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AKIRA SUZUKI
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TAKAHIDE MORI
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TOSHIO NISHIMURA
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Rabbits were injected with human chorionic gonadotrophin (HCG), and slices of developing corpora lutea taken from the ovaries 15, 18, 24, 48, 72 and 96 h after injection were incubated with [1-14C]sodium acetate at 37 °C for 3 h. The incorporation of labelled acetate into ten steroids, including progestagens, androgens and oestrogens, was analysed.

In the initial step of corpus luteal formation, the specific incorporation (incorporation of [1-14C]acetate/100 mg tissue) increased sharply. The major steroidal products were progesterone, 17-hydroxyprogesterone and 20α-hydroxypregn-4-en-3-one. Between 18 and 48 h, the increase in specific incorporation was more gradual than in the initial step. Although the pattern was also dominated by progestagens, a temporary increase in the incorporation of acetate into androgens and oestrogens was observed. In the final step, a sharp rise in the total incorporation (incorporation of [1-14C]acetate/corpus luteum) was found, whereas the specific incorporation increased only slightly. The principal steroids produced were progesterone, pregnenolone and 20α-hydroxypregn-4-en-3-one. Incorporation into C19 steroids declined markedly and that into C18 steroids could not be detected. This profile of steroidogenesis 96 h after injection of HCG was similar to that of the corpus luteum in pregnancy.

Thus marked quantitative and qualitative changes have been demonstrated during the period of formation of corpora lutea in the rabbit.

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Koichi Suzuki
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Akira Kawashima
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Aya Yoshihara
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Takeshi Akama
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Mariko Sue
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Akio Yoshida Laboratory of Molecular Diagnostics, Division of Regenerative Medicine and Therapeutics, Division of Bioimaging Sciences, Department of Mycobacteriology, National Institute of Infectious Diseases, Leprosy Research Center, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 189-0002, Japan

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Hiroaki J Kimura Laboratory of Molecular Diagnostics, Division of Regenerative Medicine and Therapeutics, Division of Bioimaging Sciences, Department of Mycobacteriology, National Institute of Infectious Diseases, Leprosy Research Center, 4-2-1 Aoba-cho, Higashimurayama, Tokyo 189-0002, Japan

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Thyroid function is tightly regulated by TSH. Although individual follicles are exposed to the same blood supply of TSH and express relatively homogenous levels of the TSH receptor, the function of individual follicles is variable. It was shown that thyroglobulin (Tg), stored in the follicular lumen, is a potent negative feedback regulator of follicular function. Thus, physiological concentrations of Tg significantly suppress thyroid-specific gene expression and antagonize the TSH-mediated stimulation that induces expression of thyroid-specific genes. Tg coordinately regulates both basal and apical iodide transporters in thyroid follicular cells. Recently, it was also reported that Tg could induce thyroid cell growth in the absence of TSH. These results indicate that Tg is an essential autocrine regulator of physiological thyroid follicular function that counteracts the effects of TSH.

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Hayato Shimada
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Hitoshi Okamura
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Yōichi Noda
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Akira Suzuki
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Shimpei Tōjō
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Akikazu Takada
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To elucidate the possible roles of increased plasminogen activator (PA) in follicular rupture and to investigate whether prostaglandins participate in ovarian PA synthesis in vivo, enzyme activities were sequentially measured by a method using the chromogenic substrate S-2251 in immature rat ovaries primed with pregnant mare serum gonadotrophin (PMSG) followed by human chorionic gonadotrophin (hCG) either alone or with a concurrent injection of indomethacin.

Before hCG injection PA activity was 0·006 ± 0·006 (s.d.) μmol/1·6 mg ovarian tissue (wet wt) per 30 min: PA activity of a saline-treated group remained at low levels (<0·018 ± 0·003 μmol/1·6 mg tissue per 30 min). In contrast, PA activity of animals given hCG alone increased after the treatment, reaching a peak value of 0·112 ± 0·071 μmol/1·6 mg tissue per 30 min 12 h later, before decreasing to 0·023 ± 0·014 μmol/1·6 mg tissue per 30 min at 32 h. Contrary to expectations, a dose of indomethacin which completely blocked ovulation had no effect on either the magnitude or the time-course of PA synthesis after hCG administration (P>0·05).

These results indicate that prostaglandins are not involved in the preovulatory synthesis of PA induced by hCG in rat ovaries and that PA is not a primary proteolytic enzyme for follicular rupture. It is suggested that PA has possible roles in cumulus detachment and/or proliferation of granulosa cells during the ovulatory process.

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Chun-Mei Li Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Shinji Takahashi Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Shinji Taneda Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Chie Furuta Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Gen Watanabe Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Akira K Suzuki Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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Kazuyoshi Taya Department of Basic Veterinary Science, The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan
Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
Ecological Effect Research Team, Dioxin and Environmental Endocrine Disrupter Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
Toxicology and Effects Research Team, PM2.5/DEP Research Project, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

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The effects of 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust, on reproductive function were investigated in adult male Japanese quail. The quail were treated with a single i.m. dose of PNMC (78, 103 or 135 mg/kg body weight), and trunk blood and testes were collected 1, 2 or 4 weeks later. Various levels of testicular atrophy were observed in all groups treated with PNMC. Sperm formation, cloacal gland area, and plasma LH and testosterone concentrations were also reduced in birds with testicular atrophy. To determine the acute effect of PNMC on gonadotrophin from the pituitary, adult male quail were administrated a single i.m. injection of PNMC (25 mg/kg), and plasma concentrations of LH were measured at 1, 3 and 6 h. This dose significantly lowered plasma levels of LH at all three time points. These results suggest that PNMC acts on the hypothalamus–pituitary axis, by reducing circulating LH within a few hours of administration and subsequently reducing testosterone secretion. In addition, in order to investigate the direct effects of PNMC on the secretion of testosterone from testicular cells in quail testes, cultured interstitial cells containing Leydig cells were exposed to PNMC (10−6, 10−5 or 10−4 M) for 4, 8 or 24 h. These quantities of PNMC significantly reduced the secretion of testosterone in a time- and dose-dependent manner. The present findings also suggest a direct effect of PNMC on the testis to reduce testosterone secretion. This study clearly indicates that PNMC induces reproductive toxicity at both the central and testicular levels, and disrupts testicular function in adult male quail.

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Makoto Ito Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Akira Gomori Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Jun Suzuki Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Shigeharu Tsujioka Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Minoru Sasaki Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Masao Matsuda Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Maria A Bednarek Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Masahiko Ito Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Akane Ishihara Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Hisashi Iwaasa Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Douglas J MacNeil Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Akio Kanatani Tsukuba Safety Assessment Laboratories, Metabolic Disorders, Tsukuba Research Institute

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Blockade of brain melanin-concentrating hormone 1 receptor (MCH1R) significantly ameliorates fatty liver as well as obesity. However, the mode of action of this effect is unknown. This study examined the effect of a MCH1R antagonist in murine steatohepatitis models with and without obesity and clarified whether these pharmacological effects were attributed to anti-obesity effects. Steatohepatitis with concomitant obese phenotypes was developed after 52-week exposure to a high-fat diet, and steatohepatitis with reduced body weight was developed by exposure to a methionine- and choline-deficient diet for 10 days. Chronic intracerebroventricular infusion of a peptidic MCH1R antagonist reduced hepatic triglyceride contents and ameliorated steatohepatitis on histological observations in both mice models. Improvement of steatohepatitis was concomitant with amelioration of obese phenotypes such as hyperinsulinemia and hyperleptinemia in the case of the obese model, whereas body weight reduction was not associated with amelioration of steatohepatitis by the antagonist in the lean model. Reduction of hepatic gene expressions encoding cytochromes P450 4A was identified by treatment with the antagonist in both the obese and lean models. These results suggest that brain blockade of MCH1R could alleviate steatohepatitis independently from anti-obesity effects. In conclusion, MCH1R antagonist could have a new therapeutic potential for the treatment of human nonalcoholic steatohepatitis.

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Koji Y Arai Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Hisashi Kishi Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Satoshi Onodera Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Wanzhu Jin Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Gen Watanabe Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Akira K Suzuki Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Shinji Takahashi Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Toshihiko Kamada Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Toshio Nishiyama Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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Kazuyoshi Taya Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Laboratory of Animal Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
Air Pollutants Health Effect Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan
Ecological Effect Research Team, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

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To elucidate changing patterns of inhibin/activin subunit mRNAs in the ovary of the golden hamster (Mesocricetus auratus) during the oestrous cycle, inhibin/activin subunit cDNAs of this species were cloned and ribonuclease protection assay and in situ hybridization were carried out. Inhibin α-subunit mRNA was localized in granulosa cells of primary, secondary, tertiary and atretic follicles throughout the 4-day oestrous cycle. It was also expressed in luteal cells on days 1 (oestrus), 2 (metoestrus) and 3 (dioestrus). βA-subunit mRNA was localized in granulosa cells of large secondary (>200 μm) and tertiary follicles throughout the oestrous cycle. βB-subunit mRNA was confined to granulosa cells of large secondary and tertiary follicles. Both α- and βA-subunit mRNAs were also found in ovarian interstitial cells and theca interna cells of tertiary and atretic follicles in the evening of day 4 (pro-oestrus). A striking increase in βA-subunit mRNA levels was also observed during the preovulatory period. The expression pattern of βA-subunit mRNA during the preovulatory period is unique and not found in other species. An i.v. injection of anti-luteinizing hormone-releasing hormone (LHRH) serum before the LH surge abolished the expression of α- and βA-subunit mRNAs in ovarian interstitial cells and theca interna cells. The treatment also abolished the preovulatory increase in βA-subunit mRNA. Furthermore, administration of human chorionic gonadotrophin (hCG), which followed the injection of anti-LHRH serum, restored the expression patterns of α- and βA-subunit mRNAs. The present study revealed that the golden hamster showed a unique expression pattern of βA-subunit mRNA in response to the LH surge.

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