Analysis of gene expression of reactive oxygen species-related enzymes in rat fetus.

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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.