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Dose-response curves were obtained for the production of androgen-binding protein (ABP) by Sertoli cells prepared from testes of 20-day-old rats and treated in culture with either FSH or testosterone (T). FSH stimulated ABP production by up to 3.5 times control levels. For NIH-FSH-S11, the ED50 was 3 ng/ml, and for highly purified ovine FSH, the ED50 was 0.066 ng/ml. Addition of T produced a stimulation of up to 3 times control levels; half-maximal response was obtained at a dose of 4 nM. The presence of small numbers of contaminating Leydig cells in some preparations resulted in production of endogenous T, especially when high doses of NIH-FSH, which contains some LH, were employed. A modified preparation method involving exposure of the cells to distilled water reduced the endogenous T production to low levels. In cultures of cells prepared in this way, addition of the antiandrogen cyproterone acetate at a concentration high enough to reduce fractional occupancy of androgen receptors by endogenous T to 0.014 or less had no effect on the stimulation by FSH of ABP production in the cultures. In contrast, cyproterone acetate inhibited stimulation by T of ABP production. We conclude that FSH and T act independently on Sertoli cells from immature rats to increase the secretion of ABP.

Seminiferous tubules isolated from normal adult rats converted (14C) testosterone to (14C) androstanediol and (14C) androstenedione as the major metabolites; (14C) dihydrotestosterone and (14C) androsterone were produced in lesser amounts. Tubules from immature rats (26-28 days of age) converted a higher proportion of (14C) testosterone to 5alpha-reduced products than did tubules from adult rats. Spermatocyte-enriched preparations contain 5alpha-reductase. The lower level of 5alpha-reductase activity in spermatid-spermatocyte preparations indicates that this enzyme is low or absent in spermatids. Sertoli cell-enriched preparations contain 5 alpha-reductase and a high level of 3alpha-hydroxysteroid dehydrogenase. The results show that spermatocytes and Sertoli cells have the capacity to metabolise (14C) testosterone to 5alpha-reduced products; dihydrotesterone is the major product formed by spermatocytes, whereas in Sertoli cells further metabolism to 5alpha-androstane-3alpha, 17beta-diol occurs.

A two-chamber assembly has been employed to investigate influences of peritubular cells (PC) and extracellular matrix (Matrigel) on barrier formation by Sertoli cells (SC) in culture and on transferrin production. The kinetics of transport of [3H]inulin across a Millipore filter were essentially the same in the presence or absence of Matrigel or PC. In contrast, a SC monolayer retarded the diffusion of [3H]inulin, increasing the estimated time for 50% equilibration from about 4 h to approximately 12 h. Matrigel and PC each independently further increased the equilibration time, with the largest effects elicited by the presence of Matrigel (approximately 21 h). Data have been interpreted to indicate that these two components, especially extracellular matrix, facilitate the formation of a functional barrier by SC in the two-chamber system. PC assume a more important role than Matrigel in the modulation of transferrin secretion by SC. Transferrin concentrations were higher in the inner chamber, corresponding to those in the adluminal compartment, but transferrin masses were higher in the outer chamber under the conditions described. We report the effects of the presence and absence of Matrigel, PC, and FSH on levels of transferrin secreted by SC. Addition of FSH resulted in increased transferrin secretion by SC maintained under all conditions examined. We compare our data with those previously reported by others and attempt to provide a basis for the differences observed. We discuss the properties of the system and outline major advantages and limits of the two-chamber assembly in investigations on the polarity and properties of SC in culture.

Factors have been identified that influence the integrity of the barrier generated by Sertoli cells (SC) in culture in a two-chambered assembly. The permeability of the barrier was assessed by determining rates of equilibration of [3H]methoxyinulin or [86Rb]Cl across the Sertoli cell monolayer. The complete system consisted of a confluent monolayer of SC maintained on an extracellular matrix (Matrigel)-coated filter together with peritubular cells on the opposite side of the filter. In confirmation of previous results, levels of plasminogen activator (PA) activity secreted were increased by treatment of SC with FSH or with cAMP derivatives [(Bu)2cAMP (dbcAMP)]. PA levels in the culture medium were inversely related to times required for 50% equilibration of [3H]methoxyinulin across the SC monolayer. Thus, elevated PA levels, elicited by stimulation with FSH or dbcAMP, were associated with a decreased integrity of the barrier generated by SC preparations maintained in serum-free medium in the complete system. The increase in permeability of the barrier in SC elicited by FSH dbcAMP could be prevented, however, by the addition of various antiproteases. FSH actions on barrier function were complex. Effects of FSH that favored barrier integrity were most readily detected when proteolytic activity was inhibited. The addition of intact serum increased the integrity of the barrier, but acid-treated serum depleted of antiproteases had no such effect. We advance the hypothesis that proteases are implicated in modulation of the formation and maintenance of the seminiferous tubule barrier by SC.

BY FOLLOWING the heavy nitrogen concentrations in tissue proteins from rats which had previously been fed N15-glycine and maintained on a low protein diet, Shemin and Rittenberg (1944) demonstrated that the N15 concentration of liver proteins decreased rapidly with time, while that of carcass proteins increased. These authors subsequently postulated that the N15 concentration of any tissue protein would approach an “average isotope concentration,” which was the calculated specific activity if the incorporated N15 were homogeneously distributed through all tissue proteins. This concept inferred that a decrease in N15 concentration of relatively highly labeled tissue proteins resulted primarily from a dilution by nitrogen transferred from other, less enriched tissue proteins, all of which interacted with constituents of a general protein metabolic pool. The present study, undertaken initially to examine the effects of the adrenal cortex on protein metabolism, has demonstrated that under conditions which pro...

The in vitro effects of FSH and LH on cyclic AMP levels in various types of preparations from rat testes were investigated. LH but not FSH stimulated cyclic AMP production in isolated interstitial cells. Furthermore, FSH did not act synergistically with LH to elevate cyclic AMP levels in interstitial cell preparations. In isolated seminiferous tubules freed of interstitial cells, FSH but not LH stimulated adeny-late cyclase activity. In tubules from normal adult rats, an effect of FSH on cyclic AMP accumulation was detectable only in the presence of theophylline. In tubule preparations from fully regressed hypophysectomized or cryptorchid rats, cyclic AMP levels were elevated by the addition of either FSH or theophylline. When added together, FSH and theophylline acted synergistically to increase cyclic AMP levels. The latter preparations from regressed testes were enriched in Sertoli cells and spermatogonia. Exposure of normal adult testes to irradiation (300 rads) in vivo caused a dramatic reduction in ...