Enhancement of Leydig cell testosterone secretion by isolated seminiferous tubules during co-perifusion in vitro: comparison with static co-culture systems

The aim of this study was to identify an in-vitro test system for the reproducible demonstration of a modulatory effect of isolated seminiferous tubules (s-tubules) on testosterone production by purified rat Leydig cells. Co-incubation of s-tubules with various numbers of Leydig cells had no significant effect on basal and hCG-stimulated testosterone production over 4-24 h incubation. In contrast, addition of s-tubule conditioned medium (STCM) to Leydig cells enhanced both basal and hCG-stimulated testosterone production over 5 h, but this effect was variable in magnitude and was not completely reproducible. Co-perifusion of isolated s-tubules with Percoll-purified Leydig cells for 6 h produced significant and consistent increases in Leydig cell testosterone secretion compared with Leydig cells perifused on their own. In six experiments, s-tubules enhanced Leydig cell testosterone secretion by 26 +/- 5% (P less than 0.001) in the absence of LH stimulation and by 48 +/- 11% following pulsatile stimulation with 1 ng/ml ovine LH (oLH). The presence of s-tubules enhanced (P less than 0.01-0.001) testosterone secretion by Leydig cells in response to pulses of oLH at doses ranging from 0.1 to 10 ng/ml, but the magnitude of enhancement was greatest with 0.1 and 1 ng/ml doses. These stimulatory effects were not explained by Leydig cell contamination or by testosterone leakage from the isolated s-tubules. Co-perifusion of Leydig cells with isolated epididymal tubules as a control tissue had no significant effect on LH-stimulated Leydig cell testosterone production. Stimulatory effects of s-tubules on Leydig cell testosterone secretion were observed at a 'physiological' ratio of s-tubules to Leydig cells (200 cm tubules/3 million cells) and was mediated by a humoural agent(s), since perifusion of s-tubules and Leydig cells in series gave similar results to co-perifusion of these tissues. This system proved to be robust and, in contrast to static culture systems, gave highly reproducible results, which should allow detailed investigation of the dynamic interactions between s-tubules and Leydig cells and the hormonal control of these events.