Expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in rat testes

We investigated the expression of reactive oxygen species (ROS)-related enzyme mRNA to clarify the role of ROS in reproduction. In the present study, we investigated the expression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in rat testes by in situ hybridization. To prepare digoxigenin-labeled probes, we obtained 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). Then, both oligonucleotides were annealed and cloned into a pGEM4Z vector. The resultant plasmid DNA was linearized with EcoR I or Hind III and used as a template for the synthesis of labeled sense or antisense riboprobes. PHGPx mRNA was expressed stage-specifically during spermatogenesis in adult rats. It was not expressed during spermatogonia, but its expression first appeared in stage VII pachytene spermatocytes, became evident in stage VIII pachytene spermatocytes and increased gradually until becoming diplotene spermatocytes. It decreased slightly during the metaphase of meiosis, and then gradually increased as the spermatids differentiated. The expression was marked in spermatids between steps 7 and 12, and the maximum expression was observed in step 9-10 spermatids. Expression was diminished completely in step 18 spermatids. These results suggest that PHGPx plays an essential role in spermatogenesis.