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

此前，我们检测了大鼠生殖器官中锰超氧化物歧化酶mRNA的表达，发现锰超氧化物歧化酶参与了卵巢中类固醇的合成。为了通过整体原位杂交研究其他抗氧化酶在大鼠胚胎中的表达，我们采用多重标记的方法制备了磷脂氢谷胱甘肽过氧化物酶(PHGPx)的RNA探针。综上所述，我们设计了含有正反义序列(93-mer)的5‘-磷酸化寡核苷酸(93-mer)，其中EcoR I或Hind III酶切位点作为突起的粘连末端(共99-mer)。这些都是作为纯化和冻干产品(&gt；99%)获得的。结果表明，PHGPx在成年大鼠卵巢中未检测到PHGPx基因的表达，而在出生后的睾丸组织中检测到PHGPx基因的表达，该基因在精子发生过程中有阶段特异性表达，在第7至13步的精子细胞中未检测到表达，提示PHGPx基因在精子发生过程中参与精子发生，同时也参与清除活性氧的作用。

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.