| Budget Amount *help |
¥10,700,000 (Direct Cost: ¥10,700,000)
Fiscal Year 1998: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1997: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Research Abstract |
The present study has been performed to clarify nitric oxide (NO) synthesis and its function in the mammalian ovaries using in vitro culture systems of porcine granulosa cells and cumulus-oocyte complexes (COC) as follows: (1) measurements of NO metabolites (nitrite and nitrate), (2) effects of NO donor (NOC18) and NO antagonist (carboxy-PTIO) on the expression of luteinizing hormone receptor (LH-R) gene, (3) detection of NO synthases by Western blots, and (4) fluorescence observation of NO synthesis. The granulosa cells prepared from the porcine ovarian follicles are matured by treatment with follicle-stimulating hormone (FSH) for 48 h. Nitrite and nitrate levels increase to 2-fold during 40 h to 48 h that is consistent with an increase of guanosine 3',5'-cyclic monophosphate. The Western blots reveal that endothelial NO synthase is detected in the developing granulosa cells, but not the inducible isoform, indicating NO is derived from the endothelial isoform. The cells are exposed to
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either NO antagonist or NO donor before or after NO generation (30 h or 46 h), then the cells are stimulated at 48 h with luteinizing hormone (LH). Removal of endogenous NO induces serious impairment in the LH-induced synthesis of progesterone, whereas NO donor has no significant effect on the synthesis. In the semiquantitative reverse transcriptase-PCR of the transcription factor genes, removal of endogenous NO results in a 50% reduction of c-fos expression, a 250% increase of c-jun and no changes of ATF-4. In contrast, NO donor has no significant effects on the expression of transcription factor genes. Consequently, the transient generation of NO may have critical roles in the expression of transcription factor genes and thereafter transformation of the granulosa cell to the luteal cell. In addition to granulosa cells, NO is also synthesized by the oocytes. The oocyte-surrounding cumulus cells are proliferated dose dependently by FSH stimulation and its proliferation reaches a maximum level 3 days after FSH stimulation. Two isoforms of No synthase are dectected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. Both the endothelial and inducible isoforms are down-regulated during development, and especially a drastic reduction of the inducible isoform are seen in the presence or absence of FSH. In the fluorescence observation of NO synthesis, oocyte NO is rapidly synthesized by addition of 1 mM L-arginine (agonist), but not L-NNA (antagonist). These observations indicate the oocyte is a major source of NO and that NO may be associated with oocyte maturation. Less
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