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M Masuda, T Kubota and T Aso

BACKGROUND: We have previously demonstrated that nitric oxide (NO) inhibits steroidogenesis via a cGMP-independent process, by inhibiting P450 aromatase activity in porcine granulosa cells (PGCs) derived from medium-sized (3--5 mm) ovarian follicles (M-PGC). OBJECTIVE: To determine whether the NO/NO synthase (NOS) system exerts any significant effects on steroidogenesis in PGCs derived from small follicles (<3 mm) (S-PGC) in comparison with those derived from medium follicles. DESIGN AND METHODS: PGCs, namely S-PGC and M-PGC, were incubated with the NO donor, NOC18, and a competitive blocker of NOS, N(3)-monomethyl-l-arginine (LNMMA), either alone or in the presence of FSH (200 ng/ml) or hCG (5 IU/ml). RESULTS: NOC18 significantly (P<0.01--0.001) suppressed basal (unstimulated) and gonadotropin-stimulated estradiol (E2) release from both S-PGC and M-PGC in a 2-h culture. NOC18 significantly (P<0.01--0.001) decreased basal and gonadotropin-stimulated progesterone release from S-PGC, but not from M-PGC. In addition, NOC18 significantly (P<0.05--0.001) inhibited aromatase activity in S-PGC. LNMMA had a significantly (P<0.01--0.001) stimulatory effect on the basal release of E2 and progesterone from M-PGC; however, it had no significant effect on basal steroidogenesis in S-PGC in a 24-h culture. In the presence of gonadotropin, LNMMA significantly (P<0.01--0.001) stimulated the release of E2 and progesterone from both S- and M-PGC, and this stimulatory effect was weaker in S-PGC than in M-PGC. These results demonstrate that NO inhibits E2 secretion by directly inhibiting the aromatase activity in S-PGC, as in M-PGC. It has been shown that the NO system suppresses the differentiation of S-PGC; however, the extent of suppression decreased with the progression of follicular growth. In addition, the activity of NOS in S-PGC was weaker than that in M-PGC. CONCLUSION: We strongly suggest that the NO/NOS system in PGC regulates steroidogenesis differently during different phase of follicular development.

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T Kubota, M Taguchi, K Kobayashi, M Masuda and T Aso

The study was undertaken to investigate the interaction between the release of endothelin-1 (ET-1) and prolactin (PRL) from human decidualized endometrial cells, and the effects of transforming growth factor (TGF)-beta 1 on the release of ET-1 and PRL from human decidual cells in the early stage of pregnancy. Stromal cells derived from human endometrial tissues were cultured on a type-1 collagen membrane in serum-free medium with or without 100 nmol/l oestradiol and 1 mumol/l medroxyprogesterone acetate (MPA) for 12 days. In addition, decidual cells of early pregnancy were cultured with or without TGF-beta 1 for 48 h. PRL and ET-1 levels in the media were measured by a specific enzyme immunoassay and RIA respectively. In decidualized endometrial cells induced by ovarian steroid hormones in vitro, PRL release increased and ET-1 release decreased in a time-dependent manner. Ovarian steroid hormones significantly attenuated ET-1 release. In the decidual cells of early pregnancy, TGF-beta 1 significantly attenuated the PRL release, whereas this growth factor dose-dependently increased ET-1 release. There was a significant negative correlation between the release of PRL and that of ET-1. These results demonstrate that the regulation of PRL release is quite different from that of ET-1 release in the human decidualized endometrial cells, and suggest that TGF-beta 1 has significant effects on PRL and ET-1 release in the human decidua of early pregnancy.

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Z Lin, T Kubota, M Masuda and T Aso

The aim of this study was to investigate the influence of the nitric oxide/nitric oxide synthase (NO/NOS) system on the release of endothelin-1 (ET-1) in human endometrial cells. Human endometrial stromal cells in secretory phase were incubated for 72 h in serum-free RPMI 1640 medium in the absence or presence of different concentrations of interleukin-1beta (IL-1beta) and NG-monomethyl-L-arginine (LNMMA), a specific competitive inhibitor of NOS. ET-1 released from the cultured cells into the medium was determined by specific RIA. In all the experiments at various times, IL-1beta significantly increased the release of ET-1. LNMMA significantly attenuated the release of ET-1 when the cells were cultured with both IL-1beta and LNMMA, but LNMMA alone had no effect on ET-1 release. These results suggest that the NO/NOS system in human endometrium is involved in the regulation of ET-1 release via IL-1beta secretion. It can also be inferred that NO and ET-1 control the functions of endometrium in close association with IL-1beta.

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K Kobayashi, T Kubota, T Aso, Y Hirata, T Imai and F Marumo

Adrenomedullin (AM) is a novel vasorelaxant peptide, isolated from human pheochromocytoma. Although AM may be involved in the regulation of the cardiovascular system, a number of other mechanisms are also involved. The present study was undertaken to confirm the presence of AM in human maternal circulation and in placental function during pregnancy. Immunoreactive (ir) AM concentrations in maternal plasma were 3.4+/-0.7fmol/ml (mean+/-s.e. m.) in the first trimester, 3.3+/-1.1fmol/ml in the second trimester, 7.3+/-2.8fmol/ml in the third trimester, 4.1+/-1.9fmol/ml in early puerperium and 3.0+/-0.4fmol/ml in non-pregnant periods; the concentration in the third trimester was significantly greater than those in other periods. Plasma concentrations of estradiol (E(2)), progesterone, human placental lactogen (hPL) and human chorionic gonadotropin (hCG) were also measured, using RIA kits. Significant correlations have been demonstrated between the concentrations of irAM and those of E(2), progesterone and hPL. We therefore examined the expression of AM within the placental tissues using immunohistochemistry and northern blot analysis in order to demonstrate a correlation between the presence of AM in the placenta and maternal plasma. Using immunohistochemistry, we detected AM in the amnion at term and the expression of AM mRNA in human placental tissues using cloned human (h) AM complementary DNA as a probe. This study demonstrates the immunoreactivity of human hAM in maternal plasma during pregnancy, and suggests that hAM in maternal plasma is generated partly from placental tissue.