Ovulation and superoxide

在氧自由基的研究中，超氧化物歧化酶作为清除酶的作用一直受到重视。为了研究排卵过程中其他抗氧化酶的表达，我们采用多重标记的方法制备了磷脂过氧化氢谷胱甘肽过氧化物酶(PHGPx)的RNA探针。总之，我们设计了两个99碱基的5‘-磷酸化寡核苷酸，含有正义和反义序列(93-mer)，以EcoR I或Hind III酶切位点为突出的粘连末端(5’-pAATTC-TTCGAp-5‘)，得到纯化和冻干的产物(&gt；99%)。然后，将这两个寡核苷酸退火热处理后克隆到pGEM4Z载体中。用EcoRI或Hind Ill线性化后，以此为模板进行转录。如果有关于目标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。与其他方法相比，该方法能更有效地将报告分子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方面的专业知识。PHGPx在成年大鼠卵巢中没有检测到，但在出生后的睾丸中通过原位杂交检测到，在精子发生中具有阶段特异性，并在第7至13步的精子细胞中有标记。这些结果表明，PHGPx参与了精子发生和清除活性氧的过程。

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.