In vivo and in vitro effects of flutamide and diethylstilbestrol on fetal testicular steroidogenesis in the rat.

Prenatal testosterone surge is considered crucial for physiological masculinization of male progeny. Disorders in sex steroid hormone balance during the fetal development may interfere with male reproductive health later in life. In this study, we have investigated in utero and in vitro effects of flutamide (FLU) and diethylstilbestrol (DES) on fetal rat testicular steroidogenesis. In utero exposure to FLU 25mg/kg or DES 0.02mg/kg had no obvious effects on ED 19.5 rat testicular testosterone and progesterone production, StAR protein or AR protein expression. However, when ED 19.5 rat testis were cultured for 180min in the presence of 0.1, 1, 10 and 100mg/l of FLU or DES, the highest doses of both compounds were capable of disturbing steroidogenesis. To study the rate of the changes seen in testicular steroidogenesis after 180min, time-series experiments, in which intact testes were cultured with FLU 100mg/l or DES 100mg/l for 30, 60 or 120min, were performed. In vitro time-series experiments revealed that changes in steroidogenesis occur very fast. Experiments with FLU brought further evidence to the hypothesis that ARs have negative autocrine role in developing Leydig cells.

Effects of 4-tert-octylphenol, 4-tert-butylphenol, and diethylstilbestrol on prenatal testosterone surge in the rat.

In the present study, we evaluated the effects that 4-tert-octylphenol (OP) and 4-tert-butylphenol (BP) had on the prenatal testicular testosterone surge at embryonic day (ED) 19.5 in the rat. In utero exposure to alkylphenols (0.1-100 mg/kg maternal weight) on EDs 13.5, 15.5, and 17.5 did not decrease testicular testosterone content, whereas exposure to diethylstilbestrol (DES) caused a significant depression in testosterone synthesis and secretion. The depression was maintained during ex vivo tissue culture. In order to elucidate the observed differences in the in vivo effects between alkylphenols and DES, the exposures were also carried out in tissue culture of intact ED 19.5 testes. Basal testosterone, progesterone, cAMP production and hCG-induced testosterone levels were determined during and after a 3-h culture period. DES (100 mg/l) did not alter testosterone production but caused a two-fold increase in progesterone. OP (10, 100, 500 mg/l) and BP (100 mg/l) significantly increased testosterone and progesterone levels by up to seven-fold. In the presence of BP 100 mg/l, however, the intratesticular testosterone content did not correlate with the significantly increased fraction of secreted, or leaked, testosterone. The latter was correlated with tissue damage observed at electron microscopic level. Consistent with this, BP 500 mg/l elevated testicular testosterone level slightly during the first hour in the culture but the level subsequently returned to the control value. At the electron microscopic level, alkylphenols caused most severe changes in Leydig cell membrane structures and lipid droplets. In the DES-treated testes, membrane vesicle formation around the lipid droplets and increased mitochondrial pleiomorphy were observed. Altogether, the present in vivo and in vitro analyses confirm different effects of alkylphenols and DES on fetal rat steroidogenesis and tissue structure.