111/j.1600-0749.2004.00179.x

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1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

DOI： 10.1038/sj.bjp.0705900

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The present study was designed to clarify the role of the agouti gene in the regulation of the proliferation and differentiation of mouse epidermal melanocytes using serum-free primary culture of epidermal melanocytes from 0.5-d-old black (a/a; C57BL/10JHir) mice and congenic, agouti (A/A; C57BL/10JHir-A/A) mice. There was no significant difference in the proliferation or differentiation of melanocytes between a/a and A/A mice. However, the content of pheomelanin in culture media from A/A melanocytes was increased by L-tyrosine compared with a/a melanocytes. In addition, the content of the pheomelanin precursor, 5-S-cysteinyldopa, in culture media from A/A melanocytes was dramatically increased by L-tyrosine. Moreover, pheomelanin content in the epidermis from 3.5- and 5.5-d-old A/A mice was much higher than in a/a mice. Analysis of the A gene using reverse transcription-polymerase chain reaction revealed that cultured keratinocytes and melanocytes do not express the A gene. Moreover, the A gene was expressed in the A/A dermis of 0.5-, 3.5- and 5.5-d-old mice, but not in the a/a dermis nor in the A/A or a/a epidermis. These results suggest that A/A epidermal melanoblasts are influenced by the A gene from the dermis of neonatal mice, and are capable of synthesizing pheomelanin in the culture. Pheomelanin production in the epidermis from 3.5- and 5.5-d-old A/A mice may be induced by the expression of the agouti gene in the dermis.

DOI： 10.1111/j.1600-0749.2004.00176.x

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Alpha-melanocyte-stimulating hormone (alpha-MSH) stimulates prolactin (PRL) release and mammotrope proliferation in mouse anterior pituitary glands. The present study investigated the regulation of melanocortin-3 receptor (MC3-R) mRNA levels in mice using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Relative MC3-R mRNA levels in the anterior lobes of female mice increased between 20 and 45 days of age, and a significant difference in MC3-R mRNA expression between sexes was seen at 45 days. Ovariectomy decreased MC3-R mRNA expression in the female anterior lobes, and estradiol-17beta (E(2)) treatment increased MC3-R mRNA levels in ovariectomized mouse anterior lobes and cultured anterior pituitary cells. E(2) treatment increased proopiomelanocortin (POMC) mRNA levels in ovariectomized mouse neurointermediate lobes and cultured neurointermediate pituitary cells. On the other hand, E(2) treatment did not affect POMC mRNA expression in mouse anterior lobes or cultured anterior pituitary cells. These results suggest that alpha-MSH directly stimulates PRL release and mammotrope proliferation through MC3-Rs expressed in mammotropes, while estrogen stimulates MC3-R gene transcription in the anterior pituitary cells and POMC gene transcription in the intermediate lobes. In lactating mice, POMC mRNA levels in the neurointermediate lobes were elevated compared with in non-lactating mice. The present study suggests that alpha-MSH is involved in augmented PRL secretion by estrogen and during lactation.

DOI： 10.1159/000082355

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The intermediate lobe of rodent pituitaries is involved in the regulation of prolactin (PRL) secretion from the anterior lobe. In a previous study, we demonstrated the stimulatory effect of a-melanocyte-stimulating hormone (alpha-MSH) on PRL release and the expression of melanocortin-3 receptors (MC3-Rs) in cultured mouse pituitary cells. The aim of the present study was to clarify whether a-MSH directly stimulates PRL release through the MC3-Rs by determining the cell type of MC3-R-expressing cells in the mouse pituitary anterior lobe. Northern blot analysis revealed a 2.7-kb transcript for MC3-R mRNA in the anterior and neurointermediate lobes of pituitary glands of adult male and female mice. Dual cellular localization of MC3-R mRNA and PRL or growth hormone (GH) in the mouse pituitary glands was performed by in situ hybridization analysis of MC3-R mRNA followed by immunocytochemical detection of PRL or GH. MC3-R mRNA was detected in most mammotropes and some somatotropes. alpha-MSH increased PRL release and stimulated DNA replication in mammotropes, and these effects were blocked by SHU9119, an antagonist of MC3-R and MC4-R. These results indicate that a-MSH stimulates PRL release and proliferation of mammotropes through MC3-Rs, and suggest that a-MSH from intermediate lobes can regulate mammotrope functions in the mouse pituitary. Copyright (C) 2003 S. Karger AG, Basel.

DOI： 10.1159/000071965

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC ENDOCRINOLOGY  

IGF-I is expressed in somatotrophs, and IGF-I receptors are expressed in most somatotrophs and some corticotrophs in the mouse pituitary gland. Our recent study demonstrated that IGF-I stimulates the proliferation of corticotrophs in the mouse pituitary. These results suggested that somatotrophs regulate corticotrophic functions as well as somatotrophic functions by the mediation of IGF-I molecules. The present study aimed to clarify factors regulating pituitary IGF-I expression and also the roles exerted by IGF-I within the mouse anterior pituitary gland. Mouse anterior pituitary cells were isolated and cultured under serum-free conditions. GH (0.5 or 1 mug/ml), ACTH (10(-8) or 10(-7) M), GH-releasing hormone (GHRH; 10(-8) or 10(-7) M), dexamethasone (DEX; 10(-8) or 10(-7) M) and estradiol-17beta (E2; 10(-11) or 10(-9) M) were given for 24 h. IGF-I mRNA levels were measured using competitive RT-PCR, and GH and proopiomelanocortin (POMC) rnRNA levels were measured using Northern blotting analysis. GH treatment significantly increased IGF-I mRP4A levels (1.5- or 2.1-fold). ACTH treatment did not alter GH and IGF-I mRNA levels. IGF-I treatment decreased GH mRNA levels (0-7or 0.5-fold), but increased POMC mRNA levels (1.8-fold). GH treatment (4 or 8 mug/ml) for 4 days increased POMC rnRNA levels. GHRH treatment increased GH rnRNA levels (1.3-fold), but not IGF-I mRNA levels. DEX treatment significantly decreased IGF-I mRNA levels (0.8-fold). E2 treatment did not affect IGF-I mRNA levels. GH receptor mRNA, probably with GH-binding protein mRNA, was detected in somatotrophs, and some mammotrophs and gonadotrophs by in situ hybridization using GH receptor cDNA as a probe. These results suggested that IGF-I expression in somatotrophs is regulated by pituitary GH, and that IGF-I suppresses GH expression and stimulates POMC expression at the transcription level. Pituitary IGF-I produced in somatotrophs is probably involved in the regulation of somatotroph and corticotroph functions.

DOI： 10.1677/joe.0.1780071

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The melanocortin receptors (MCR) belong to the superfamily of G-protein-coupled receptors that participate in both peripheral and central functions, including regulation of energy balance. Genomic clones of the five chicken (GGA) MCRs were isolated and used to find the chromosomal location of each of the loci. The genes encoding MC2R and MC5R mapped to the middle part of the long arm of chromosome 2 (GGA2q22-q26) and MC4R proximally on the same chromosome arm, close to the centromere (2q12). This arrangement seems to be conserved on chromosome 18 in the human (HSA18). The MC1R and MC3R genes mapped to different microchromosomes that also appear to share homology with the respective human localization. The conserved synteny of the MC2R, MC5R, and MC4R cluster in chicken (GGA2), human (HSA18), and other mammals suggests that this cluster is ancient and was formed by local gene duplications that most likely occurred early in vertebrate evolution. Analysis of conserved synteny with mammalian genomes and paralogon segments prompted us to predict an ancestral gene organization that may explain how this family was formed through both local duplication and tetraploidization processes. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0888-7543(03)00028-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.20.639

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e(+R), e(+B&D), e(wh)/e(y), E-Rfayoumi gave receptors to which melanocortin stimulating hormone (alpha-MSH) bound with similar IC50 values and responded to alpha-MSH by increasing intracellular cAMP levels in a dose-dependent manner. Three of the cMC1 receptors; e(b), E and E-R, did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E-R alleles are associated with black feather colour in chicken while the e (b) allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the 'E92K switch' mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.

DOI： 10.1046/j.1432-1033.2003.03506.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.20.83

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：NEW YORK ACAD SCIENCES  

The interest in the physiological role of alpha-MSH in birds has been limited because they lack the intermediate lobe of the pituitary, the main source of circulating alpha-MSH in most vertebrates. Recent studies have improved our understanding of the avian melanocortin system. We have cloned and characterized all five MC-R subtypes, POMC, and AGRP in chicken. Analyses of the tissue distribution of expression of these genes revealed widespread expression throughout the body, corresponding to the situation in mammals in which alpha-MSH exerts a multiplicity of effects in different tissues by acting as a local mediator. We showed that the extended black locus controlling feather pigmentation in the chicken encodes MC1-R. Moreover, black chickens carrying the dominant allele, the extended black, express the MC1-R with ligand-independent activity as the somber-3J black mice. alpha-MSH and AGRP were expressed in the infundibular nucleus of POMC and NPY neurons, respectively, in the brain of Japanese quail. Furthermore, fasting stimulated AGRP expression and lowered POMC expression. These data indicate that at least two of the major melanocortin systems reported in mammals, that is, regulation of pigmentation and energy homeostasis, was developed in a common ancestor to chicken and mammals at least 300 million years ago. Furthermore, alpha-MSH peptide was identified in developing chicken eye, suggesting a possible involvement of alpha-MSH in regulation of ocular development. Collectively, the data reviewed here indicate that alpha-MSH is produced locally and acts as an antocrine/paracrine hormone in birds.

DOI： 10.1111/j.1749-6632.2003.tb03201.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Research in mammals has established the existence of a neuronal network that lies within the hypothalamus and that regulates energy homeostasis. However, it is unknown whether this system has been evolutionarily conserved. The objective of the present study was therefore to examine the influence of the agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), prepro-orexin, and vasoactive intestinal polypeptide (VIP) genes on energy balance in birds by quantifying the effect of a 24-h fast on their expression in the hypothalamus of the Japanese quail. In situ hybridization revealed strong signals for AGRP and POMC mRNAs in the infundibular nucleus (IN), for prepro-orexin in the lateral hypothalamic area (LHy) and periventricular hypothalamic nucleus, and for VIP in the LHy. POMC mRNA was co-localized with alpha-melanocyte-stimulating hormone-like immunoreactivity in individual IN neurons. Compared with the ad-libitum-fed state, a 24-h fast resulted in a 2.2-fold increased expression of AGRP mRNA in the IN. However, fasting did not induce changes in POMC, prepro-orexin, or VIP mRNAs. The results suggest an involvement of the central melanocortin system in the regulation of energy balance in birds, as in mammals. In contrast, orexins in birds may be primarily involved in the control of physiological functions other than energy homeostasis.

DOI： 10.1007/s00441-003-0755-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.19.789

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The neural mechanisms involved in the compensatory hyperphagia exhibited by many vertebrate species after a fast are not fully understood but, in mammals, appear to involve nutritionally-sensitive neurons that co-express neuropeptide Y (NPY) and agouti-related protein (AGRP) in the infundibular hypothalamus. We investigated whether these neurons have been evolutionarily conserved in a non-mammalian vertebrate, the Japanese quail. Birds exhibited compensatory hyperphagia I h after return of food following a 24-h fast. We addressed a potential regulatory role for NPY, first, by using in situ hybridisation (ISH) to map NPY gene expression in the hypothalamus. This revealed a strong signal in the infundibular nucleus (IN). Secondly, we quantified NPY gene expression in 24-h fasted birds compared to ad libitum fed controls using two independent methods. In whole hypothalamus, measured by ribonuclease protection assay, NPY mRNA increased 1.5-fold in fasted birds. A similar, 1.7-fold, increase was observed specifically in the IN when analysed by ISH. No differences in NPY expression between fed and fasted birds were observed in other brain regions. To determine whether NPY neurons in the avian IN co-express AGRP, we cloned a fragment of the quail AGRP gene and used it to localise AGRP mRNA by ISH. The gene was expressed exclusively in the hypothalamus, specifically in the IN, where its distribution matched that of NPY. Double-label ISH revealed that the majority of NPY neurons in the IN co-express AGRP mRNA. Collectively, these data indicate that this cell type has been neuroanatomically and functionally conserved during vertebrate evolution. (C) 2002 Elsevier Science BY All rights reserved.

DOI： 10.1016/S0169-328X(02)00145-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOCIETY REPRODUCTION & DEVELOPMENT-SRD  

Active angiogenesis occurs during early luteal development. Angiogenesis in the corpus luteum (CL) is regulated by various growth factors in many species. The purpose of the present study was to determine the effect of hepatocyte growth factor (HGF) on the proliferation of the microvascular endothelial cells derived from developing bovine CL. The expression of HGF and HGF receptor (c-met) mRNAs in cultured bovine endothelial cells was also observed by reverse transcription-polymerase chain reaction analysis. The cells were exposed to HGF (50 ng/ml) for 1, 2, 4, and 6 days. HGF significantly increased the total DNA in endothelial cells at all exposure times (P<0.05). When the endothelial cells were exposed to HGF (1-50 ng/ml) for 4 days, total DNA was increased in a dose-dependent manner (P<0.05). Moreover, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) also increased the total DNA. However, when the endothelial cells were simultaneously exposed to HGF (50 ng/ml) with bFGF (50 ng/ml) or VEGF (50 ng/ml), the effect of HGF was not augmented. Furthermore, the mRNA expressions of both HGF and c-met were determined in the endothelial cells. The overall results suggest that HGF is involved in luteal angiogenesis by stimulating proliferation of endothelial cells in cattle.

DOI： 10.1262/jrd.48.49

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Authorship：Last author   Language：English   Publishing type：Research paper (international conference proceedings)  

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor α (TGF-α) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-α (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-α binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17β (E2, 10-9M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-α antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-α mRNA and growth hormone by in situ hybridization and fluorescenceimmunocytochemistry demonstrated that TGF-α mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-α-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-α produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.

DOI： 10.1076/apab.110.1.34.895

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

GH is considered to play a role in the pathogenesis of diabetic retinopathy, causing neovascularization in the retina. The present study was conducted to assess the possibility that GH may play a role in ocular development by determining whether GH is expressed in the eye of the chicken during development. In the 17-d-old embryo, immunocytochemistry detected immunoreactive GH in retinal pigment epithelial (RPE) cells. Characterization of GH mRNA expressed in the eye by RT-PCR and rapid amplification of cDNA 5'-ends revealed it to be a novel GH mRNA transcribed from the middle of the intron 3 of the chicken GH (cGH) gene. The deduced protein, designated small GH isoform (s-cGH), was a cytosolic protein of 16.5 kDa with 140 amino acid (aa) residues, lacking the signal peptide and the N-terminal 71 aa residues of 22-kDa cGH, replacing them with 20 aberrant aa residues, and identical to 22-kDa cGH for the C-terminal 120-aa residue portion. Western blotting determined the molecular size of immunoreactive GH in RPE cells to be 80-84 kDa, similar to the computed molecular mass of s-cGH/GH receptor complex. Furthermore, RT-PCR demonstrated that GH receptor mRNA, but not s-cGH mRNA, was expressed in RPE cells. These results suggest that RPE cell is one of the target cells of s-cGH in the eye. During embryonic development, the immunoreactivity for s-cGH in RPE cells was initially observed on embryonic d 10, and the staining intensity increased and peaked on embryonic d 17. By hatching, s-cGH immunoreactivity in RPE cells was gradually decreased, and it was not detectable after hatching. This ontogenetic staining pattern correlates well with the pattern of the production of alpha MSH in RPE cells. The cell type expressing s-cGH remains to be identified; however, our findings imply a possible involvement of GH in the regulation of ocular development by acting on the intraocular melanocortin system in the chicken.

DOI： 10.1210/en.142.12.5158

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2<alpha> secretion in epithelial, but not stromal cells. RT-POR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion. Mol. Reprod. Dev. 60: 472-480, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/mrd.1112

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC ENDOCRINOLOGY  

The presence and possible physiological roles of alpha -melanocyte-stimulating hormone (alpha -MSH) in the peripheral tissues of birds have not been established. By a combination of RT-PCR, immunocytochemistry and in situ hybridization, we have examined alpha -MSH expression in the eye of the chicken during development. In the 1-day-old chick, alpha -MSH was expressed in the retinal pigment epithelial (RPE) cells, and also at a lower level in the cone cells. The melanocortin receptor subtypes, CMC1, CMC4 and CMC5, were expressed in the layers of the choroid and the neural retina, but not in the RPE cells. It is probable that the RPE cells secrete alpha -MSH to exert paracrine effects on the choroid and neural retina. During embryonic development, alpha -MSH immunoreactivity in the RPE cells was initially detected at embryonic day 10, and increased in intensity as development proceeded. No cone cells were stained with anti-alpha -MSH antiserum in any of the embryonic stages tested. The immunoreactivities for two prohormone convertases, PC1 and PC2, were co-localized to the RPE cells with a pattern of staining similar to that of alpha -MSH. Despite containing alpha -MSH immunoreactivity, the RPE cells in 1-day-old chicks expressed no immunoreactivity for the endoproteases. Furthermore, in a 3-day-old chick, proopiomelanocortin mRNA was detectable by in situ hybridization only in the, photoreceptor layer and not in the RPE cells. These results suggest that the RPE cells and the cone cells are intraocular sources of alpha -MSH in the embryonic and postnatal life of the chicken respectively. Embryonic expression of alpha -MSH in the RPE cells implies a possible role for the peptide in ocular development.

DOI： 10.1677/joe.0.1680527

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.17.661

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC ENDOCRINOLOGY  

Oestrogen stimulates the proliferation of pituitary cells. The present study was designed to clarify the involvement of transforming growth factor-alpha (TGF-alpha) in the oestrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF) receptor, this action may be exerted through the EGF receptor. Oestradiol-17 beta (OE2, 10(-9) M) stimulated mammotrophic and corticotrophic cell proliferation. RG-13022, an EGF receptor inhibitor, inhibited the cell proliferation induced by EGF or OE2, showing that the EGF receptor was involved in the growth response in mammotrophs and corticotrophs. Treatment with antisense TGF-alpha oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by OE2, but treatment with antisense EGF ODN did not. RT-PCR analysis revealed that OE2 stimulated TGF-alpha mRNA and EGF receptor mRNA expression. These results indicate that TGF-alpha mediates the stimulatory effect of oestrogen on the pituitary cell proliferation in a paracrine or autocrine manner, and that EGF receptor expression is stimulated by oestrogen.

DOI： 10.1677/joe.0.1650493

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

The 5' upstream regulatory region of the mouse tyrosinase gene contains a long (GA)n sequence, that may be capable of adopting a triple-helical conformation (triplex), We analyzed protein-DNA interactions in a part of the 5' upstream region containing the (GA)n sequence by gel retardation analysis and found evidence for a cell type-specific protein(s) that bound to this region. We also found a (TC)(10) sequence about 100 bp downstream from a polyadenylation site of the gene. Examination of tyrosinase cDNAs and Northern analysis indicated that this sequence is transcribed and removed during 3' end-processing of the mRNA, Based on the hypothesis that the (TC)(10) sequence binds to the (GA)n sequence and forms an intermolecular tripler, we performed the same gel retardation assay in the presence of the 3' non-coding RNA fragments containing the (UC)(10) sequence. The probe DNA failed to interact,vith the cell type-specific protein(s), These results suggest a novel hypothesis for the regulation of the mouse tyrosinase gene, i.e. that the 3' non-coding RNA fragments of mouse tyrosinase transcripts suppress its own expression at the transcriptional level. This might occur by preventing cell type-specific protein factor(s) from binding to the regulatory cis-elements in the 5' upstream region of the gene, possibly through a tripler formation, although this hypothesis remains to be proven.

DOI： 10.1034/j.1600-0749.2000.130209.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Agouti-related protein (AGRP) is a naturally occurring antagonist of melanocortin action. It is expressed mainly in the arcuate nucleus where it plays an important role in the hypothalamic control of feeding and energy homeostasis by antagonism of central melanocortin 4 receptors in mammals. Besides in the brain, the melanocortin 4 receptor is expressed in numerous peripheral tissues in the chicken. To examine whether or not the peripheral melanocortin 4 receptor signaling could be regulated by AGRP, we cloned and localized the expression of the AGRP gene in the chicken. The chicken AGRP gene was found to encode a 154 or 165 amino acid protein, depending on the usage of two alternative translation initiation sites. The coding sequence consisted of three exons, like that of mammalian species. The C-terminal cysteine-rich region of the predicted AGRP displayed high levels of identity to mammalian counterparts (78-84%) and all 10 cysteine residues conferring functional conformation of AGRP were conserved; however, other regions showed apparently no homology, suggesting that biological activities of AGRP are located in its C-terminal region. RT-PCR analysis detected the AGRP mRNA in all tissues examined: the brain, adrenal gland, heart, liver, spleen, gonads, kidney, uropygial gland, skeletal muscle and adipose tissues. Interestingly, the skin also expressed the AGRP mRNA, where Agouti, another melanocortin receptor antagonist regulating hair pigmentation, is expressed in rodents. Most of those AGRP-expressing tissues have been demonstrated to express melanocortin 4 receptors and/or other subtypes of melanocortin receptor whose mammalian counterparts can bind AGRP. These results imply the possibility that some peripheral melanocortin systems could be regulated by the functional interaction between melanocortins and AGRP at melanocortin receptors in the chicken. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4889(00)00022-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Epidermal growth factor (EGF) produced within the pituitary gland is associated with the growth of pituitary cells in rats. The aim of the present study was to localize EGF- and EGF receptor-expressing cells, and to clarify the involvement of EGF in DNA replication in 2-month-old male mouse pituitary cells, In situ hybridization of the pituitaries of these mice demonstrated that EGF mRNA was expressed in the anterior and intermediate lobes. Within the anterior pituitary, EGF mRNA-expressing cells were medium-sized and round, and made up 40% of the total number of secretory cells. EGF receptor mRNA was only detected in anterior pituitary cells. Forty-seven percent anterior pituitary cells expressed EGF receptor mRNA, An immunocytochemical study showed that most somatotropes and some mammotropes expressed EGF mRNA, When anterior pituitary cells were enzymatically dissociated and cultured in serum-free medium, RT-PCR demonstrated both EGF mRNA and EGF receptor mRNA expression. Treatment with EGF (1 and 10 ng/ml) for 5 days stimulated DNA replication in mammotropes and corticotropes, These results indicate that the DNA replication in mammotropes and corticotropes is regulated by the paracrine and/or autocrine activity of EGF produced at least in part by these cell types themselves. Copyright (C) 2000 S. Karger AG, Basel.

DOI： 10.1159/000054532

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17 beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum-supplemented medium (control) showed that about 70% of all GH3 cells were GH-immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL-immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH-ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells.

DOI： 10.1007/s004410050021

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Somatotrophs (GH cells) were classified immunoelectron microscopically into three types mainly on the basis of the size of secretory granules in the mouse pituitary of ICR strain. Type I cells contained large secretory granules. Type II cells contained both large secretory granules and small secretory granules. Type III cells contained small secretory granules. All three types of GH cells were found from the neonatal ages to adult. The relative proportion of three types did not change with age, and no sex differences in the relative proportion of the cell types were detected. Type I cells predominate in all age groups observed. In 60-day-old male mice percentages of each type were as follows: type I 93.7 ± 0.1%, type II 5.4 ± 0.8%, and type III 0.9.0 ± 0.4% (n = 5), and in 60-day-old female mice type I 95.2 ± 0.1%, type II 2.7 ± 0.5%, and type III 2.1 ± 0.9% (n = 5). The maximum diameters of the large secretory granules increased from 7 to 60 days of age. The small secretory granules similarly increased in size in female mice, but those in male mice did not change. In the diabetic female mice of the nonobese diabetic (NOD) strains, GH cells in diabetic mice became smaller, and the number and size of secretory granules decreased, indicating diminished GH secretion. However, the relative proportion of each subtype of GH cells did not differ irrespective of the occurrence of diabetes. Copyright (C) 2000 S. Karger AG, Basel.

DOI： 10.1159/000016706

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The gene for pro-opiomelanocortin (POMC), a common precursor of melanocortins, lipotropins and beta-endorphin, was isolated in the chicken first among avian species. The chicken POMC gene was found to be a single copy gene and appeared to show the same structural organization as that of other species of different classes. The predicted POMC displayed the highest identity to Xenopus POMC(A) (60.1%), and consisted of 251 amino acid residues with nine proteolytic cleavage sites, suggesting that it could be processed to give rise to all members of the melanocortin family, including adrenocorticotropic hormone and alpha-, beta- and gamma-melanocyte-stimulating hormones, as well as the other POMC-derived peptides. RT-PCR analysis detected the POMC mRNA in the brain, adrenal gland, gonads, kidney, uropygial gland and adipose tissues, each of which has been demonstrated to express melanocortin receptors. These results suggest that melanocortins act in a paracrine and/or autocrine manner to control a variety of functions both in the brain and in the peripheral tissues in the chicken. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4889(99)00046-4

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The melanocortin 3 receptor (MC3-R) in the melanocortin receptor family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. We report here the isolation of the chicken gene for MC3-R, CMC3, displaying different tissue distribution from mammalian counterparts. The CMC3 gene was found to be a single copy gene encoding a 325 amino acid protein, sharing 75.3-76.8% identity with mammalian counterparts. When assessed by RT-PCR, the CMC3 mRNA was not detected in the brain but was exclusively expressed in adrenal glands, where Agouti-related protein/Agouti-related transcript (AGRP/ART), a newly identified endogenous antagonist of MC3-R, is expressed in mammals, raising the possibility that the CMC3 plays a role in complicated regulation of the gland function by melanocortins and AGRP/ART in the chicken. Noteworthy, MC1-R gene was found to be a quite unique member of the chicken MC-R family with regard to GC content and codon usage. It may reflect as yet unidentified evolutionary pressure operating specifically on the gene. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4889(98)00165-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

DOI： 10.1023/A:1009269830117

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Two receptor genes belonging to the melanocortin receptor (MC-R) family were isolated in the chicken, the CMC4 and CMC5, each of which is a chicken homologue of the mammalian MC4-R and MC5-R, respectively. The CMC4 encodes a 331 amino acid protein, sharing 86.4-88.1% identity with mammalian analogs, and the CMC5 encodes a 325 amino acid protein, which is 72.3-79.1% identical to mammalian counterparts. Both genes contain no intron in their coding regions and exist in the chicken genome as single copy genes. Reverse transcription-PCR analysis revealed that the CMC4 mRNA is expressed in a nide variety of peripheral tissues, including the adrenal, gonads, spleen, and adipose tissues, as well as in the brain, where mammalian counterparts are exclusively expressed in the brain, indicating that the regulation of MC4-R gene expression differs between mammals and chickens. The CMC5 mRNA, on the other hand, is expressed in the liver, gonads, adrenal, kidney brain, and adipose tissues as well as in the uropygial gland. These findings raise the possibility that melanocortins affect a variety of functions both in the brain and in the peripheral tissues of the chicken. (C) 1998 Academic Press.

DOI： 10.1006/gcen.1998.7167

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.15.567

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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.

DOI： 10.2108/zsj.15.573

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Neural cell adhesion molecules (NCAMs) were immunocytochemically detected in the pituitary glands of female ICR mice and in the pituitary tumor cell lines (GH(3) cells and AtT-20 cells) using the antibody against mouse NCAM. Immunoreactive NCAMs (ir-NCAMs) were detected in the cell membranes of mouse pituitary cells, GH(3) cells and AtT-20 cells. Fifty four percent of anterior pituitary cells, 58% of neurointermediate cells (cells of both posterior and intermediate lobes), 13% of GH(3) cells and 43% of AtT-20 cells expressed ir-NCAMs. Anterior pituitary cell types of NCAM-expressing cells were identified. Most of somatotrophs, gonadotrophs, and thyrotrophs showed NCAM-immunoreactivity, while parts of mammotrophs and corticotrophs did. The cell-type-specific pattern of NCAM expression in the mouse pituitary may be closely associated with physiological roles of pituitary NCAMs. For clarifying roles of pituitary NCAM, AtT-20 cells were treated with antisense oligodeoxynucleotide (ODN) which was complementary to the translation initiation site of NCAM mRNA, or with sense ODN which corresponded to the sense sequence for 7 days. Antisense ODN treatment (100 nM) decreased the percentage of NCAM-expressing cells, and surprisingly increased the cell number of AtT-20 cells compared with sense ODN treatment and non-treatment. This result suggests that NCAM is involved in the growth control of AtT-20 cells.

DOI： 10.2220/biomedres.19.269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：J ENDOCRINOLOGY LTD  

IGF-I is synthesized in the human and rat anterior pituitary glands. The present study was designed to clarify the growth-promoting action of IGF-I on mouse pituitary cells in a primary serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of bromodeoxyuridine (BrdU). BrdU labelling in the nucleus was found in all types of secretory cells: corticotrophs, thyrotrophs, gonadotrophs (LH cells and FSH cells), somatotrophs and mammotrophs. IGF-I (75 ng/ml) stimulated the proliferation of corticotrophs and mammotrophs among the pituitary secretory cells. IGF-I receptor mRNA was detected in the cultured pituitary cells using reverse transcription (RT)-PCR, indicating that mouse pituitary cells expressed IGF-I receptors. Insulin (100 ng/ml) or IGF-I (7.5 ng/ml) failed to increase the percentage of BrdU-labelled cells. However, treatment with insulin (100 ng/ml) plus IGF-I (7.5 ng/ml) increased the percentage of BrdU-labelled cells in a synergistic-like manner. Genistein, a tyrosine kinase specific inhibitor, decreased the IGF-I-induced cell proliferation, indicating that IGF-I acts through IGF-I receptors. IGF-I mRNA was also detected in the cultured pituitary cells by RT-PCR, and its peptides were immunocytochemically detected. The present results demonstrate that all types of pituitary secretory cells have the ability to proliferate in our serum-free culture system. IGF-I synthesized in the pituitary gland may stimulate the growth of pituitary cells, in particular corticotrophs and mammotrophs, by an autocrine or paracrine mechanism.

DOI： 10.1677/joe.0.1570053

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The chicken melanocortin 2-receptor (MC2-R) gene was isolated. It is found to be a single copy gene encoding a 357 amino acid protein, sharing 65.8-68.7% identity with mammalian counterparts. The chicken MC2-R mRNA is expressed in the adrenal and spleen, suggesting that the receptor mediates both endocrine and immunoregulatory functions of ACTH in the chicken. The amino acid sequence of the chicken MC2-R is collinear with those of other subtypes of MC-R, whereas all cloned mammalian MC2-Rs contain a gap in the third intracellular loop, suggesting that mammalian MC2-R molecules have evolved by lacking a part of the domain which determines the specificity of signal transduction in G-protein coupled receptors. Interestingly, the codon usage differs dramatically between MC1-R and MC2-R in the chicken the GC-contents at the third codon position in MC1-R and MC2-R are 94.6 and 50.6%, respectively. It may reflect selective constraints on the usage of synonymous codons. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4889(98)00022-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE INC  

Estrogens stimulate proliferation and differentiation of uterine epithelial cells in vivo, Mitogenic action of estrogens may be mediated by growth factors such as insulin-like growth factor-I (IGF-I), This study was designed to determine whether IGF-I and insulin affect proliferation of uterine epithelial cells obtained from 3 to 4-week-old immature female mice in a serum-free culture system, The epithelial cell number on Day 5 in culture was significantly increased by adding IGF-I (10 and 100 ng/ml) or insulin (100 and 1000 ng/ml) to the culture media, indicating that IGF-I is more effective than insulin in inducing the epithelial growth, The epithelial DNA syn thesis was significantly stimulated by IGF-I (1 and 10 ng/ml), suggesting that both the epithelial proliferation and their detachment from substratum are stimulated by 1 ng/ml of IGF-I, but that the former is more accelerated than the latter by 10 ng/ml of IGF-I, These results demonstrate that both IGF-I and insulin directly stimulate the growth of uterine epithelial cells, and suggest that insulin may act via IGF-I receptors, IGF-I immunoreactivity was detected in the cytoplasm of the cultured cells, indicating that the cells synthesize IGF-I, Estradiol-17 beta (E-2) at lower concentrations (0.001-0.1 nM) tended to increase the number of epithelial cells, while E-2 at higher concentrations (1 to 100 nM) did not affect it, It is highly probable that IGF-I produced in endometrial cells induces their proliferation by an autocrine or paracrine mechanism.

DOI： 10.3181/00379727-215-44152

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DOI： 10.11477/mf.2425901240

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The Extended black (E) locus on chromosome 1 acts within the melanocyte to regulate feather color pigmentation in the chicken. Several alleles exhibiting different pigmentation have been described and their phenotypes are similar to those of the murine extension locus which encodes melanocortin 1-receptor (MC1-R), the receptor for alpha-melanocyte-stimulating hormone (alpha-MSH). To investigate whether the MC1-R gene is responsible for E-locus function, we examined the structure of MC1-R in E-locus mutants by RFLP analysis and genomic DNA sequencing. In the most recessive allele (e(y)), which exhibits a uniformly red-yellow pigmentation, MC1-R was found to contain amino acid substitutions possibly causing functional deficiency. On the other hand, in the most dominant allele (E), which confers a uniformly black pigmentation, MC1-R possessed mutation responsible for a constitutively active MC1-R and resultant black coat color in mice. Our finding that the structure of MC1-R was affected by individual E-locus alleles strongly suggests that MC1-R is associated with the E-locus. Furthermore, since the same mutation of MC1-R was found in mice and chickens that exhibit the same pigmentation, it is possible that the regulatory mechanism of MC1-R function is shared in chickens and mammals.

DOI： 10.1016/0167-4781(96)00100-5

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The chick melanocortin 1-receptor gene was isolated. It is found to be an intronless gene encoding a 314 amino acid protein, sharing 64% identity with mammalian counterparts. A cis-element responsible for melanocyte-specific transcription is found in its 5' upstream region. It is probable that the expression of the receptor in melanocytes is closely correlated with that of melanogenesis-related genes.

DOI： 10.1016/0167-4781(96)00026-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP-1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5' flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5' upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5' upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence-coding regions.

DOI： 10.1111/j.1600-0749.1992.tb00551.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED RES FOUND  

Structural changes and changes in volume of three lobes of the pituitary gland and of degenerative regions of the anterior pituitary were investigated 3, 5, 7, 14, 21 and 28 days after pituitary hemorrhage induced by intraperitoneal injection of 9% NaCl solution at a dose of 0.03 ml/g body weight in mice. The necrotic changes in the anterior lobe was most extensive on day 3 of bleeding. A huge mass of necrosis was observed protruding into the distended hypophyseal lumen on days 5 to 7 of hemorrhage. On days 14 to 28, the cellular elements and necrotic debris in the lumen were almost eliminated. These findings suggest a new role for the hypophyseal lumen as a site of storage and digestion of waste materials migrated from the anterior pituitary. Among the three lobes of the pituitary gland, the anterior lobe decreased in volume after hemorrhage showing a minimum on day 14 (43.2% of the control) followed by a slight recovery on day 28 (50.1% of the control). This atrophy of the anterior pituitary after hemorrhage is ascribed to the shrinkage of necrosis and to the lack of regeneration of unaffected normal cells of the anterior lobe during the time examined.

DOI： 10.2220/biomedres.13.253

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The strain-dependent expression of murine serum amyloid P-component (SAP) has been known to be linked to the Sap locus. We have quantified the SAP mRNA in several inbred strains including DBA/2 and C57BL/6 mice which represent high and low producers of SAP at resting state, respectively, and found that the mRNA levels correlated well with the amount of SAP protein. Interestingly, the SAP mRNA level of F1 mouse between DBA/2 and C57BL/6 was low and similar to that of C57BL/6. Primer extension and ribonuclease (RNAase) protection analyses demonstrated that a single type of transcript was generated from the SAP gene and that the cap sites were identical regardless mouse strains tested under unstimulated and stimulated (by lipopolysaccharide (LPS) or interleukin-6 (IL-6)) conditions. To investigate possible structural difference of the SAP gene including 5′ flanking region, we have cloned, sequenced and compared the SAP genes from DBA/2 and C57BL/6 mice. Sequence analyses revealed that the 5′ flanking regulatory regions, as well as the coding regions, were well-conserved between the two strains. These results demonstrate that the strain-dependent SAP expression occurs at the transcriptional level but seems to be affected by neither different type of the transcripts nor structural difference of the 5′ flanking and coding regions of the SAP gene. It was suggested that a possible transcription factor with suppressive activity, which is encoded by a gene linked to Sap, may be involved. © 1992.

DOI： 10.1016/0167-4781(92)90024-T

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The strain-dependent expression of murine serum amyloid P-component (SAP) has been known to be linked to the Sap locus. We have quantified the SAP mRNA in several inbred strains including DBA/2 and C57BL/6 mice which represent high and low producers of SAP at resting state, respectively, and found that the mRNA levels correlated well with the amount of SAP protein. Interestingly, the SAP mRNA level of F1 mouse between DBA/2 and C57BL/6 was low and similar to that of C57BL/6. Primer extension and ribonuclease (RNAase) protection analyses demonstrated that a single type of transcript was generated from the SAP gene and that the cap sites were identical regardless mouse strains tested under unstimulated and stimulated (by lipopolysaccharide (LPS) or interleukin-6 (IL-6)) conditions. To investigate possible structural difference of the SAP gene including 5' flanking region, we have cloned, sequenced and compared the SAP genes from DBA/2 and C57BL/6 mice. Sequence analyses revealed that the 5' flanking regulatory regions, as well as the coding regions, were well-conserved between the two strains. These results demonstrate that the strain-dependent SAP expression occurs at the transcriptional level but seems to be affected by neither different type of the transcripts nor structural difference of the 5' flanking and coding regions of the SAP gene. It was suggested that a possible transcription factor with suppressive activity, which is encoded by a gene linked to Sap, may be involved.

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An electron microscopic and morphometric study on pars intermedia (PI) cells of the pituitary was undertaken in male mice given either a liquid diet consisting of 3% powdered milk in a 5% glucose solution or the identical liquid diet supplemented with 1% urea for 10 days. Three parameters representing the secretory activity of PI cells were examined. The percent area of the cytoplasm occupied by the rough endoplasmic reticulum (an index of protein synthesis) increased to 124% and 172% of the control value in mice maintained on the liquid diet alone and on the liquid diet containing 1% urea, respectively; the numerical density of immature Golgi granules (an index of granule formation) also increased to 143% and 185% of the control value in those given the liquid diet alone and the liquid diet supplemented with 1% urea, respectively. In contrast, the numerical density of secretory granules (an index of granule storage) decreased to 74% and 62% of the control value in animals fed the liquid diet alone and the liquid diet supplemented with 1% urea, respectively. These results indicate that the balance between synthesis and release of PI hormones is in equilibrium at a higher level in mice fed the liquid diet with urea than in animals given the liquid diet without urea.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

We introduced a mouse tyrosinase minigene, mg-Tyrs-J, in which the authentic genomic 5' non-coding flanking sequence was fused to a mouse tyrosinase cDNA, into fertilized eggs of albino mice. Of the 25 animals that developed from the injected eggs, four mice exhibited pigmented hair and eyes. Histological analysis of the transgenic mice revealed that the melanogenesis was restricted to hair bulbs and eyes. These results suggest that this minigene encodes active tyrosinase protein and that its 5' flanking region contains the sequences regulating expression of mouse tyrosinase gene. This is the first report of a successful expression of tyrosinase gene and of pigment production in transgenic mice.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

An attempt was made in the present study to express mouse tyrosinase cDNAs fused with the authentic genomic 5′ non-coding flanking sequence in cultured albino melanocytes. One of the cDNA sequences, which expressed successfully and produced melanin pigments, was analyzed with respect to deduced amino acid sequence. Sequencing of the tyrosinase genomic gene revealed the existence of several sets of a characteristic structure which consists of a chain of two successive stem structures, CCAAT-homology and TATA box at its 5′ non-coding region. It seems possible that this region represents the regulatory element of the tyrosinase gene. Unusually long GA cluster at 5′ upstream region was also found. © 1989, The Genetics Society of Japan. All rights reserved.

DOI： 10.1266/jjg.64.121

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

In order to study the molecular aspects of albinism, we analyzed the expression of tyrosinase gene in some amelanotic mutant mice by Northern blotting using mouse tyrosinase cDNA as a probe. It hybridized with a species of 2.1 kilobase RNA prepared from the skin of wild-type mice and two albino strains in the same quantity. In black-eyed white mouse, however, no RNA transcript encoding tyrosinase was detected. Our results suggest that these albinism is due to a point mutation in the structural region of the tyrosinase gene, not to a deficiency of tyrosinase gene expression and that the black-eyed white mouse has a deficiency in the gene expression possibly related to melanocyte differentiation. © 1988 Academic Press, Inc.

DOI： 10.1016/S0006-291X(88)81110-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GENETICS SOC JAPAN  

A cDNA library was constructed from poly (A)+ mRNA from mouse melanocytes and screened using anti-tyrosinase antiserum and oligonucleotide probes corresponding to amino acid sequence of tyrosinase. Sequencing of some cDNA clones positive in these screenings gave a nucleotide sequence of 1838 nucleotides including a open reading frame of 1344 nucleotides. © 1987, The Genetics Society of Japan. All rights reserved.

DOI： 10.1266/jjg.62.271

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子宮における性ステロイドホルモンによる細胞増殖は、子宮において産生される成長因子によって仲介されると考えられている。本研究においては、子宮内膜で発現するインスリン様成長因子I （IGF-I）に着目し、マウス子宮内膜間質細胞のDNA合成に及ぼすIGF-Iの効果を調べた。21-23日齢の未成熟マウス子宮から子宮内膜間質細胞をトリプシン処理により単離し培養した。 R-PCR解析により、子宮内膜間質細胞にはIGF-ImRNA及びIGF-Iの1型受容体ｍRNAが発現していることがわかった。 IGF-ImRNAはesradiol-17β投与により増加した。 IGF-I投与は、用量依存的に子宮内膜間質細胞のDNA合成を促進した。このIGF-IによるDNA合成の促進作用は、チロシンキナーゼの阻害剤gensiein、MAPK系の阻害剤PD098059により阻害された。これらの結果よりマウス子宮内膜で産生されるIGF-Iは、子宮内膜間質細胞の増殖に関与すること並びに、子宮内膜間質細胞のIGF-IによるDNA合成の制御にはMAPK系が関与する可能性が示唆された。

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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Language：English   Publisher：WILEY-LISS  

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Language：Japanese   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

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