卵巣・顆粒膜細胞と子宮内膜からの黄体機能調節機構解明へのアプローチ(シンポジウム1 黄体機能の調節とその異常)
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The amount of hormones secreted from the corpus luteum in the luteal phase has been shown to be largely influenced by the process of follicle maturation and early luteinization. Additionally, the full maturation of secretory endometrium is affected by various, factors, especially sex steroids secreted from the corpus luteum and some physiological active substances. Recently, some vasoactive substances derived from vascular endothelial cells have been implicated in a variety of physiological and pathological processes. The present study was undertaken to investigate the effects of endothelin (ET), that is the potent vasoconstrictive peptide, and nitric. oxide (NO) that is the free radical gas believed to be endothelium-derived relaxing factor, on the luteinization induced by gonadotropin (Gn) in granulosa cells and on the differentiation induced by sex steroid hormones in human endometrial cells of the secretory phase in order to clarify the control mechanism of luteal function. At first, human luteinized granulosa cells (HGC) were obtained by follicular aspiration from women undergoing IVF-ET, and porcine granulosa cells (PGC) were obtained from small (<3mm) (S-PGC) and medium-sized follicles (3-5mm) (M-PGC) by needle aspiration method. Secondly, human endometrial tissues at different stages of regular menstrual cycle were scraped from the uterus at hysterectomies. Informed consent was obtained from each women. These tissues were used for the experiments of molecular biology and also used for the cell-culture experiment of endometrial stromal cells enzymatically dispersed. In the first experiment, a large amount of ET-1 was identified in human follicular fluids, and ET-1 levels in them obtained from small follicles (<15mm) were significantly higher than those of the larger follicles. ET-1 attenuated basal (unstimulated) progesterone (P) secretion in HGC and inhibited Gn-stimulated P secretion in HGC and PGC. ET-1 significantly enhanced cell proliferation and intracellular Ca@S12+@E1 concentrations in HGC and PGC, and decreased intracellular cAMP accumulation in Gn-stimulated PGC. Messenger RNAs for prepro-ET-1, ET@S2A@E2 and ET@S2B@E2 receptor were present in HGC. We also investigated the effect of NO/NO synthase (NOS) on the control of luteinization in cultured PGC. An NO donor, NOC 18, significanly suppressed basal and Gn-stimulated P release from S-PGC. In contrast, NOC18 did not significantly change the basal and Gn-stimulated P release from M-PGC. NOC18significantly suppressed the estradiol (E@S22@E2) release by inhibiting P450 aromatase activity in basal and Gn-stimulated S-PGC and M-PGC. A competitive blocker of NOS, N-monomethy L-arginine (LNMMA), significantly stimulated the basal P and E@S22@E2 release in M-PGC, but no significant effect on the basal release in S-PGC. In the second experiments using human endometrium, Northern blot analysis revealed expression of prepro-ET-1 mRNA in endometrial tissues, and the concentration of the secretory phase was less than in the ovulatory and proliferative phases. Immunoreactive ET-1 was secreted into the medium, and E @S22@E2 plus P significantly attenuated ET-1 release in cultured endometrial stromal cells. The concentrations of ET@S1A@E1 and ET@S1B@E1 receptor mRNA in the secretory phase were least among the stages of menstrual cycle. In decidualized endometrial cells induced by E@S22@E2 and P in vetro, ET-1 release decreased and PRL release increased in a time-dependent manner, and there was a significant negative correlation between the release of ET-1 and that of PRL. WE also investigated the effect of NO/NOS on the differentiation of human endometrial cells. Nested RT-PCR produced one intense band of human inducible (i) NOS mRNA and human endothelial (e) NOS mRNA from total RNA extracted from endometrium and the cultured endometrial stromal cells. However, Northern blot analysis revealed no expression of iNOS mRNA in human endmetrium of normal menstrual cycle. On t
- 1998-08-01
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