Background: The dynamic cross-talk between epididymal cells is hormonally regulated and, in part, through direct cell-to-cell interactions. To date, no information is available regarding possible impact of anti-androgens on the proteins involved in the gap junctional communication within the boar epididymis. Thus, a question arised whether prenatal or postnatal exposure to an anti-androgen flutamide alters the expression of gap junction protein - connexin43 (Cx43) and androgen receptor (AR) expression in the caput, corpus and cauda epididymis and leads to delayed effects on morphology and function of adult pig epididymis.Methods: First two experimental groups received flutamide prenatally on gestational days 20-28 and 80-88 (GD20 and GD80) and further two groups were exposed to flutamide postanatally on days 2-10 and 90-98 after birth (PD2 and PD90). Epididymides were collected from adult boars. Routine histology was performed using hematoxylin-eosin staining. The expression of Cx43 and AR were analyzed using immunohistochemistry and Western blotting. Both analyses were supported by quantitative approaches to demonstrate the variations of the expression levels following the treatment. Apoptotic cells were identified using TUNEL assay.Results: Histological examination revealed differences in epididymal morphology of flutamide-exposed boars when compared to controls. Scarce spermatic content were seen within the corpus and cauda lumina of GD20, PD2 and PD90 groups. Concomitantly, frequency of epididymal cell apoptosis was significantly higher (p< 0.05) after exposure to flutamide at GD20. Moreover, in GD20, PD2, and PD90 groups, significantly lower AR expression (p< 0.05) was found in the principal and basal cells of the corpus and cauda regions, while in the stromal cells AR expression was significantly reduced (p< 0.05) along the epididymal duct. Concomitantly, a decrease in Cx43 expression (p< 0.05) was noticed in the stromal cells of the cauda region of GD20 and PD2 groups. This indicates high sensitivity of the stromal cells to androgen withdrawal.Conclusions: The region-specific alterations in the epididymis morphology and scarce spermatic content within the lumina of the corpus and cauda indicate that flutamide can induce delayed effects on the epididymal function of the adult boar by decrease in AR protein levels that results in altered androgen signaling. This may cause disturbances in androgen-dependent processes including Cx43 (de)regulation, however, we can not exclude the possibility that in response to flutamide decreased Cx43 expression may represent one mechanism responsible for functional disturbance of the boar epididymis.

The aim of this study was to show whether the connexin43 (Cx43) expression in gonads is affected by an anti-androgen action. To test this, pigs were prenatally (on gestational days 20-28 and 80-88; GD20, GD80), and postnatally (on days 2-10 after birth; PD2) exposed to flutamide that was given in five doses, every second day and its effect was observed in prepubertal gilts and boars. Morphology and expression of Cx43 was investigated in testes and ovaries by means of routine histology, immunohistochemistry, Western blotting, and RT-PCR, respectively. Qualitative analysis of immunohistochemical staining for Cx43 was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density of diaminobenzidine deposits. There were statistically significant differences in Cx43 signal intensity between interstitial tissue of control and GD20 pigs (**p less than 0.01), between seminiferous tubules of control and PD2 boars (**p less than 0.01), between granulosa cells of preantral follicles of control and GD20 and PD2 pigs (**p less than 0.01 and *p less than 0.05, respectively), and between theca cells of control and GD80 and PD2 gilts (**p less than 0.01). In Western blotting Cx43 appeared as a band of 43 kDa, whereas the size of the PCR-amplified product was 232 bp in all gonad tissue samples. Since we demonstrated changes in gonad morphology and in the expression of Cx43 at the level of protein of prepubertal boars and gilts, it seems possible that flutamide through blocking androgen action, causes delayed gonadal maturation in later postnatal life and, among other factors, may be involved in the regulation of Cx43 gene expression in pig gonads.