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The synthesis and release of prolactin in rats were significantly higher at oestrus than at any other stage of the cycle, when measurements were made between 10.00 and 11.00 h (Ieiri, Akikusa & Yamamoto, 1971). Since considerable evidence indicates that stimulation of prolactin release begins on the afternoon of the day of pro-oestrus (Ieiri et al. 1971), synthesis and release were measured in the afternoon of the day of each stage of the oestrous cycle to determine the fluctuations of these parameters in the course of the cycle.

Adult female rats of the Wistar strain showing regular 4-day cycles were used. Animals were killed by decapitation between 16.00 and 17.00 h. Each isolated anterior pituitary was incubated with [14C]leucine. All the experimental conditions were the same as described previously (Ieiri et al. 1971).

The anterior pituitary functions were estimated by counting the radioactivity of [14C]leucine incorporated into prolactin

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T. Kurabayashi, T. Fujimaki, M. Yasuda, Y. Yamamoto, and K. Tanaka


This study was carried out (1) to compare the time-course of the change in bone metabolism in rats administered gonadotrophin-releasing hormone agonist (GnRHa) and bilaterally ovariectomized (OVX) rats and (2) to investigate the changes in bone metabolism after discontinuance of GnRHa.

Seventy female Sprague–Dawley rats, aged 90 days, were divided into four groups. Group 1 underwent a sham operation, group 2 was surgically ovariectomized and group 3 was given a GnRHa (leuprorelin acetate for depot suspension) s.c. injection every 30 days. Group 3 was further divided into three subgroups: rats were administered GnRHa for 12 months (GnRHa 12M), 6 months (GnRHa 6M) or 3 months (GnRHa 3M). Group 4 served as a basal control. The bone mineral density (BMD) of lumbar vertebrae and femoral bone, measured by dual-energy X-ray absorptiometry, and the serum bone metabolic parameters were determined every 45–90 days. The bone histomorphometry of lumbar vertebra was measured on days 180 and 360 after surgery.

GnRHa 12M rats showed significantly lower BMD of vertebrae and femoral bone, lower bone volume and higher bone turnover compared with sham-operated rats and those with secondary hyperparathyroidism on days 180 and 360. Their time-course for changes in bone metabolism was almost the same as that of OVX rats. GnRHa-discontinued rats showed a recovery of bone turnover. The recovery of BMD in GnRHa 6M rats was smaller than that of GnRHa 3M rats after GnRHa discontinuance. The bone volume for GnRHa 6M rats was significantly lower than that for GnRHa 3M on day 360.

Journal of Endocrinology (1993) 138, 115–125

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H. Okamura, K. Yamamoto, S. Hayashi, A. Kuroiwa, and M. Muramatsu


A rat oestrogen receptor-β-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct. Scatchard analysis of [3H]oestradiol-17β binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1·49 nmol/l. The relative molecular weight (M r) of the fusion protein was determined as 180 000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor.

The protein was isolated by means of SDS-PAGE and subsequent electroblotting. By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit. Binding of [3H]oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner. The antibody was also able to recognize the oestrogen receptor occupied by [3H]oestradiol. Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone. In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of M r 67 000, the size of the oestrogen receptor, was detected by the antibody. Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain. The immunoreactivity was restricted exclusively to cell nuclei in both tissues.

These results demonstrate that the polyclonal antibody obtained in the present study was specific to the oestrogen receptor, and that it would be a powerful tool to detect and analyse the receptors in various target tissues for oestrogen.

Journal of Endocrinology (1992) 135, 333–341

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Y Koshihara, K Hoshi, R Okawara, H Ishibashi, and S Yamamoto

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.

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H Ikawa, K Yamamoto, Y Takahashi, N Ueda, Y Hayashi, S Yamamoto, K Ishimura, M Irahara, and T Aono


Arachidonate 12-lipoxygenase, which oxygenates positions 12 and 13 of arachidonic and linoleic acids, is present in porcine anterior pituitary cells. Colocalization of the 12-lipoxygenase with various pituitary hormones was examined by immunohistochemical double-staining using antibodies against 12-lipoxygenase and various anterior pituitary hormones. Under light microscopy, approximately 7% of the cells producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were positive for 12-lipoxygenase, whereas the enzyme was detected in less than 2% of the cells producing thyrotrophin, prolactin, growth hormone (GH), and adrenocorticotrophin. In an attempt to examine the participation of 12-lipoxygenase metabolites in pituitary hormone release, we incubated the primary culture of porcine anterior pituitary cells with 12-hydroperoxy-arachidonic acid or 13-hydroperoxy-linoleic acid. Significant stimulation of LH and FSH release by these hydroperoxides was observed at 10 μm in a time-dependent manner. At doses around 10 μm these compounds produced responses of similar magnitude to 1 nm gonadotrophin-releasing hormone (GnRH), but higher concentrations (30 μm) of the compounds were required for GH release. In contrast, 12-hydroxy-arachidonic and 13-hydroxy-linoleic acids were almost ineffective. Furthermore, the gonadotrophin release by 1 nm GnRH was inhibited by nordihydroguaiaretic acid (a lipoxygenase inhibitor) with an IC50 of about 5 μm. Thus, the hydroperoxy (but not hydroxy) products of 12-lipoxygenase may be involved in the release of pituitary hormones especially LH and FSH.

Journal of Endocrinology (1996) 148, 33–41

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T Kotani, K Umeki, I Yamamoto, H Maesaka, K Tachibana, and S Ohtaki

In this study we describe a novel mutation of the thyroid peroxidase (TPO) gene that resulted in a total iodide organification defect. TPO activity and thyroxine formation in thyroglobulin in the thyroid gland of the patient were below the limits of detection. However, TPO mRNA was detectable at a similar size and concentration as compared with normal thyroid tissues when measured by Northern blot analysis. Sequence analysis of the TPO gene showed the presence of two mutations, a missense mutation in exon 7 and C insertion in exon 14. These mutations were heterozygous and located in different alleles. The latter mutation has already been reported as one of the mutations of the TPO gene resulting in total iodide organification defect. The former mutation was further analysed by mRNA transfection studies in which mutated mRNA was transfected to CHO-K1 cells by electroporation. The results of transfection studies showed that the cells transfected with mutated mRNA expressed similar size TPO molecules to those of cells transfected with wild-type mRNA but that they lacked TPO activity. The two mutations of the TPO gene resulting in the total iodide organification defect in the patient cosegregated from her parents.

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K Ono, T Akatsu, T Murakami, M Nishikawa, M Yamamoto, N Kugai, K Motoyoshi, and N Nagata

Of various PGs, PGE1 and PGE2 are shown to be the most potent stimulators of osteoclastogenesis in vitro. PGE receptors have been classified into four subtypes, EP1-EP4. Little is known about PGE receptors functioning in bone cells. In this study, using mouse marrow culture, we investigated which PGE receptors are important in osteoclast-like cell (OCL) formation induced by PGE. 11-deoxy-PGE1 (EP2, EP3 and EP4 agonist) stimulated OCL formation potently. Butaprost (EP2 agonist) stimulated it slightly, while sulprostone (EP1 and EP3 agonist) and ONO-AP-324-01 (EP3 agonist) did not. AH23848B (EP4 antagonist) inhibited PGE2-induced OCL formation in a dose-dependent manner. The expression of EP4 mRNA in mouse bone marrow was confirmed by RT-PCR. The results indicate an important role of EP4 in PGE2-induced OCL formation in marrow cultures and suggest therapeutic potential of EP4 antagonists in some clinical conditions with accelerated bone resorption.

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T Takeda, H Kurachi, T Yamamoto, Y Nishio, Y Nakatsuji, K Morishige, A Miyake, and Y Murata

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

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


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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T Takeda, M Sakata, R Minekawa, T Yamamoto, M Hayashi, K Tasaka, and Y Murata

Breast milk has non-nutritional protective effects on recipient infants. It has been speculated that bioactive substances present in human milk have important roles in protecting infants. However, the mechanisms by which such substances protect newborns are unclear. Therefore, we analyzed the growth-promoting activity of human milk and the intracellular signaling mechanism thereof using human fetal small intestinal (FHS 74 Int) cells. Epidermal growth factor (EGF) stimulated the proliferation of these cells. However, this stimulation was less effective than that of aqueous milk (5% vol/vol). The bioactivity of human milk was heat stable but protease sensitive. EGF receptor tyrosine kinase inhibitor did not repress the milk-induced growth-promoting effect on fetal small intestinal cells. Regarding the intracellular signaling pathway, the milk-induced cell proliferation pathway was tyrosine kinase dependent but was neither mitogen-activated protein (MAP) kinase nor phosphatidylinositol-3 (PI-3) kinase dependent. On the other hand, EGF-induced cell proliferation was tyrosine kinase, MAP kinase, and PI-3 kinase dependent. Rapid tyrosine phosphorylation of several intracellular proteins was detected after milk stimulation. Furthermore, the time course of phosphorylation induced by milk was different from that induced by EGF. The sizes of the proteins phosphorylated in response to milk were different from those of the Shc proteins phosphorylated in response to EGF. These results suggest that human milk induces fetal intestinal cell proliferation through a unique tyrosine kinase pathway different from the EGF receptor signaling pathway.