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.

在此之前，我们研究了锰超氧化物歧化酶mRNA在大鼠生殖器官中的表达，发现Mn-SOD参与了卵巢中的类固醇合成。为了研究其他抗氧化酶在大鼠胎儿的整体原位杂交表达，我们用多重标记法制备了磷脂氢谷胱甘肽过氧化物酶（PHGPx）mRNA的RNA探针。简而言之，我们从文献中报道的cDNA序列设计了含有有义和反义序列（93-mer）的5 ′-磷酸化寡核苷酸，其中EcoR I或Hind III限制性位点作为突出的粘性末端（总99-mer）。这些作为纯化和冻干的产物获得（>99%）。PHGPx mRNA在成年大鼠卵巢中未检测到，但在出生后的睾丸中检测到，在精子发生过程中，PHGPx mRNA的表达具有阶段特异性，在第7 ~ 13个精子细胞中均有表达，结果表明，PHGPx参与精子发生过程，并具有清除活性氧自由基的功能。

项目成果

Futami、J.: "Tissue-specific expression of pancreatic-type RNases and RNase inhibitor in humanS." DNA and Cell Biology. 16・4. 413-419 (1997)

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）。

佐々木順造: "「電子顕微鏡基礎技術と応用1997-ナノ世界への道」In situ ハイブリダイゼーション法" 学際企画, 240 (1997)

佐佐木淳三：《电子显微镜基础技术及应用1997-通向纳米世界之路》原位杂交方法，跨学科规划，240（1997）

Nishizaki K,Anniko M,Orita Y,Masuda Y,Yoshino T,Kanda S and Sasaki J: "Programmed cell death in the development of the mouse external auditory canal." Anat Rec. 252. 378-382 (1998)

Nishizaki K、Anniko M、Orita Y、Masuda Y、Yoshino T、Kanda S 和 Sasaki J：“小鼠外耳道发育过程中的程序性细胞死亡。”