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

一氧化氮对哺乳动物卵巢的作用及其信号转导的分子生物学方法

• 批准号: 09460139
• 负责人: HATTORI Masa-aki
• 金额: $ 6.85万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460139/
• 关键词: granulosa cells; oocyte; nitric oxide; nitric oxide synthases; transcription factor genes; gene expression; RT-PCR; cell differentiation; 果位膜細胞; 酸化窒素貿易; PT=PCP; 顆粒膜細胞培養; 転写調節因子; 成長因子; 細胞情報伝達

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

本研究利用猪颗粒细胞和卵丘卵母细胞复合物 (COC) 的体外培养系统，阐明哺乳动物卵巢中一氧化氮 (NO) 的合成及其功能，具体如下：(1) NO 代谢物（亚硝酸盐和硝酸盐）的测量，(2) NO 供体 (NOC18) 和 NO 拮抗剂的作用 （羧基-PTIO）对黄体生成素受体（LH-R）基因表达的影响，（3）Western blotting检测NO合酶，（4）NO合成荧光观察。从猪卵巢卵泡制备的颗粒细胞通过卵泡刺激素（FSH）处理48小时而成熟。 40 小时至 48 小时期间，亚硝酸盐和硝酸盐水平增加至 2 倍，这与 3',5'-环单磷酸鸟苷的增加一致。蛋白质印迹显示，在发育中的颗粒细胞中检测到内皮NO合酶，但未检测到诱导型同工型，表明NO源自内皮同工型。在 NO 生成之前或之后（30 小时或 46 小时），将细胞暴露于 NO 拮抗剂或 NO 供体，然后在 48 小时用黄体生成素 (LH) 刺激细胞。内源性 NO 的去除会严重损害 LH 诱导的孕酮合成，而 NO 供体对合成没有显着影响。在转录因子基因的半定量逆转录酶 PCR 中，去除内源性 NO 会导致 c-fos 表达减少 50%，c-jun 增加 250%，而 ATF-4 没有变化。相比之下，NO供体对转录因子基因的表达没有显着影响。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发挥关键作用。除颗粒细胞外，卵母细胞也合成 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，并且其增殖在FSH刺激后3天达到最大水平。通过免疫印迹，在卵母细胞中检测到 No 合酶的两种亚型，但在增殖的卵丘细胞中未检测到。内皮同工型和诱导同工型在发育过程中都会下调，尤其是在存在或不存在 FSH 的情况下，诱导同工型会急剧减少。在NO合成的荧光观察中，通过添加1mM L-精氨酸（激动剂），卵母细胞NO迅速合成，但L-NNA（拮抗剂）则不合成。这些观察结果表明卵母细胞是 NO 的主要来源，并且 NO 可能与卵母细胞成熟有关。较少的

项目成果

Hattori M-A, Nishida N, Takase K, Kato Y & Fujihara N: "Nitric oxide synthesis by porcine oocytes: its regulation and function during follicular development."Biol Reprod (Suppl). 60. 445 (1999)

服部 M-A、西田 N、高濑 K、加藤 Y

Fujihara N,Inada S,Hasebe M,YamaguchiH & Hattori M-A: "In Vivo gene transfer to chick embryos via spermatozoa and primordial germ cells." Nadanishi Printing Co.(H.Miyamoto,N.Manabe,eds), 3 (1998)

藤原 N、稻田 S、长谷部 M、山口 H

Takasue K,Nishida N,Hattori M-AandFujihara N: "Fluorecent observation of nitric oxide synthesized by the porcine oocyte"Biology of Reproduction,(Supplement 1). 60. 447 (1999)

Takasue K，Nishida N，Hattori M-A和Fujihara N：“猪卵母细胞合成一氧化氮的荧光观察”生殖生物学，（补充1）。

Fujihara N,Inada S,Hasebe M,Yamaguchi H&Hattori M-A: "In Vivo gene transfer to chick embryos via spermatozoa and primordial germ cells."Nakanishi Printing Co.(H.Miyamoto,N.Manabe,eds). 3 (1998)

藤原 N、稻田 S、长谷部 M、山口 H

服部眞彰: "卵巣顆粒膜細胞の成熟分化に伴って発現するガングリオシドGM3:細胞認識リガンドあるいは細胞内シグナリング調節因子として機能?" 化学と生物. 36(9). 561-562 (1998)

Masaaki Hattori：“神经节苷脂 GM3 在卵巢颗粒细胞的成熟和分化过程中表达：它是否充当细胞识别配体或细胞内信号调节剂？” 36(9) (1998)。