IDENTIFICATION AND CHARACTERIZATION OF ANTIOXIDANT ENZYME MESSENGER-RNA IN THE RAT EPIDIDYMIS

Spermatozoa are highly sensitive to oxidative stress. The epididymis, a natural sperm reservoir, has maturational and storage functions. The epididymis may also protect spermatozoa from oxidative injury by elab orating scavengers of reactive oxygen species (ROS). Therefore, we hav e evaluated the mRNA expression of antioxidant enzymes in the normal r at epididymis and the effects of efferent duct ligation on the express ion of these enzymes. Adult rat epididymides were harvested, divided i nto caput, corpus and cauda and processed for RNA extraction. Addition al adult rats were subjected to unilateral efferent duct Ligation and the epididymides harvested at 1, 4, 8, 16 or 28 days after the procedu re. Antioxidant enzyme mRNA expression was assessed by Northern blot a nalysis using P-32- labelled DNA probes derived from known cDNA sequen ces for classical cellular glutathione peroxidase (GSHPx), phospholipi d hydroperoxide glutathione peroxidase (PHGPX), secretory epididymal g lutathione peroxidase (E-GPX), copper-zinc superoxide dismutase (SOD), secretory epididymal superoxide dismutase (E-SOD) and catalase. Speci fic mRNA levels were measured, with gene expression evaluated relative to total RNA, not per organ. Variations in lane loading were controll ed by measuring the levels of 28S ribosomal RNA. GSHPx, PHGPX, SOD and catalase mRNA were detected in the caput, corpus and cauda epididymis . E-GPX mRNA was only detected in the caput, whereas E-SOD mRNA was pr imarily detected in the corpus. At 28 days after efferent duct ligatio n, epididymal weight decreased by 34% relative to controls (p < 0.05). With the exception of PHGPX, the relative mRNA levels of the antioxid ant enzymes studied did not change after efferent duct ligation. This study demonstrates that mRNAs for multiple antioxidant enzymes are exp ressed in the epididymis and that the relative expression of these enz ymes remains largely unchanged in response to efferent duct ligation. Taken together, these results suggest that antioxidant enzymes may pla y an important, region-specific role in epididymal function. Expressio n of the secretory antioxidant enzymes E-SOD and E-GPX is region-speci fic, indicating that the need for antioxidant enzymes may vary along t he length of the epididymis.