The aim of this study was to identify potential androgen-regulated proteins (ARP) that might mediate the supportive effects of testosterone on spermatogenesis. Adult rats were injected with ethane dimethane sulphonate (EDS) to destroy Leydig cells and thus induce complete testosterone withdrawal. Other EDS-treated rats were injected with 25 mg testosterone esters (TE) every 3 days to maintain quantitatively normal spermatogenesis. A timeframe for the study of androgen action on spermatogenesis was deduced from enumeration of degenerating germ cells at stage VII of the spermatogenic cycle in perfusion-fixed testes from rats in the early stages (4 to 8 days) after EDS treatment. Based on this data and changes in testicular interstitial fluid volume, long seminiferous tubule segments were isolated from control rats and from EDS-treated rats (+/- TE-supplementation) at stages II-V, VI-VIII, or IX-XII, 2 days to 6 days after EDS treatment. Seminiferous tubule segments were incubated for 22 hours with 60 microCi 35S-labelled methionine. Incorporation into newly synthesized proteins in the seminiferous tubule culture medium (= secreted proteins) or in seminiferous tubule lysates (= intracellular proteins) was determined by trichloroacetic acid-precipitation followed by analysis using two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis. In control rats, incorporation of 35S-methionine into proteins secreted by isolated seminiferous tubules was more than twice as great at stages VI-VIII than at stages II-V or IX-XII. This doubling in methionine incorporation into stages VI-VIII secreted proteins was abolished, however, 4 days after EDS treatment (when germ cell degeneration at stage VII was only just evident). A similar change occurred 4 days after testosterone withdrawal induced by immunoneutralization of luteinizing hormone. In the latter case and after EDS treatment, TE-supplementation of rats from day 0 maintained the normal control pattern of methionine incorporation into seminiferous tubule secreted proteins, although 6 days after EDS and TE treatment, incorporation into stages VI-VIII secreted proteins was 19% lower (P less than 0.05) than in the control group. In contrast, incorporation of methionine into proteins secreted by seminiferous tubules at stages II-V and IX-XII was unaffected by EDS and TE pretreatment, as was incorporation into intracellular proteins at all stages.(ABSTRACT TRUNCATED AT 400 WORDS)