The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles

Abstract

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7 )M), 2-Hf (1.7 × 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3β-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3β-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary.

Fig. 1

The effect of 24 h antiandrogen treatment on the immunostaining for 3β-HSD (a–d), P450c17 (e–h) and P450arom (i–l) after whole porcine follicles incubations. Control cultures (a, e, i), cultures under T influence (b, f, j), cultures under 2-Hf influence (c, g, k), cultures under T+2-Hf influence (d, h, l). Cytoplasmic staining of steroidogenic enzymes in granulosa cells (white arrowheads), cytoplasmic staining in theca cells (black arrows). Quantitative analysis of the intensity of steroidogenic enzymes staining expressed as relative optical density (ROD) of diaminobenzidine brown reaction products in follicle cultures (a, b, c) exposed to an androgen agonist (T), an antagonist (2-Hf) or both (T+2-Hf) versus respective controls. Values are mean ± SEM. Asterisks indicate significant differences between control and experimental cultures. Significant differences from control values are denoted as *p < 0.05, **p < 0.01, Mann–Whitney’s test. Bar = 50 μm (a–d, g–j), bar = 20 μm (e, f, k, l)

Fig. 2

Representative Western blot analysis of 3β-HSD a, P450c17 b and P450arom c protein expression levels in homogenates of control, testosterone (T), 2-hydroxyflutamide (2-Hf), testosterone and 2-hydroxyflutamide (T+2-Hf) treated follicles after 24 h cultures. Data obtained from three separate analyses are expressed as mean ± SEM (n = 3). Significant differences from control values are denoted as *p < 0.05 and **p < 0.01, Mann–Whitney’s test

Fig. 3

Relative expression of mRNA for 3β-HSD (HSD3B1) a, P450c17 (CYP17A1) b and P450arom (CYP19A1) c in control, testosterone (T), 2-hydroxyflutamide (2-Hf), testosterone and 2-hydroxyflutamide (T+2-Hf) treated whole follicles cultured for 24 h, using quantitative real-time PCR analysis. RQ (relative quantification) is expressed as mean ± SEM. Asterisks indicate statistically significant differences in the expression of steroidogenic enzyme genes between control and experimental groups (*p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney’s test)

Fig. 4

Progesterone, androgens and estradiol (mean ± SEM) secretion by whole follicles cultured for 24 h, respectively (n = 3 independent experiment). Statistical differences between control and experimental cultures were determined by Student t test. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01). C control, T testosterone, 2-Hf 2-hydroxyflutamide, T+2-Hf testosterone +2-hydroxyflutamide