BackgroundThe 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.MethodsFirst 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.ResultsHistological 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.ConclusionsThe 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.

Spermatogenesis, the process of spermatozoa production, is regulated by several endocrine factors, including testosterone, follicle stimulating hormone, luteinizing hormone and estradiol 17β. For spermatogenesis to reach completion, developing germ cells must traverse the seminiferous epithelium while remaining transiently attached to Sertoli cells. If germ cell adhesion were to be compromised for a period of time longer than usual, germ cells would slough the seminiferous epithelium and infertility would result. Presently, Sertoli-germ cell adhesion is known to be mediated largely by classical and desmosomal cadherins. More recent studies, however, have begun to expand long-standing concepts and to examine the roles of other proteins such as intercellular adhesion molecules. In this review, we focus on the biology of intercellular adhesion molecules in the mammalian testis, hoping that this information is useful in the design of future studies.

Several recent studies have indicated that androgen disruption induced by the administration of anti-androgen flutamide during critical developmental stages results in various reproductive abnormalities, mainly in rodents. However, scarce data are available regarding the alterations caused by this toxicant on cell-cell adhesion molecules. Of note, there is no report on the expression and regulation of tight and adherens junction proteins in the boar. Therefore, the purpose of this study was to analyse whether foetal and neonatal exposure to flutamide affects the expression and distribution of ZO-1, occludin, β-catenin, and N-cadherin in testes of adult pigs. Moreover, to evaluate whether androgen signal was altered in the boar, testicular levels of testosterone and oestradiol and the expression of androgen receptor were examined. Flutamide (50 mg/kg bw) was injected into pregnant gilts during gestational days 20-28 and 80-88 (GD20, GD80), and into male piglets on postnatal days 2-10 (PD2). In the testes of all flutamide-exposed boars, expressions of ZO-1, N-cadherin and β-catenin were significantly decreased at mRNA and protein level, whereas expression of occludin was unchanged when compared with the controls. In addition, in severely damaged seminiferous tubules of PD2 pigs, mislocalization of ZO-1, N-cadherin and β-catenin was observed. Changes in junction protein expressions were accompanied by disturbed intratesticular androgen-oestrogen balance, although androgen receptor expression was not altered. Taken together, these results demonstrate that blockade of androgen action by flutamide during both gestational and neonatal periods affects the expression of ZO-1, N-cadherin and β-catenin in adult pig testes. Of concern, neonatal window seems to be most critical for the organization of BTB and consequently for normal spermatogenesis in the boar. It is likely that altered expression of junction proteins is related to insufficient testosterone production and/or excessive oestradiol synthesis, which may result from impaired Leydig cell function.

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The mammalian blood-testis barrier (BTB) restructures throughout spermatogenesis, thereby allowing developing germ cells to enter the adluminal compartment of the seminiferous epithelium. Previous studies have shown pro-inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1α to be important regulators of Sertoli cell barrier/BTB function in vitro and in vivo. In this study, the effects of TNFα on Sertoli cell barrier function were assessed, with emphasis on changes in proteases and cell adhesion molecules following treatment. By immunoblotting and immunohistochemistry, MMP9 was found to be present in germ cells, localizing by and large to spermatocytes and spermatids in the adult rat testis. Following treatment of Sertoli cells with physiologically relevant consecutive doses of recombinant human TNFα (25 ng/ml), the steady-state levels of active-matrix metalloprotease 9 (MMP9), membrane-bound intercellular adhesion molecule (mICAM-1) and androgen receptor increased significantly. TNFα also downregulated the steady-state level of occludin, in agreement with earlier results that showed TNFα to disrupt Sertoli cell barrier/BTB function. In addition, TNFα affected the filamentous actin cytoskeleton in Sertoli cells, which appeared to be mediated by cortactin, a regulator of actin dynamics. Taken collectively, these findings imply that germ cells may be involved in BTB restructuring via the localized production of TNFα. These results also illustrate that barrier restructuring correlated with an increase in Sertoli cell mICAM-1, suggesting that it may be critical for adhesion as germ cells traverse the “opened” BTB.