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.
Highlights
Female reproduction depends on a succession of multiple integrated processes in which the ovary has two important roles: during follicular development produces eggs for fertilization and is an important site of steroids production whose concentrations are tightly regulated by feedback control mechanisms [1,2,3]
In all carried out variants of follicular cultures, the granulosa layer displayed a gradient of cytoplasmic 3b-hydroxysteroid dehydrogenase (3b-hydroxysteroid dehydrogenases (HSDs)) immunoreaction intensity (Fig. 1a–d) with the strongest immunolabelling in the antral granulosa cells, and a much weaker one in the cells lying in closer proximity to the theca interna layer that is mural granulosa cells
The most intense 3b-HSD immunolabeling was observed in the theca interna cells of control follicles (Fig. 1a) in contrast to the follicles incubated with the addition of T and 2-Hf (T?2-Hf) (Fig. 1d) which demonstrated a quite weak 3b-HSD immunolabeling
Summary
Female reproduction depends on a succession of multiple integrated processes in which the ovary has two important roles: during follicular development produces eggs for fertilization and is an important site of steroids production whose concentrations are tightly regulated by feedback control mechanisms [1,2,3]. Androgens can modulate follicular function by interactions with various factors to promote granulosa cells (GC) differentiation [4]. The agonistor antagonist- bound steroid hormone receptors regulate gene expression by binding to the regulatory elements in promoters of susceptible genes, in a tissue-dependent manner. FLU was used to treat prostate cancer, where it competes with testosterone (T) for binding to AR, thereby reducing the growth of cancer cells [11]. It reduced fecundity in the fathead minnow by decreasing oocytes maturation in females and causing spermatocyte degeneration and necrosis in the testis [12]. The antiandrogenic activity of 2-Hf and FLU has been well established [13,14,15,16], what is interesting it has been demonstrated that 2-Hf is approximately 1–5 times more potent than its parental compound, FLU [17]
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