Analysis of gene expression of reactive oxygen species-related enzymes in rat fetus.

大鼠胎儿活性氧相关酶基因表达分析。

• 批准号: 09670013
• 负责人: NOMURA Takako
• 金额: $ 1.73万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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-09670013/
• 关键词: whole-mount; in situ hybridization; fetus; superoxide; PHGPx; synthetic probe; digoxigenin; testis; Mn-SOD

Previously, we examined the expression of Mn-superoxide dismutase mRNA in rat reproductive organs and found that Mn-SOD was involved in the steroidogenesis in the ovary. To investigate the expression of other antioxidant enzymes in rat fetus by whole-mount in situ hybridization, we prepared RNA probes for phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA using a multiple-labeling method. In brief, we designed 5'-phosphorylated oligonucleotides containing sense and antisense sequences (93-mer) with EcoR I or Hind III restriction sites as protruding cohesive ends (total 99-mer) from reported cDNA sequences in the literature. These were obtained as purified and lyophilized products (>99%). Then, both oligonucleotides were annealed and inserted into vectors and used as templates for in vitro transcription.PHGPx mRNA was not detected in the ovary of adult rat, but was detected in the testis after birth by ISH and whole-mount ISH.The expression was stage-specifically in spermatogenesis and was marked in step 7 to 13 spermatids, The expression in the fetus was not detected.These results suggest that PHGPx is involved in spermatogenesis as well as scavenging reactive oxygen species.

此前，我们检测了大鼠生殖器官中Mn-超氧化物歧化酶mRNA的表达，发现Mn-SOD参与了卵巢中类固醇的生成。为了通过整体原位杂交研究大鼠胎儿中其他抗氧化酶的表达，我们使用多重标记方法制备了磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）mRNA的RNA探针。简而言之，我们根据文献中报道的 cDNA 序列设计了包含有义和反义序列（93 聚体）的 5'-磷酸化寡核苷酸，其中 EcoR I 或 Hind III 限制位点作为突出的粘性末端（总共 99 聚体）。这些是作为纯化和冻干产品获得的（>99%）。然后，将两条寡核苷酸退火并插入载体中，作为体外转录的模板。成年大鼠卵巢中未检测到PHGPx mRNA，但出生后通过ISH和全贴装ISH在睾丸中检测到PHGPx mRNA。在精子发生阶段特异，并在第 7 至 13 步精子细胞中被标记，在胎儿中未检测到表达。这些结果表明 PHGPx 参与精子发生和清除活性氧。

项目成果

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Futami, J.：“人类 DNA 中胰腺型 RNase 和 RNase 抑制剂的组织特异性表达”，16・4（1997 年）。

Junzo Sasaki, Takako Nomura, Hideki Mori, Junko Matsuura, Mayumi Honda, Hitoshi Yamamoto and Hiroki Watanabe: Ovulation and superoxide. (Japanese) Free Radicals in Clinical Medicine. Nihon Igakukan, Inc., Tokyo, (1997)

Junzo Sasaki、Takako Nomura、Hideki Mori、Junko Matsuura、Mayumi Honda、Hitoshi Yamamoto 和 Hiroki Watanabe：排卵和超氧化物。

Sasaki J: "Multiple-labeling of oligonucleotide probes for in situ hybridization." Acta Histochem Cytochem. 31・4. 275-279 (1998)

Sasaki J：“用于原位杂交的寡核苷酸探针的多重标记。Acta Histochem Cytochem”275-279（1998）。

Hanada,H.: "Do reactive oxygen species underlie the mechanism of apoptosis in the tadpole tail?" Free Radical Biology and Medicine. 23・2. 294-301 (1997)

Hanada, H.：“活性氧是蝌蚪尾部细胞凋亡机制的基础吗？” 23・2 (1997)。

Akihiko Kashiwagi, Hideki Hanada, Munehisa Yabuki, Tomoko Kanno, Rumi Ishisaka, Yoshiki Takehara, Junzo Sasaki, Masayasu Inoue and Kozo Utsumi: Molecular mechanism of cell death in the amphibian metamorphosis.SEIMEI TANJOU TO SEIBUTU NO SEIZON SENRYAKU (J

Akihiko Kashiwagi、Hideki Hanada、Munehisa Yabuki、Tomoko Kanno、Rumi Ishisaka、Yoshiki Takehara、Junzo Sasaki、Masayasu Inoue 和 Kozo Utsumi：两栖动物变态细胞死亡的分子机制。SEIMEI TANJOU TO SEIBUTU NO SEIZON SENRYAKU (J