Flutamide induces alterations in the cell-cell junction ultrastructure and reduces the expression of Cx43 at the blood-testis barrier with no disturbance in the rat seminiferous tubule morphology

Abstract

BackgroundPresent 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).MethodsFlutamide (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.ResultsSeminiferous 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.ConclusionsShort-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.

Flutamide Alters the Expression of Chemerin, Apelin, and Vaspin and Their Respective Receptors in the Testes of Adult Rats

“…The question arises whether adipokines may modulate Leydig cell functioning through a mechanism that has not yet been defined. It is worth adding that our previous morphometric analysis [ 40 ] revealed a marked increase of interstitial volume, accompanied by a decreased number of Leydig cells per 1 mm 2 of the interstitial space, indicating the occurrence of Leydig cell hypertrophy after flutamide exposure. Elucidation of whether a compensatory mechanism within Leydig cells exists will require further study at the ultrastructural level.…”

“…As has been stated in the introduction, short-term flutamide exposure influences morpho-functional aspects of spermatogenesis in the rat [ 40 , 45 ]. On the other hand, it has been shown that AR-mediated androgen signalling plays an important role in male metabolism by affecting the energy balance [ 46 ].…”

“…A dose of flutamide was selected as high enough to produce changes in the expression of cell–cell junction protein levels without generating testicular germ cell loss from the seminiferous tubules. Treatment protocol was evaluated according to the literature and our previously published data [ 40 , 45 , 71 ]. The animals were housed in a fully controlled standard environment (temperature 22 °C, relative humidity 55% ± 5%, light:dark cycle of 12:12 h), with access to water and food ad libitum (Agropol, Motycz, Poland).…”

“…We previously showed that flutamide produced diverse effects on different cellular targets within adult rat testes. Short-term flutamide treatment had no effect on the morphology of the seminiferous epithelium; however, it caused an apparent enlargement of the interstitial tissue in which Leydig cells are located [ 40 ]. In the light of these findings, we proposed that the tubular compartment and the intertubular compartment are variously sensitive to androgens and a different set of cellular factors may be stimulated (or inhibited) by flutamide in various cell types.…”

Changes in porcine cauda epididymal fluid proteome by disrupting the HPT axis: Unveiling potential mechanisms of male infertility

“…Other studies, using flutamide as a antiandrogen, describe that it affects the expression of junction proteins and junction complex structure, not only by inhibiting androgen receptor activity, but also by modulating protein kinase‐dependent signaling in testicular cells (Chojnacka et al, 2016; Hejmej & Bilinska, 2018). As expected, the epididymis is also affected by flutamide exposure, with increased epididymal cells apoptosis, lower androgen receptor expression, concomitant with decreased Cx43 (gap junction protein connexin43; Lydka et al, 2011).…”