Rat Testicular Extracellular Superoxide Dismutase: Its Purification, Cellular Distribution, and Regulation¹

^c Population Council, Center for Biomedical Research, New York, New York 10021 ^d Department of Zoology, University of Hong Kong, Hong Kong, People's Republic of China ^e Institute of Pharmacology and Pharmacognosy, University of Rome "La Sapienza," 00185 Rome, Italy

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 ( chain) and 33 kDa (ß chain) on SDS-polyacrylamide gel running under reducing conditions. Partial N-terminal amino acid sequence analysis of these two monomers revealed sequences of NH₂-DXGESGVDLADRL (SOD_EX-) and NH₂-XXDTGESGVDLADXL (SOD_EX-ß), which are identical to rat extracellular superoxide dismutase (SOD_EX) with the exceptions that SOD_EX- and SOD_EX-ß 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-1 but not by either interferon- 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.

¹ This work was supported in part by grants from the Rockefeller Foundation (PS9528, PS9601, PS9721), Conrad Program (CIG96-05), Noopolis Foundation, NIH (HD-13541), and Hong Kong Research Grant Council (HKU7235/97M). This work was derived from a dissertation to be submitted by D.M. to the University of Hong Kong for the partial fulfillment for the requirements of Doctor of Philosophy.

² Correspondence: C. Yan Cheng, Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021. FAX: (212) 327-7678; yan{at}popcbr.rockefeller.edu

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