| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 100 === The environmental hormone 17 alpha-ethynylestradiol (EE2) is a derivative of estradiol released into the environment via urine and feces from people who took it as a medication. EE2 has been used in almost all modern formulations of combined oral contraceptive pills. Effects of EE2 on the development of the reproductive organs have been well described. However, the action mechanisms of EE2 on testosterone biosynthesis are still unknown. In the present study, we examined the effect and action mechanism of EE2 on steroidogenesis of testosterone. In the in vivo study, male rats were injected subcutaneously with EE2 (0, 10, 20 or 40 g/ml/kg body weight) once per day for 3 days.
Blood samples (0.3 ml each) were collected at different time intervals after intravenous injection of human chorionic gonadotropin (hCG, 5 IU/ml). The plasma testosterone level in rats pre-treated with EE2 was lower as compared with control rats at corresponding time intervals after hCG challenge. Acute administration of EE2 in male rats reduced either basal or hCG-evoked levels of testosterone. In the in vitro study the enzymatically dispersed rat Leydig cells (1x105 cells/0.5 ml) were prepared and challenged with different concentrations of EE2 (10-10 - 10-6 M) in the presence or absence of hCG (0.05 IU/ml), 8-bromo-cyclic 3’:5’-adenosine monophosphate (8-Br-cAMP, a permeable analog of cAMP; 10-4 M), forskolin (an adenylyl cyclase activator, 10-5 M) or A23187 (a calcium ionophore, 10-5 M) and 25-OH-cholestrol (25-OH-C, 10-5 M) to evaluate their effects on testosterone production. The basal release of testosterone from Leydig cells was inhibited by EE2 in a dose-dependent manner. Moreover, EE2 also attenuated evoked release of testosterone. We then investigated the activity of steroidogenic rate-limiting enzyme P450 side-chain cleavage (P450scc). Rat Leydig cells were incubated with the serial doses of EE2 (10-8 - 10-6 M) in the presence or absence of 25-OH-C (10-5 M) and trilostane (a blocker of 3β-hydroxysteroid dehydrogenase, 10-5 M) to examine the activity of P450scc by measuring the levels of medium pregnenolone. The production of pregnenolone in response to 25-OH-C and trilostane treatments revealed that administration of EE2 attenuated P450scc activity as compared with control group. Moreover, rats treated with EE2 (0, 10, 20 or 40 g/ml/kg body weight) once per day for 7 days.
In all treated groups the prostate and seminal vesicles were atrophy, and the mobility of epididymal sperm was poor. The basal and evoked release of testosterone from Leydig cells was suppressed by pretreatment of EE2 at any dose. The activities of P450scc and the protein expression of another key protein in steroidogenesis, the steroidogenic acute regulatory (StAR) protein were decreased in all treated groups as compared with control group. These results suggested that EE2 diminished testosterone production via a reduction of P450scc activity, and StAR protein expression during steroidogenesis in rat Leydig cells.
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