Action of nitric oxide on the mammalian ovary and molecular biological approach on its signal transduction

本研究利用猪颗粒细胞和卵丘-卵母细胞复合体(COC)体外培养系统，研究了哺乳动物卵巢中一氧化氮(NO)的合成及其功能：(1)NO代谢产物(亚硝酸盐和硝酸盐)的测定；(2)NO供体(NOC18)和NO拮抗剂(Carxy-PTIO)对黄体激素受体(LH-R)基因表达的影响；(3)Western blotting检测NO合成酶；(4)NO合成的荧光观察。猪卵泡颗粒细胞经卵泡刺激素(FSH)处理48h后成熟，亚硝酸盐和硝酸盐水平在40~48h内增加2倍，这与鸟苷3‘，5’-环单磷酸的增加是一致的。免疫印迹显示，在发育中的颗粒细胞中检测到内皮型一氧化氮合酶，但未检测到诱导型异构体，这表明一氧化氮来自内皮型异构体。这些细胞暴露在…中在不产生之前或之后(30h或46h)不加拮抗剂或不加供体，然后在48h用黄体生成素(LH)刺激细胞。去除内源性NO会严重影响黄体生成素的合成，而NO供体对黄体生成素的合成无明显影响。在转录因子基因的半定量逆转录聚合酶链式反应中，去除内源性NO可导致c-fos表达降低50%，c-jun表达增加250%，ATF-4表达无变化。相反，没有供体对转录因子基因的表达没有显著影响。因此，NO的瞬时产生可能在转录因子基因的表达以及随后颗粒细胞向黄体细胞的转化中起关键作用。除了颗粒细胞外，卵母细胞也合成了NO。卵母细胞周围的卵丘细胞在FSH刺激下呈剂量依赖性增殖，在FSH刺激后3d达到最大值。免疫印迹法在卵母细胞中检测到一氧化氮合酶的两种异构体，但在增殖的卵丘细胞中未检测到。在发育过程中，内皮细胞和可诱导的异构体都被下调，尤其是在存在或不存在FSH的情况下，可诱导的异构体急剧减少。在NO合成的荧光观察中，加入1 mM的L-精氨酸(激动剂)可使卵母细胞迅速合成NO，而加入拮抗剂L-NNA则不能。这些观察表明，卵母细胞是NO的主要来源，NO可能与卵母细胞成熟有关。较少

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 … More 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