Flutamide alters the distribution of c-Src and affects the N-cadherin-β-catenin complex in the seminiferous epithelium of adult rat

Abstract

Flutamide alters the distribution of c-Src and affects the N-cadherinb-catenin complex in the seminiferous epithelium of adult rat SUMMARYThis study was undertaken to explore interactions between c-Src kinase and the N-cadherin-b-catenin complex in seminiferous tubules of flutamide-treated rats. An anti-androgen flutamide (50 mg/kg bw) was injected daily into adult rats from postnatal days 82 to 88. Testes from 90-day-old control and flutamide-treated rats were used for experiments. Flutamide did not affect testis morphology, but impaired connexin43 immunoexpression between Sertoli cells at the blood-testis barrier (BTB) region, indicating the BTB as a sensitive target for flutamide. Real-time RT-PCR and western blot analyses revealed upregulation of N-cadherin at the mRNA and protein level after flutamide exposure (p < 0.05), whereas no changes in b-catenin and c-Src expression were observed. Notably, membranous b-catenin immunolocalization indicated its involvement in the cell adhesion complex rather than its contribution to the Wnt signaling pathway. As we used an exposure regime which avoided germ cell loss, it is likely that changes in the N-cadherin-b-catenin complex are a primary effect of androgen signaling disruption by flutamide. Immunohistochemistry revealed a diffusion of N-cadherin and b-catenin signals away from the BTB with concomitant disruption of c-Src staining pattern. As detected by immunofluorescence and coimmunoprecipitation, flutamide promoted disassembly of the N-cadherin-ß-catenin complex, induced N-cadherin to dissociate from c-Src at the BTB site, and altered interactions between the cell junction proteins and/or c-Src. Equally important, increased levels of p-N-cadherin-Tyr860 and p-b-catenin-Tyr654 (p < 0.05) pointed to a mechanism related to adhesion complex disassembly and suggested a potential role of c-Src in the control of the protein-protein dynamics. Overall, for the first time we have shown that flutamide alters the distribution of c-Src and affects N-cadherin-b-catenin interactions at the BTB. Understanding mechanism(s) by which anti-androgens can affect intercellular adhesion within the testis is relevant for predicting and preventing reproductive disorders affecting male fertility.

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

“…Thus in experimental studies, flutamide is often used to study the role of androgen receptor signaling in physiological and pathological processes. Taking into account our previous studies showing affected spermatogenesis in boars treated with flutamide neonatally [ 13 , 19 ] and recent observations showing altered immunoexpression of Cx43 at the BTB level after flutamide exposure of adult rat [ 20 ] and in primary rat Sertoli cells in vitro [ 21 ], we hypothesize that limited exposure to flutamide may have a direct impact on the Cx43 gene expression in the testis, which may precede an impact on the tissue histology. To assess this we applied short, seven-day-treatment with flutamide and examined rat seminiferous tubule morphology and Cx43 expression at the mRNA and protein level.…”

“…According to our hypothesis, to discriminate a primary effect of androgen signaling disruption by flutamide, it was important to select a dose, frequency, and time of flutamide treatment which was high enough to induce changes in the expression of intercellular junction proteins within seminiferous epithelium without causing alterations in testicular cell morphology and producing a toxic effect in Sertoli cells in vitro . Thus treatment protocol was based on the literature data and our previous studies [ 20 – 22 ].…”

“…Equal amounts of protein were resolved by SDS-PAGE under reducing conditions, transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany) and analyzed by Western blotting with: rabbit polyclonal antibodies against Cx43 (1 : 12 000; Sigma-Aldrich) or caspase 3 (1 : 1000; Cell Signaling Technology, Beverly, MA, USA), or mouse monoclonal antibodies against ZO-1 (1 : 500; Invitrogen) or PCNA (1 : 500; Merck Millipore), as previously reported [ 19 ]. The presence of the primary antibody was revealed with horseradish peroxidase-conjugated secondary antibodies diluted 1:3000 (Vector Lab., Burlingame, CA, USA) and visualized with an enhanced chemiluminescence detection system as previously described [ 20 ]. Actin served as a loading control.…”

“…Real-time RT-PCR was performed using the StepOne Real-Time PCR system (Applied Biosystems) and optimized standard conditions as described previously [ 20 ]. The mRNA expression levels of the Cx43 and ZO-1 were quantified in each sample using TaqMan Gene Expression Assays (Applied Biosystems) as follows: for Cx43 assay ID, Rn01433957_m1; for ZO-1 assay ID, Rn02116071_s1; GAPDH levels were determined as an endogenous control assay (Applied Biosystems, assay ID, Rn01775763_g1).…”

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

“…injections of flutamide (50 mg/kg bw) daily for 7 days as described previously [21] or single injection of ethanedimethane sulphonate (EDS; 75 mg/kg bw) as described previously [22,23]. Flutamide reduces androgen action by inhibiting AR transcriptional activity, whereas EDS selectively destroys Leydig cells, the main source of androgens, leading to significant decrease in testosterone production [24,25].…”