RAT TESTICULAR EXTRACELLULAR-SUPEROXIDE DISMUTASE - ITS PURIFICATION,CELLULAR-DISTRIBUTION, AND REGULATION

Using multiple HPLC steps, we have identified and purified a 68-kDa po lypeptide las estimated by gel permeation HPLC) to apparent homogeneit y, from primary Sertoli cell-enriched culture medium, that consisted o f two monomers of 35 (alpha chain) and 33 kDa (beta chain) on SDS-poly acrylamide gel running under reducing conditions. Partial N-terminal a mino acid sequence analysis of these two monomers revealed sequences o f NH2-DXgESGVDLADRL (SODEX-alpha) and NH2-XXDTGESGVDLADXL (SODEX-beta) , which are identical to rat extracellular superoxide dismutase (SODEX ) with the exceptions that SODEX-alpha and SODEX-beta are missing, res pectively, four (Trp-Thr-Met-Ser) and two (Trp-Thr) amino acids from t heir N-termini, compared to rat SODEX, suggesting that the cleavage si tes of the SODEX gene in the testis are different from that of other o rgans. Studies by sequential use of reverse transcription and polymera se chain reaction (PCR) using two SODEX primers have demonstrated the expression of SODEX in the heart, brain, lung, kidney, epididymis, tes tis, Sertoli, and germ cells, with low expression in the liver and ova ry and no expression in the uterus, spleen, or thymus. Nucleotide sequ ence analysis of this 447-base pair PCR product from Sertoli cells rev ealed that its sequence is equivalent to the sequence of previously pu blished rat SODEX. During testicular maturation, the SODEX steady-stat e mRNA level increased significantly from 20 to 60 days of age and the n declined at 90 days of age. Such an increase in the testicular SODEX expression during maturation is not likely a result of an up-regulati on by germ cells, since germ cells isolated from either 20- or 60-day- old rats when cocultured with Sertoli cells failed to elicit an increa se in SODEX expression in the cocultures. Using primary Sertoli cell c ultures in vitro, it was found that Sertoli cell SODEX expression was stimulated by interleukin-alpha but not by either interferon-gamma or basic fibroblast growth factor. These results illustrate that Sertoli cells as well as germ cells synthesize and/or secrete a testicular var iant of SODEX that may provide essential clues to understanding supero xide radical-mediated damage in the gonad.