Ovulation and superoxide

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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.