Testosterone and Spermatogenesis Identification of Stage-Specific, Androgen-Regulated Proteins Secreted by Adult Rat Seminiferous Tubules

: 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 μCi 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 polyacrimide 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—VIM 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 < 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. Seven proteins were identified as putative ARP's on the basis that: 1) all showed major, repeatable changes at stages VI—VIII after EDS treatment (in most cases, the proteins virtually disappeared) that were reversed by TE-supplementation; and 2) comparison of secreted proteins by seminiferous tubules from control rats at stages VI—VIII with those at stages II—V and IX—XII revealed that (with the exception of ARP-5) these ARP's were barely evident at stages II—V or IX-XII, but were more prominent at stages VI-VIII. The cellular source of the ARP's is uncertain, although ARP-5, which is regulated negatively by testosterone, is probably a highly acidic form of sulfated glycoprotein-2, a major Sertoli cell product. Other identified Sertoli cell products (sulfated glycoprotein-1 and cyclic protein-2) were unaffected by testosterone withdrawal or replacement. The other putative ARP's do not correspond to previously identified testicular proteins, although ARP-6 and ARP-7 are of similar molecular weight to that reported for the two forms of P-Mod-S, and ARP-1 and ARP-2 are in the molecular weight range of many growth factors. This study shows for the first time that testosterone exerts major general and specific effects on proteins secreted by seminiferous tubules at stages VI-VIII, but not on those secreted at stages II—V or IX—XII, of the spermatogenic cycle. While it remains to be established that the identified ARP's are regulated primarily by testosterone, these studies could pave the way for identifying, at both the biochemical and molecular level, how testosterone drives spermatogenesis.