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K Tamura, T Kawaguchi, and H Kogo

Interleukin-6 (IL-6) and its receptor components have been shown to be present in rat follicular granulosa cells. The present study was designed to examine the effect of this cytokine on changes in expression of the luteinizing hormone receptor (LHR) messenger RNA and of the steroidogenic enzyme, CYP11A1 (cytochrome P450 scc) in an in vitro model of granulosa cell maturation. Ovarian granulosa cells harvested from immature rats 2 days after treatment with equine chorionic gonadotropin were cultured for 48 h in media containing 10% fetal bovine serum. They were then transferred to a chemically defined serum-free medium and cultured for an additional 72 h. Within 24 h of transfer, the expressions of LHR and CYP11A1 mRNA increased significantly and remained increased for 72 h. The cells responded to exposure to FSH, but not LH, by an increase in production of cAMP before the additional 72 h of culture. The cAMP response to LH was attained within 24 h and persisted for 72 h, whereas the response to FSH decreased continuously with time. Inclusion of IL-6 in the culture medium caused a dose-dependent decrease in expression of LHR mRNA, in addition to a decrease in the cAMP response to LH. Immunoneutralization of endogenous granulosa cell IL-6 resulted in an increase in expression of LHR mRNA, but not CYP11A1 mRNA. The results are consistent with the view that IL-6 may have a physiological role in the maturation of ovarian follicles by modulating the attainment of the LHR in granulosa cells.

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T Sakurai, K Tamura, and H Kogo

Vascular endothelial growth factor (VEGF) is known to be necessary for the vascularization of the developing corpus luteum. Our recent data suggested that cyclooxygenase-II (COX-II) may play a role in the formation of vascular plexuses in developing corpora lutea of the rat. Here we examined the relationship between VEGF and the expression of prostaglandin (PG)- metabolizing enzymes in rat ovarian luteal cells. VEGF treatment caused a dose-dependent increase in the expression of COX-II and membrane-associated PGE synthase (mPGES) mRNA in cultured rat luteal cells. However, pretreatment of the luteal cells with a selective COX-II inhibitor, NS-398, abolished the VEGF-enhanced mPGES mRNA expression. VEGF also increased PGE2 secretion. Conversely, PGE2 dose-dependently stimulated VEGF mRNA expression. Furthermore, VEGF induced VEGF mRNA expression, but this effect was abolished by NS-398 pretreatment. These findings suggest that VEGF enhances PGE2 production by stimulating COX-II and mPGES expression in rat corpus luteum and that the effect of VEGF on luteal cells may be partially mediated by this stimulation of PGE2 production.

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K. Mikami, M. Omura, Y. Tamura, and S. Yoshida


The site of action of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) in ACTH-induced stimulation of steroidogenesis was examined in rat adrenocortical fasciculata cells. Prior addition of AA861, a specific inhibitor of 5-lipoxygenase, had no significant effect on cyclic AMP-dependent protein kinase activity and cholesterol esterase activities, when stimulated by ACTH in adrenocortical cells, compared with that stimulated by ACTH alone. Cholesterol accumulation in the mitochondria of cells treated with ACTH and cycloheximide was also not altered by pretreatment with AA861. We found, however, that pregnenolone formation, stimulated by ACTH, decreased in a dose-dependent manner when cells were pretreated with AA861. The inhibition of ACTH-stimulated pregnenolone formation by treatment with AA861 was restored only by prior addition of 5-HPETE. Furthermore, addition of AA861 also did not affect the conversion of pregnenolone into corticosterone.

In conclusion, 5-HPETE may act at the level of the mitochondria in ACTH-induced steroidogenesis in rat adrenal fasciculata cells.

Journal of Endocrinology (1990) 125, 89–96

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N. Sugino, H. Tamura, Y. Nakamura, K. Ueda, and H. Kato


The present study investigated possible sites through which ACTH or corticosterone inhibit progesterone secretion in pregnant rats, and the role of placental factors in blocking the inhibitory effect. The number of conceptuses was adjusted to one (1C group) or more than ten (FC group) on day 7 of pregnancy by aspirating the desired number. Serum concentrations of progesterone, testosterone and oestradiol were significantly (P<0·01) lower on day 15 in the 1C group than in the FC group. Corpora lutea (CL) obtained on day 15 were incubated for 6 h with corticosterone or ACTH. Corticosterone (1 μmol/l) significantly (P<0·05) inhibited progesterone secretion in the IC group but not in the FC group. The inhibitory effect of corticosterone in the IC group was completely blocked by co-addition of 1 μmol testosterone/l or 1 μmol oestradiol/l but not by 1 μmol dihydrotestosterone/l. ACTH (1 μg/l–1 mg/l) had no direct effect on progesterone secretion in either the IC or the FC groups, although ACTH apparently decreases progesterone secretion in vivo. Placentae obtained from rats of the FC group on day 15 were incubated for 24 h with or without ACTH (1 mg/l). The supernatant after placental incubation without ACTH significantly (P<0·01) increased progesterone secretion by the CL in both the IC and FC groups, and also eliminated the inhibitory effect of corticosterone in the IC group. The supernatant after placental incubation with ACTH also increased progesterone secretion in the FC group as effectively as the supernatant from the control incubation, but it had no effect in the IC group. It is concluded that corticosterone directly inhibits progesterone secretion by the CL, whereas the inhibitory effect of ACTH is mediated through the placenta. The results indicate that these inhibitory effects of corticosterone or ACTH are eliminated if the CL has been exposed to enough placental hormones before day 15 of pregnancy.

Journal of Endocrinology (1991) 129, 405–410

Free access

K Tamura, N Yokoyama, Y Sumida, T Fujita, E Chiba, N Tamura, S Kobayashi, M Kihara, K Murakami, M Horiuchi, and S Umemura

This study examined whether type 1 angiotensin II receptor (AT1) and angiotensin-converting enzyme (ACE) mRNAs are regulated during dietary salt loading in angiotensinogen gene-knockout (Atg-/-) mice which are genetically deficient in endogenous production of angiotensin II. Wild-type (Atg+/+) and Atg-/- mice were fed a normal-salt (0.3% NaCl) or a high-salt (4% NaCl) diet for 2 weeks. The mRNA levels were measured by Northern blot analysis. In Atg+/+ mice, concentrations of plasma angiotensin peptides were decreased by salt loading, whereas the treatment increased the brainstem, cardiac, pulmonary, renal cortex, gastric and intestinal AT1 mRNA levels. Salt loading also enhanced renal cortex ACE mRNA levels in Atg+/+ mice. Although plasma angiotensin peptides and urinary aldosterone excretion were not detected in Atg-/- mice, salt loading increased blood pressure in Atg-/- mice. In Atg-/- mice, pulmonary, renal cortex, gastric and intestinal AT1, and renal cortex and intestinal ACE mRNA levels were higher than those in Atg+/+ mice. However, salt loading upregulated AT1 mRNA expression only in the liver of Atg-/- mice, and the treatment did not affect ACE mRNA levels in Atg-/- mice. Furthermore, although the levels of ACE enzymatic activity showed the same trend with the ACE mRNA levels in the lung, renal cortex and intestine of both Atg-/- and Atg+/+ mice, the results of radioligand binding assay showed that cardiac expression of AT1 protein was regulated differently from AT1 mRNA expression both in Atg-/- and Atg+/+ mice. Thus, expression of AT1 and ACE is regulated by salt loading in a tissue-specific manner that appears to be mediated, at least partly, by a mechanism other than changes in the circulating or tissue levels of angiotensin peptides.

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Y Nakamura, H Tamura, M Ono, K Shimamura, N Sugino, F Numa, K Ueda, and H Kato


The purpose of this study was to examine the possible mechanism through which RU486 induces luteolysis during the late-luteal phase in pseudopregnant (PSP) rats. PSP rats received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight) or sesame oil alone once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10, 11 and 12 and assayed for progesterone content. To examine the possible action of RU486 through a uterine and/or a pituitary (prolactin-dependent) mechanism, PSP rats and chronic hysterectomized PSP rats which had been hysterectomized before PSP induction received a subcutaneous injection of RU486 in sesame oil (5 mg/kg body weight), sesame oil alone, prolactin in 50% polyvinylpyrrolidone (15 IU/day), or RU486 and prolactin once a day between day 9 and day 11 of pseudopregnancy. Serial blood samples were collected on days 5, 9, 10 and 11 and assayed for progesterone content. Blood samples were also collected at 0400 h on day 12 and used for prolactin and progesterone determinations. To examine the direct effect of RU486 on corpus luteum and/or pituitary, hysterectomized rats underwent hypophysectomy and pituitary autotransplantation on dioestrus 1 and received a subcutaneous injection of RU486 in sesame oil or sesame oil alone for 3 days between day 21 and day 23 after surgery. Serial blood samples were collected on days 10, 21, 22, 23 and 24 and assayed for progesterone and prolactin contents.

In ordinary PSP rats, serum progesterone levels were significantly (P<0·01) lower in the RU486-treated group than in the control group (9 ± 1 vs 53 ± 7 ng/ml; mean ± s.e.m.) on day 11. Serum prolactin levels at 0400 h on day 12 of pseudopregnancy were significantly (P<0·05) lower in the RU486-treated group than in the control group (16 ±4 vs 154 ±44 ng/ml; mean ± s.e.m.). The concomitant prolactin treatment reversed the luteolytic effects of RU486 on day 11 of pseudopregnancy. In hysterectomized PSP rats, RU486 also suppressed serum prolactin levels, and the concomitant prolactin treatment again reversed the luteolytic effects of RU486. In hysterectomized rats which were hypophysectomized and pituitary autotransplanted, RU486 treatment did not induce any significant changes in serum progesterone and prolactin levels.

These results indicated that RU486 induced luteolysis during the late-luteal phase in PSP rats by suppressing prolactin secretion via a hypothalamic mechanism.

Journal of Endocrinology (1996) 150, 93–98

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F. Miyauchi, H. Kato, H. Yamashita, K. Ueda, H. Tamura, T. Mano, and T. Torigoe


The effects of a conceptus-derived substance on the activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-HSD in the ovary were studied in the rat. On day 7 of pregnancy (day 1 = insemination), rats were laparotomized and the desired number of conceptuses was aspirated from the uterus; thus, rats carrying one, two, three, four, five to seven or eight to ten conceptuses were prepared. They were autopsied on day 15 and 3β-HSD and 20α-HSD activity in the corpus luteum (CL) or non-luteal ovarian tissue (NLO) was determined. Conceptus number was directly related to 3β-HSD and inversely related to 20α-HSD activity in the CL. The serum progesterone level and CL weight were also directly related to conceptus number. Neither 3β-HSD nor 20α-HSD activity in the NLO was affected by conceptus number. These results indicated that 3β-HSD and 20α-HSD in the CL are probably regulated by placental hormone secreted in proportion to the number of conceptuses; in the NLO these enzymes may be controlled by a different mechanism.

J. Endocr. (1984) 101, 285–288

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K. Tamura, M. Kobayashi, S. Suzuki, Y. Ishii, S. Koyama, H. Yamada, K. Hashimoto, M. Niwa, and F. Shibayama


Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1–25 μg/l, compared with the range of 1–50 μg/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.

Journal of Endocrinology (1990) 125, 327–335