权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Ovulation and superoxide

排卵和超氧化物

基本信息

批准号：
09671683
负责人：
SASAKI Junzo
金额：
$ 1.6万
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-09671683/
关键词：
in situ hybridization synthetic probe ovulation superoxide PHGPx testis Mn-SOD PHGRx 排卵酵素

项目摘要

The role of superoxide dismutases as scavenger enzymes has been emphasized in oxygen radical studies. However, we reported that Mn-SOD or reactive oxygen species were involved in cellular metabolism inciuding steroidogenesis in the ovaries, To investigate the expression of other antioxidant enzymes during ovulation, we prepared RNA probes for phospholipid hydroperoxide glutathione peroxidase (PHGPx) using a multiple-labeling method. in brief, We designed two 99-base 5'-phosphorylated oligonucleotides, complementary to each other, containing sense and antisense sequences (93-mer) with EcoR I or Hind III restriction sites as protruding cohesive ends (5'-pAATTC-----------A-3', 3'-G-----------TTCGAp-5'), These were obtained as purified and lyophilized products (>99%). Then, both oligonucleotides were annealed and cloned into the pGEM4Z vector. The resultant plasmid DNA was linearized with EcoRI or Hind Ill and used as a template for invitrotranscription. If information regarding the target cDNA sequence is available. synthetic oligonucleotides can be obtained from commercial sources at low cost. This method allowed more efficient incorporation of reporter molecules such as ^<35>S-UTP or digoxigenin-UTP into the oligonucleotide probes than other methods and exhibited excellent results without requiring expertise in molecular biological techniques.PHGPx mRNA was not detected in the ovary of adult rat, but was detected in the testis after birth by in situ hybiridization, The expression was stage-specific in spermatogenesis and was marked in step 7 to 13 spermatids.These results suggest that PHGPx is involved in spermatogenesis as well as scavenging reactive oxygen species.

超氧化物歧化酶作为清除酶的作用在氧自由基的研究中一直受到重视。然而，我们报道Mn-SOD或活性氧参与卵巢的细胞代谢，包括类固醇生成。为了研究排卵期间其他抗氧化酶的表达，我们用多重标记法制备了磷脂氢谷胱甘肽过氧化物酶（PHGPx）的RNA探针。简而言之，我们设计了两个99个碱基的5 ′-磷酸化寡核苷酸，它们彼此互补，含有有义和反义序列（93-mer），EcoR I或Hind III限制性位点作为突出的粘性末端（5 ′-pAATTC-然后，将两种寡核苷酸退火并克隆到pGEM 4 Z载体中。用EcoRI或HindIII线性化所得质粒DNA，并用作体外转录的模板。如果关于靶cDNA序列的信息可用。合成的寡核苷酸可以从商业来源以低成本获得。与其它方法相比，该方法能更有效地将报告分子如β-<35>S-UTP或地高辛-UTP掺入寡核苷酸探针中，且不需要分子生物学技术的专业知识，显示出优异的结果。PHGPx mRNA在成年大鼠卵巢中未检测到，但通过原位杂交在出生后的睾丸中检测到，他的表情是阶段性的-PHGPx在精子发生过程中具有特异性，在第7 ~ 13步精子细胞中有表达，提示PHGPx参与精子发生过程，并具有清除活性氧的功能。