Mechanism of the Inhibitory Action of Epidermal Growth Factor on Testicular Androgen Biosynthesis in Vitro*

Abstract

The mechanism whereby epidermal growth factor (EGF) inhibits gonadotropin-stimulated production of testosterone by primary cultures of testicular cells was investigated. Testicular cells from adult hypophysectomized rats were maintained for 8 days. Media were changed, and appropriate treatments were administered for 48 h before the collection of media for steroid RIA. Highly purified LH or hCG stimulated testosterone production by these cells, whereas concomitant EGF treatment inhibited LH and hCG action. In contrast, the administration of fibroblast growth factor or nerve growth factor did not affect hCG action. The inhibitory effect of EGF upon hCGstimulated testosterone production was detected 12 h after treatment and was associated with increases in cellular protein, but not DNA, content. Both cholera toxin (10 ng/ml) and (Bu)₂cAMP (0.5 mg/ml) stimulated testosterone production, whereas EGF inhibited the actions of these stimulatory agents, with ED₅₀ values of 0.2 and 0.3 ng/ml, respectively. EGF also inhibited the hCG-stimulated production of androstenedione (88% decrease) and 17α-hydroxyprogesterone (66% decrease), but increased the accumulation of progesterone (18-fold) and pregnenolone (6-fold). The addition of exogenous progesterone or 17α-hydroxyprogesterone augmented testosterone production by control and hCG-treated cultures, whereas concomitant treatment with EGF inhibited the conversion of these substrates into testosterone. Furthermore, EGF also inhibited the conversion of exogenous 17α-hydroxyprogesterone to androstenedione. These observations suggested that EGF decreased hCG-stimulated testosterone production via inhibition of 17α-hydroxylase and 17,20-lyase. In an enriched interstitial cell preparation, EGF also inhibited hCG- and (Bu)₂cAMP-stimulated testosterone production. The possible direct action of EGF on Leydig cells was reinforced by the demonstration of high affinity (K_d ∼ 3.5 × 10^-10 M), low capacity (∼24,000 sites/cell) [I¹²⁵]iodo-EGF-binding sites in these cells. These in vitro studies demonstrate that EGF binds to testicular binding sites and inhibits gonadotropin-stimulated testosterone production through decreasing the activities of 17α-hydroxylase and 17,20-lyase