Summary: 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.

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.

Adipokines influence energy metabolism and have effects on male reproduction, including spermatogenesis and/or Sertoli cell maturation; however, the relationship between these active proteins and androgens in testicular cells is limited. Here, we studied the impact of short-term exposure to flutamide (an anti-androgen that blocks androgen receptors) on the expression of chemerin, apelin, vaspin and their receptors (CCRL2, CMKLR1, GPR1, APLNR, GRP78, respectively) in adult rat testes. Moreover, the levels of expression of lipid metabolism-modulating proteins (PLIN1, perilipin1; TSPO, translocator protein) and intercellular adherens junction proteins (nectin-2 and afadin) were determined in testicular cells. Plasma levels of adipokines, testosterone and cholesterol were also evaluated. Gene expression techniques used included the quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) and immunohistochemistry (IHC). The androgen-mediated effects observed post-flutamide treatment were found at the gonadal level as chemerin, apelin, and vaspin gene expression alterations at mRNA and protein levels were detected, whereas the cellular targets for these adipokines were recognised by localisation of respective receptors in testicular cells. Plasma concentrations of all adipokines were unchanged, whereas plasma cholesterol content and testosterone level increased after flutamide exposure. Differential distribution of adipokine receptors indicates potential para- or autocrine action of the adipokines within the rat testes. Additionally, changes in the expression of PLIN1 and TSPO, involved in the initial step of testosterone synthesis in Leydig cells, suggest that testicular cells represent a target of flutamide action. Increase in the gene expression of PLIN1 and TSPO and higher total plasma cholesterol content indicates enhanced availability of cholesterol in Leydig cells as a result of androgen-mediated effects of flutamide. Alterations in adherens junction protein expression in the testis confirm the flutamide efficacy in disruption of androgen signalling and presumably lead to impaired para- and autocrine communication, important for proper functioning of adipokines.

Present study was designed to establish a causal connection between changes in the cell-cell junction protein expression at the blood-testis barrier and alterations in the adult rat testis histology following an anti-androgen flutamide exposure. Particular emphasis was placed on the basal ectoplasmic specialization (ES) in the seminiferous epithelium and expression of gap junction protein, connexin 43 (Cx43). Flutamide (50 mg/kg body weight) was administered to male rats daily from 82 to 88 postnatal day. Testes from 90-day-old control and flutamide-exposed rats were used for all analyses. Testis morphology was analyzed using light and electron microscopy. Gene and protein expressions were analyzed by real-time RT-PCR and Western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by radioimmunoassay. Seminiferous epithelium of both groups of rats displayed normal histology without any loss of germ cells. In accord, no difference in the apoptosis and proliferation level was found between control and treated groups. As shown by examination of semi-thin and ultrathin sections, cell surface occupied by the basal ES connecting neighboring Sertoli cells and the number of gap and tight junctions coexisting with the basal ES were apparently reduced in flutamide-treated rats. Moreover, the appearance of unconventional circular ES suggests enhanced internalization and degradation of the basal ES. These changes were accompanied by decreased Cx43 and ZO-1 expression (p < 0.01) and a loss of linear distribution of these proteins at the region of the blood-testis barrier. On the other hand, Cx43 expression in the interstitial tissue of flutamide-treated rats increased (p < 0.01), which could be associated with Leydig cell hypertrophy. Concomitantly, both intratesticular testosterone and estradiol concentrations were elevated (p < 0.01), but testosterone to estradiol ratio decreased significantly (p < 0.05) in flutamide-treated rats compared to the controls. Short-term treatment with flutamide applied to adult rats exerts its primary effect on the basal ES, coexisting junctional complexes and their constituent proteins Cx43 and ZO-1, without any apparent morphological alterations in the seminiferous epithelium. In the interstitial compartment, however, short-term exposure leads to both histological and functional changes of the Leydig cells.