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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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T. Endo, H. Watanabe, H. Yamamoto, S. Tanaka, and M. Hashimoto


While prostaglandin F (PGF) has been thought to be a natural luteolysin in non-primates, a luteolytic effect in the human corpus luteum is less evident. We therefore investigated the action of PGF on monolayer cultures of human luteal cells obtained from mid-luteal phase corpora lutea.

PGF increased basal and human chorionic gonadotrophin (hCG)-stimulated progesterone production by human cultured luteal cells. A potent tumour-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), also stimulated progesterone production by cultured human luteal cells.

Although human luteal cells were incubated for 24 h with PMA, hCG was still able to stimulate the production of progesterone by PMA-pretreated cells. However, PMA pretreatment blocked the ability of PGF to stimulate progesterone production. It is possible that the luteotrophic effect of PGF may be mediated, in part, by the activation of protein kinase C.

Addition of PGF to suspensions of human luteal cells preincubated with myo-[2-3H]inositol promoted an increase in labelled inositol phosphates. PGF also rapidly increased intracellular free Ca2+ in human luteal cells loaded with the fluorescent Ca2+ probe, fura-2.

We conclude that PGF and PMA stimulate progesterone production and that PGF increases the intracellular free calcium and inositol phosphates of human cultured luteal cells in the mid-luteal phase.

Journal of Endocrinology (1992) 133, 451–458

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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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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. Tamura, J. Kitawaki, T. Yamamoto, Y. Osawa, S. Kominami, S. Takemorit, and H. Okada


Immunohistochemical localization of 17α-hydroxylase/C17-20 lyase (P-45017α,lyase) and aromatase cytochrome P-450 (P-450arom) in polycystic ovary (PCO) syndrome was studied using specific polyclonal antibodies which had been raised against the corresponding enzymes. In the majority of follicles that were atretic and smaller than 7 mm in diameter, theca interna cells showed high P-45017α,lyase immunoreaction, while small numbers of granulosa cells showed little P-450arom immunoreaction. In some atretic follicles that were larger than 11 mm in diameter, the hyperplastic theca interna cell layer showed high immunoreaction to P-45017α,lyase, while the poorly proliferated granulosa cell layer showed a mixture of weak and negative immunoreaction to P-450arom. No immunoreaction to P-45017α,lyase or P-450arom was recognized in PCO stroma. These findings suggest that the theca interna cells and the granulosa cells from PCOs show abnormal steroidogenic function, while the localization of P-45017α,lyase and P-450arom in PCOs was essentially identical to that in the normal ovary. Theca interna cells in PCO atretic follicles are the main site of excess androgen production.

Journal of Endocrinology (1993) 139, 503–509

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T. Tamura, J. Kitawaki, T. Yamamoto, Y. Osawa, S. Kominami, S. Takemori, and H. Okada


Immunohistochemical localization of 17α-hydroxylase/C17–20 lyase (P-45017α,lyase) and aromatase cytochrome P-450 (P-450arom) in normal human ovaries during the menstrual cycle was studied using specific polyclonal antibodies which were raised against corresponding enzymes. In the follicular phase of matured follicles, P-45017α,lyase was localized in theca interna cells and P-450arom in granulosa cells. P-45017α,lyase was expressed in theca interna cells before P-450arom was expressed in granulosa cells. The corpus luteum showed immunoreactivity to both enzymes and, after menstruation, immunoreactivity decreased gradually until it could not be detected in the corpus albicans. In corpus luteum graviditatis the immunoreactivity continued to be expressed strongly. In some atretic follicles, P-45017α,lyase and/or P-450arom continued to be expressed. In the stromal layer, P-45017α,lyase was detected in secondary interstitial cells, which originated from the theca interna of atretic follicles, and P-450arom was detected in hilar cells. Immunoreactivity to both enzymes was also detected in oocytes of developing follicles. These results are consistent with the two cell theory in the human ovary. They also suggest that androgens and oestrogens are produced not only by follicles and corpora lutea but also by stroma and oocytes.

Journal of Endocrinology (1992) 135, 589–595

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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.

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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.