Androgen receptor-mediated non-genomic effects of vinclozolin on porcine ovarian follicles and isolated granulosa cells: Vinclozolin and non-genomic effects in porcine ovarian follicles

• Vnz modifies ARs immunolocalization and both, AR mRNA and protein expression.
• MAP and Akt kinase pathways are involved in the action of Vnz in the porcine GCs.
• Selective destruction of porcine follicles might be a consequence of their exposure to Vnz.
• It can lead to premature ovarian failure in affected organisms.

The present study investigated the influence of the androgen receptor (AR) agonists testosterone (T) and dihydrotestosterone (DHT), and vinclozolin (Vnz), a fungicide with antiandrogenic activity, on non-genomic signal transduction within ovarian follicles. Porcine granulosa cells (GCs) isolated from mature follicles were cultured for 48 h. For the last 24 h of culture, they were exposed to T (10−7 M), DHT (10−7 M), Vnz (1.4 × 10−5 M), T and Vnz (T + Vnz), or DHT and Vnz (DHT + Vnz) at the same concentrations. To better imitate in vivo conditions, whole follicles (4–6 mm in diameter) were incubated (24 h) in an organ culture system with the same factors. Expression of AR mRNA and protein was determined by real-time PCR and western blot analyses. To demonstrate AR localization in cultured GCs and whole follicles, immunocytochemistry and immunohistochemistry were performed, respectively. To elucidate the possible non-genomic action of Vnz in GCs, protein expression and the activity of ERK1/2 and Akt kinases were determined by western blot and ELISA analyses. The immunocytochemistry and immunohistochemistry results showed that exposure of GCs and follicles to Vnz resulted in cytoplasmic and perinuclear AR localization. Real-time PCR and western blot analysis showed that AR mRNA and protein expression increased (P ≤ 0.001) in GC cultures after combined treatment with an androgen and Vnz. In whole follicles, such treatment also increased AR mRNA with a decrease in the respective protein expression (P ≤ 0.001). Moreover, addition of T or DHT with Vnz increased the activity of ERK1/2 and Akt kinases in cultured GCs (P ≤ 0.001). The results suggest a novel mechanism for Vnz action in porcine ovarian follicles on both AR mRNA and protein levels. Thus, this environmental antiandrogen activates non-genomic signaling pathways, as indicated by the increased activity of both investigated kinases observed within minutes of Vnz addition. Given the widespread presence of Vnz in the environment, elucidation of its non-genomic action should be the subject of studies on female fertility.