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M. Mori
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I. Kobayashi
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S. Kobayashi
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ABSTRACT

We have investigated the effect of TRH on the accumulation of glycosylated TSH in the rat anterior pituitary gland. Hemipituitaries from adult male rats were incubated in medium containing [3H]glucosamine in the presence of TRH. [3H]Glucosamine-labelled TSH in media and pituitaries was measured by immunoprecipitation and characterized by isoelectric focusing after affinity chromatography. Incorporation of [3H]glucosamine into immunoprecipitable TSH in the media and pituitaries increased progressively with the period of incubation. Although the release of [3H]glucosamine-labelled and unlabelled TSH into media was stimulated by the addition of TRH in a time- and dose-dependent manner, TRH administration did not alter the amounts of labelled or unlabelled TSH in the anterior pituitary lobes. The anterior pituitaries were found, by isoelectric focusing analysis, to be composed of four major component peaks of [3H]glucosamine-labelled TSH. Administration of TRH caused profound changes in the radioactivity of these components and evoked new radioactive peaks, resulting in the appearance of six components in total. The present data provide evidence that TRH significantly changes the heterogeneity of glycosylated TSH in the anterior pituitary without altering the amount of the glycosylated form.

J. Endocr. (1986) 109, 227–231

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M Sakai
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M Kobayashi
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H Kawauchi
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Abstract

The activation of rainbow trout (Oncorhynchus mykiss) phagocytic cells by chum salmon GH, prolactin (PRL) and somatolactin (SL) was investigated in vitro. Rainbow trout kidney leucocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng of each hormone/ml and the production of superoxide anion was measured using reduction of nitroblue tetrazolium (NBT) and ferricytochrome C. Macrophages incubated with 10–100 ng GH or PRL/ml showed significantly enhanced production of superoxide anion in both the NBT and ferricytochrome C tests compared with control macrophages (without hormone). SL did not induce enhanced production of superoxide anion in macrophages. The phagocytic cells treated with GH or PRL also showed increased phagocytic activity and phagocytic index. However, the cells treated with SL showed no enhancement of phagocytic activity or index. These results indicate that GH and PRL stimulate macrophages in vitro.

Journal of Endocrinology (1996) 151, 113–118

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M. Kobayashi
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R. Nakano
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A. Ooshima
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ABSTRACT

Ovaries from 37 women with normal menstrual cycles were analysed for localization of pituitary gonadotrophins and gonadal steroids using an immunohistochemical method. In the follicular phase, FSH and oestradiol-17β localized in the granulosa layer, and LH, progesterone and testosterone localized in the internal thecal layer. In the luteal phase, gonadotrophins and steroids localized in luteal cells. Particularly in the early luteal phase, FSH and oestradiol-17β localized in large luteal cells, and LH, progesterone and testosterone localized in small luteal cells. The results of the present immunohistochemical analysis confirm the two-cell, two-gonadotrophin hypothesis of steroidogenesis in the human ovary.

Journal of Endocrinology (1990) 126, 483–488

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M. Mori
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M. Murakami
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T. Iriuchijima
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H. Ishihara
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I. Kobayashi
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S. Kobayashi
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K. Wakabayashi
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ABSTRACT

An influence of thyrotrophin-releasing hormone (TRH) on TSH heterogeneity in close association with de-novo biosynthesis was studied in rat anterior pituitary glands. Hemipituitary glands from adult male rats were incubated in Krebs–Henseleit–glucose media containing [3H]glucosamine and [14C]alanine for 3 and 6 h in the presence or absence of 10 ng TRH per ml. Fractions of TSH in the pituitary extracts were obtained using affinity chromatography coupled with an anti-rat TSH globulin. These TSH fractions were analysed by isoelectric focusing. The control pituitary glands were composed of four component peaks (isoelectric point (pI) 8·7, 7·8, 5·3 and 2·5) of [3H]glucosamine and [14C]alanine incorporated into TSH, and the amounts of radioactivity of these components were increased with the incubation time. Of these peaks, radioactive components of pI 8·7 and 7·8 coincided with the non-radioactive TSH components measured by radioimmunoassay. Addition of TRH increased incorporation of [14C]alanine into TSH in each of the components to a greater extent than that of [3H]glucosamine. In addition, new components with pI 7·2, 6·5 and 6·2, each component corresponding to each unlabelled TSH component, were demonstrated in the presence of TRH. Because addition of TRH did not change the amounts of [14C]alanine-labelled TSH in the media, the newly formed components were assumed to be connected with protein synthesis occurring in the anterior pituitary gland, which may be specific substances in response to TRH administration. These results indicate that TRH principally elicits an increase in protein synthesis in TSH at the anterior pituitary level, resulting in an alteration of TSH heterogeneity.

J. Endocr. (1984) 103, 165–171

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GW Sun
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H Kobayashi
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M Suzuki
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N Kanayama
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T Terao
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Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

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T Kobayashi
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O Ushijima
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J-T Chen
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M Shiraki
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T Ohta
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M Kiyoki
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Abstract

Hyper-release of calcitonin gene-related peptide (CGRP) plays a direct and pivotal role in the induction of menopausal hot flushes (HFs), in which a drastic increase in skin temperature occurs. However, it is not possible to investigate whether CGRP induces skin temperature increase and whether skin temperature response to CGRP changes and contributes to the occurrence of HFs in postmenopausal women who are in oestrogen deficiency. By using rats' tail skin temperature (TST), a good marker to evaluate skin temperature regulation, we examined the effects of CGRP and calcitonin (3, 10 and 30 μg/kg, i.v.) on TST in female rats and further investigated the TST change induced by CGRP (10 μg/kg, i.v.) in ovariectomized (OVX) rats compared with that in sham-operated (Sham) rats. We found that CGRP, but not calcitonin, induced a TST increase in a dose-dependent manner and that the TST change induced by CGRP (0·6 ±0·2 °C for OVX rats vs 0·3 ±0·1 °C for Sham rats, P<0·05) and also the basal TST (26·0 ± 0·2 °C for OVX rats vs 25·5 ±0·1 °C for Sham rats) were significantly greater in OVX rats (P<0·05). Furthermore, treatment with oestradiol (30 μg/kg, s.c.) for 8 days partially inhibited the augmented TST response to CGRP in OVX rats and almost completely inhibited (P<0·05) the basal TST elevation, with the concomitant recovery of the serum oestradiol level to that in Sham rats. These results suggest that the augmented skin temperature response to CGRP and the elevation of basal skin temperature that are found in OVX rats, animals which are oestradiol deficient, may also occur in menopausal women and contribute to their HFs.

Journal of Endocrinology (1995) 146, 431–437

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K Katoh Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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M Yoshida Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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Y Kobayashi Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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M Onodera Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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K Kogusa Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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Y Obara Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan

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In order to assess the biological significance of weaning and water deprivation on the control of plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, growth hormone (GH) and metabolites in response to stimulation with arginine-vasopressin (AVP) and corticotropin-releasing hormone (CRH), we carried out three experiments in which male goats before and after weaning were intravenously injected with AVP or CRH alone, or in combination with each other. In experiment 1, 17-week-old (post-weaning) goats were intravenously injected with AVP or CRH alone at the doses of 0.1, 0.3 and 1.0 nmol/kg body weight (BW). The AVP injection significantly and dose dependently increased plasma levels of ACTH, cortisol, GH and metabolites, whereas the injection with CRH did not cause significant increases in the levels of these parameters. In experiment 2, 4-week-old (pre-weaning) and 13-week-old (post-weaning) goats were injected with either AVP or CRH alone, followed by a combined injection of both secretagogues at a dose of 0.3 nmol/kg BW. Although the basal levels of the hormones and metabolites, with the exception of glucose, were greater in the 4-week-old goats, the hormone responses induced by stimulation with AVP were weaker than those induced in 13-week-old goats. Additionally, there were no responses in any hormone patterns to CRH stimulation in 4-week-old goats. In experiment 3, 13-week-old goats were injected with CRH alone followed by injection with AVP for two consecutive days of water deprivation. The animals were subjected to withdrawal of up to 20% of the total blood volume and water deprivation for up to 28 h. However, no significant differences in plasma ACTH, cortisol or GH levels were observed between days 1 and 2. Based on these results, we concluded that: (1) AVP is a more potent stimulant than CRH in terms of its ability to induce increases in plasma levels of ACTH, cortisol and GH; (2) the role of AVP as a secretagogue of hypothalamus–pituitary–adrenal hormones is strengthened, whereas the ineffective role of CRH remains unaltered, by weaning; (3) acute stress such as massive withdrawal of blood volume and subjection to water deprivation may not be sufficient burdens to alter stress-related hormone levels in young goats.

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O. Carnevali
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G. Mosconi
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K. Yamamoto
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T. Kobayashi
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S. Kikuyama
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A. M. Polzonetti-Magni
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ABSTRACT

Male and female Rana esculenta liver was induced in an in-vitro system by homologous and Rana catesbeiana pituitary to synthesize and release vitellogenin, a lipoglycophosphoprotein precursor of yolk proteins, lipovitellins and phosvitins, in oviparous vertebrates.

In the present experiments, the action of prolactin on hepatic vitellogenin synthesis and release was investigated, using ovine prolactin and Rana catesbeiana prolactin. The effects of prolactin on hepatic vitellogenin synthesis displayed different trends related to sex; male liver was found to be more responsive than female liver to both ovine and frog prolactin; moreover, the response to prolactin was dose-related (r = 0·998; P <0·05) in male but not in female liver. In both sexes, a high degree of seasonality in the responsiveness of the liver was found, since the vitellogenin levels induced by prolactin during the winter phase were significantly (P < 0·001) higher than those produced during the summer phase. Thus, there was no significant difference between the action of ovine and frog prolactin on vitellogenin synthesis; in fact, mammalian prolactins are structurally similar with regard to nucleotide and amino acid sequences.

The direct action of prolactin on hepatic vitellogenin synthesis in the frog Rana esculenta is discussed, on the basis of the role played by prolactin as an important growth modulatory hormone in fetal and adult tissues.

Journal of Endocrinology (1993) 137, 383–389

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A. Miyamoto
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S. Kobayashi
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S. Arata
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M. Ohtani
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Y. Fukui
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D. Schams
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ABSTRACT

Prostaglandin F2α (PGF2α) is a primary luteolysin in the cow. Although the mechanisms involved in luteolysis are thought to be a complex of its direct action on luteal cells and indirect effect on luteal blood flow, the detailed mechanisms remain to be elucidated. This study focuses on the possible interaction of endothelial cells-derived endothelin-1 (ET-1) with PGF2α in the rapid suppression of progesterone release from the bovine corpus luteum (CL). In in vitro microdialysis system (MDS) of CL, PGF2α acutely stimulated the release of progesterone and oxytocin during infusion and ET-1 release after infusion. Moreover, PGF2α induced slight decrease of progesterone release during the last period of the experiment (8-11 h after PGF2α exposure). Two 1 h-perfusions of ET-1 at 3 h intervals induced only a slight decrease of progesterone release after the second perfusion. This treatment also affected the oxytocin release; the first ET-1 perfusion produced an acute stimulation, whereas the second ET-1 perfusion inhibited the release to below 50%. When the CL pieces were pre-perfused with PGF2α for 2 h, the two consecutive perfusion of ET-1 at 3 h intervals induced drastic decrease in progesterone and oxytocin release only after the second ET-1 perfusion. Thus, a pre-exposure with PGF2α clearly potentiated the inhibiting activity of ET-1 in the progesterone release. These results suggest a physiological impact of PGF2α and ET-1 in the rapid cascade of functional luteolysis in vivo, and a possible interaction between endothelial cells and luteal cells.

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K Katoh Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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K Yoshioka Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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H Hayashi Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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T Mashiko Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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M Yoshida Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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Y Kobayashi Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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Y Obara Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Feed Functionality Research Laboratory, Meiji Feed Co., Kashima, Ibaraki 314-0103, Japan

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Postprandial changes in plasma concentrations of GH, insulin, IGF-I, leptin and metabolites were compared between young Holstein bull calves fed with milk alone (control group) and with milk+5′-uridylic acid (UMP) (UMP group). UMP (2 g/day) was given with milk at 0830 h and 1530 h for 11 days from the 4th to the 14th day after birth. The perirenal fat weight was significantly lower in the UMP group than in the control group, but there was no significant difference in the weights of the liver, spleen and heart between the groups. Basal GH concentrations in the UMP group were slightly higher, but the postprandial increase in plasma insulin level and the area under the curve for insulin in the UMP group were significantly lower than those in the control group. There was no significant difference in IGF-I levels between the groups. In addition, the postprandial glucose concentrations were lower in the UMP group as reflected by the insulin level, and nonesterified fatty acid concentrations were not different. In the muscle (M. longissimus thoracis) sampled at 14 days of age, the triacylglycerol (TAG) content was significantly greater but glycogen content was significantly lower in the UMP group than in the control group. From these results, we have concluded that feeding 5′-UMP at 2 g/day for 11 days significantly alters TAG accumulation in the body and plasma concentrations of GH and insulin in young bull calves.

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