Rat testicular extracellular superoxide dismutase: Its purification, cellular distribution, and regulation

Abstract

Using multiple HPLC steps, we have identified and purified a 68-kDa polypeptide (as estimated by gel permeation HPLC) to apparent homogeneity, from primary Sertoli cell-enriched culture medium, that consisted of two monomers of 35 (α chain) and 33 kDa (β chain) on SDS-polyacrylamide gel running under reducing conditions. Partial N-terminal amino acid sequence analysis of these two monomers revealed sequences of NH2-DXGESGVDIADRI (SOD(EX)-α) and NH2-XXDTGESGVDLADXL (SOD(EX)-β), which are identical to rat extracellular superoxide dismutase (SOD(EX)) with the exceptions that SOD(EX)-α and SOD(EX)β are missing, respectively, four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from their N-termini, compared to rat SOD(EX), suggesting that the cleavage sites of the SOD(EX) gene in the testis are different from that of other organs. Studies by sequential use of reverse transcription and polymerase chain reaction (PCR) using two SOD(EX) primers have demonstrated the expression of SOD(EX) in the heart, brain, lung, kidney, epididymis, testis, Sertoli, and germ cells, with low expression in the liver and ovary and no expression in the uterus, spleen, or thymus. Nucleotide sequence analysis of this 447-base pair PCR product from Sertoli cells revealed that its sequence is equivalent to the sequence of previously published rat SOD(EX). During testicular maturation, the SOD(EX) steady- state mRNA level increased significantly from 20 to 60 days of age and then declined at 90 days of age. Such an increase in the testicular SOD(EX) expression during maturation is not likely a result of an up-regulation by germ cells, since germ cells isolated from either 20- or 60-day-old rats when cocultured with Sertoli cells failed to elicit an increase in SOD(EX) expression in the cocultures. Using primary Sertoli cell cultures in vitro, it was found that Sertoli cell SOD(EX) expression was stimulated by interleukin-1α but not by either interferon-γ or basic fibroblast growth factor. These results illustrate that Sertoli cells as well as germ cells synthesize and/or secrete a testicular variant of SOD(EX) that may provide essential clues to understanding superoxide radical-mediated damage in the gonad.