Gap and tight junctions present within the basal ectoplasmic specialization are primary targets for flutamide action in adult rat testis

Present study was designed to establish a causal connection between changes in the cell-cell junction protein expression at the blood-testis barrier (BTB) and alterations in the adult rat testis histology following an anti-androgen flutamide exposure. Particular emphasis was placed on the basal ectoplasmic specialization (bES) in the seminiferous epithelium and expression of gap and tight junction proteins, connexin 43 (Cx43) and zonula occludens (ZO-1).Flutamide (50 mg/kg b.w.) was administered to male rats daily from 82 to 88 postnatal day. Testes from 90-day-old control and flutamide-treated rats were used for all analyses. Gonadal morphology was assessed by light and electron microscopy. Gene and protein expressions were analyzed by qPCR, Western blotting and immunohistochemistry, and steroid hormone concentrations determined radioimmunologically. Seminiferous epithelium of both groups of rats displayed normal histology without any morphologically recognizable loss of germ cells. Examination of semithin and ultrathin sections has shown that the length of the bES connecting neighboring Sertoli cells and the number of gap and tight junctions were apparently reduced in flutamide-exposed rats. Moreover, the appearance of unconventional circular ES indicated enhanced internalization and degradation of these junctional complexes. The changes were accompanied by decreased Cx43 and ZO-1 expression (p<0.01) and a loss of linear distribution of the proteins at the region of the BTB. 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 controls. In conclusion, short-term treatment with flutamide applied to adult rats exerts its primary effect on the bES, 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 Leydig cells, involving altered steroidogenic activity and increased Cx43 expression. Of note, these observations suggest diverse mechanisms of flutamide action in different cellular targets within the testis of adult rat.