Cross-Inhibitions of Human Chorionic Gonadotropin (hCG) and Ethynyl-Estradiol (EE2) at Pico-Molar Concentrations on Mouse Leydig Tumor Cells (mLTC) Progesterone Secretion

Abstract

P-365 Introduction: The synthetic steroidal hormone ethynyl-estradiol is the most-abundantly used œstrogen component of oral contraceptives (about 0.2–0.3 ton per year worldwide) and its concentration in sewage treatment plant influents has been found to amount 3 to 66 ng/l (around 10−11 to 2x10−10M). These concentrations appear low compared to the doses in contraceptive pills (20–50 μg/day) but they are in the order of magnitude of the free active molecule in blood. In order to point out their eventual impact on human fecundity, we test these sub-nanomolar doses in various in vivo and in vitro assays related to reproduction to determine whether they can exhibit stimulatory or inhibitory effects. In the present paper, we present data concerning EE2 interaction with gonadotropin-stimulated steroidogenesis of a mouse Leydig cell line. Methods: Mouse Leydig Tumor cells (mLTC) were preincubated for 40h with Ethynyl-Estradiol EE2 (0 or 10−11-10−8 M) and subsequently incubated for 3h with hCG (0 or 10−11 M-2.4x10−10 M). Progesterone secretion in the medium was assessed by specific RIA. Results: Even at the lowest dose tested (10−11 M), EE2 induced an inhibition of the progesterone secretion by mLTCs during subsequent incubation with hCG. Interestingly, the inhibition was almost complete on the highest doses of hCG (1.2–2.4x10−10 M) but only marginal with lowest doses of hCG (1-4x10−11 M). In the absence of hCG during the incubation period, a stimulatory effect of EE2 present during the pre-incubation period, could be revealed. Interestingly, the presence of hCG even at the lowest stimulatory concentrations tested (10−11M), exerted an inhibitory effect on the progesterone response due to EE2 (10−10M) present during pre-incubation. This response was also much lower than that of the same dose of hCG (10−11M) in the absence of EE2 during the preincubation period. Discussion and conclusions: Both hCG and EE2 when alone were shown to exert stimulatory effects on the secretion of progesterone by mLTCs. Unexpectingly, not only they did not show any additive stimulations but, in the contrary, they exhibited strong cross-inhibition of their stimulatory effects. The inhibitory effect of pre-incubation of mLTCs with EE2 on hCG stimulation of progesterone secretion might be due either to interference with the hCG stimulatory pathway or to stimulation of progesterone-binding proteins. Although the precise molecular mechanisms of these effects are not clear, the observation that EE2 can act on mLTCs at pico-molar concentrations even over short periods of time might be of significance for possible interaction with human reproduction. Further in-vitro studies on testicular Leydig cells and in vivo assays will be necessary to assess the physiological and pharmacological relevance of these data.