RAT SEMINIFEROUS TUBULAR CULTURE-MEDIUM CONTAINS A BIOLOGICAL FACTOR THAT INHIBITS LEYDIG-CELL STEROIDOGENESIS - ITS PURIFICATION AND MECHANISM OF ACTION

Seminiferous tubules prepared from adult rats cultured for 48 h in ser um-free conditions produce multiple biological factors that modulate L eydig cell steroidogenic function in vitro. Using gel filtration chrom atography, it was shown that seminiferous tubular culture medium (STCM ) contained at least three inhibitory activities designated AI, AII, a nd AIII that inhibited testosterone production by purified Leydig cell s. The factor that induced AIII activity, designated Leydig cell inhib itor (LCI), was further purified to apparent homogeneity by sequential HPLC using gel permeation, C8-, C18-, C2/C18-reversed-phase, and micr obore anion exchange columns. When this batch of purified factor was r esolved by SDS-PAGE under reducing conditions, only a single silver st ained band with an apparent M(r) of 21 000 was detected. Protein seque nce analysis using about 100 pmol of purified LCI revealed that its N- terminus was blocked. Incubation of this highly purified factor with P ercoll gradient purified Leydig cells induced a dose-dependent inhibit ion of hCG-stimulated testosterone production. LCI inhibited the basal testosterone production and hCG-stimulated cAMP production by Leydig cell dose-dependently. It also inhibited the forskolin- and cholera to xin-stimulated testosterone and cAMP production but had no apparent ef fect on the binding of I-125-labeled hCG to LH receptors. These data s uggest that this LCI exerts its inhibitory action at steps beyond the LH receptors but prior to the cAMP formation by affecting the adenylat e cyclase activity directly or indirectly through inhibition of the st imulatory G-protein (G(s)-protein); however, it is also possible that it decreases the coupling of the receptors to the G(s)-protein. LCI al so inhibited the conversion of exogenously added 22R-hydroxycholestero l, pregnenolone, progesterone, and 17 alpha-hydroxyprogesterone to tes tosterone. However, it had no effect on the conversion of dehydroepian drostenedione and androstenedione to testosterone. These data strongly suggest that LCI affects the steroidogenic enzymes metabolizing chole sterol to testosterone, the cytochrome P-450 side-chain cleavage (P-45 0(SCC)), and cytochrome P-450 17 alpha-hydroxylase/17,20-lyase (P-450( C17)). However, it has no effect on the 3 beta-hydroxysteroid dehydrog enase (3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-H SD) enzyme activities. Based on the results of the present study, it i s apparent that this LCI is distinct from other known potent Leydig ce lls inhibitors such as interleukin-1 (IL-1) and transforming growth fa ctor-beta (TGF-beta). The LCI appears to involve in the paracrine regu lation of Leydig cell function.