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

This study was undertaken to explore interactions between c-Src kinase and the N-cadherin-ß-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 ß-catenin and c-Src expression were observed. Notably, membranous ß-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-ß-catenin complex are a primary effect of androgen signaling disruption by flutamide. Immunohistochemistry revealed a diffusion of N-cadherin and ß-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-ß-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-ß-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.

Spermatogonial stem cells alone are not sufficient to re-initiate spermatogenesis in the rat testis following adjudin-induced infertility.

The blood-testis barrier (BTB) is a unique ultrastructure in the testis, which creates a specialized microenvironment in the seminiferous epithelium known as the apical (or adluminal) compartment for post-meiotic germ-cell development and for maintenance of an immunological barrier. In this study, we have demonstrated unequivocally that a functional and intact BTB is crucial for the initiation of spermatogenesis, in particular, the differentiation of spermatogonial stem cells (SSCs). It was shown that adult rats (∼300 g body weight, b.w.) treated with adjudin at 50 (low-dose) or 250 (high-dose) mg/kg b.w. by gavage led to germ-cell depletion from the seminiferous tubules and that >98% of the tubules were devoid of germ cells by ∼2 week and rats became infertile in both groups after the sperm reserve in the epididymis was exhausted. While the population of SSC/spermatogonia in the seminiferous tubules from both groups was similar to that of normal rats, only rats from the low-dose group were capable of re-initiating spermatogenesis; and by 20 weeks, greater than 75% of the tubules displayed normal spermatogenesis and the fertility of these rats rebounded. Detailed analysis by dual-labelled immunofluorescence analysis and a functional BTB integrity assay revealed that in both treatment groups, the BTB was disrupted from week 6 to week 12. However, the disrupted BTB 'resealed' in the low-dose group, but not in the high-dose group. Our findings illustrate that SSC/spermatogonia failed to differentiate into spermatocytes beyond A(aligned) spermatogonia in the high-dose group with a disrupted BTB. In short, these findings illustrate the critical significance of the BTB for re-initiation of spermatogenesis besides SSC and spermatogonia.

Polarity proteins and actin regulatory proteins are unlikely partners that regulate cell adhesion in the seminiferous epithelium during spermatogenesis.

In mammalian testis, spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, which is composed of a series of cellular events. These include: (i) spermatogonial stem cell (SSC) renewal via mitosis and differentiation of SSC to spermatogenia, (ii) meiosis, (iii) spermiogenesis, and (iv) spermiation. Throughout these events, developing germ cells remain adhered to the Sertoli cell in the seminiferous epithelium amidst extensive cellular, biochemical, molecular and morphological changes to obtain structural support and nourishment. These events are coordinated via signal transduction at the cell-cell interface through cell junctions, illustrating the significance of cell junctions and adhesion in spermatogenesis. Additionally, developing germ cells migrate progressively across the seminiferous epithelium from the stem cell niche, which is located in the basal compartment near the basement membrane of the tunica propria adjacent to the interstitium. Recent studies have shown that some apparently unrelated proteins, such as polarity proteins and actin regulatory proteins, are in fact working in concert and synergistically to coordinate the continuous cyclic changes of adhesion at the Sertoli-Sertoli and Sertoli-germ cell interface in the seminiferous epithelium during the epithelial cycle of spermatogenesis, such that developing germ cells remain attached to the Sertoli cell in the epithelium while they alter in cell shape and migrate across the epithelium. In this review, we highlight the physiological significance of endocytic vesicle-mediated protein trafficking events under the influence of polarity and actin regulatory proteins in conferring cyclic events of cell adhesion and de-adhesion. Furthermore, these recent findings have unraveled some unexpected molecules to be targeted for male contraceptive development, which are also targets of toxicant-induced male reproductive dysfunction.

In vitro regulation of extracellular superoxide dismutase in sertoli cells.

Rat Sertoli and germ cells express extracellular superoxide dismutase (SOD(EX)), however, the relative level of SOD(EX) expressed by these cells was not known. We report herein germ cells consisting largely of spermatogonia, spermatocytes, and round spermatids expressed only one-third SOD(EX) as that of Sertoli cells when examined by semi-quantitative RT-PCR. While cocultures of germ cells with Sertoli cells failed to induce any changes in SOD(EX) expression possibly due to the limited number of cells that can be supported by the in vitro culture system dissimilar to the in vivo condition, incubation of total germ cell-conditioned medium with Sertoli cells was able to significantly inhibit Sertoli cell SOD(EX) expression dose-dependently suggesting a germ cell-derived soluble factor(s) may regulate SOD(EX) in the testis. On the other hand, cytokines such as TGF-beta1, beta-NGF, or FGF and steroid hormones such as estradiol-17beta, progesterone, testosterone, and DHT were unable to effect the expression of Sertoli cell SOD(EX). However, FSH at 100 ng/dish was able to induce a significant increase in Sertoli cell SOD(EX) expression. While cytokines, the known mediators of the inflammatory response, were unable to affect Sertoli cell SOD(EX) expression, the induction of generalized inflammation in vivo was able to cause a 2- to 2.5-fold increase in testicular SOD(EX) expression concomitant with a transient increase in the liver but not in the brain. Taken collectively, these results demonstrate that while SOD(EX) is an important antioxidant enzyme protecting the testis from reactive oxygen species, the mechanism(s) regulating its expression may involve an array of molecules and is a complicated cellular event.

Sertolin is a novel gene marker of cell-cell interactions in the rat testis.

A novel testicular protein designated sertolin was cloned. The full-length sertolin cDNA consists of 853 base pairs with an open reading frame of 381 base pairs coding for a 127-amino acid polypeptide that shares limited identities with antaxin/josephin and thrombospondin proteins. Sertolin (calculated molecular mass, 13,759 daltons) has two mRNA transcripts of 2.3 and 1 kilobase. A 22-amino acid peptide based on the deduced amino acid sequence of sertolin (NH(2)-KKEHFNLFKAASVSHLVQVVPQ) was synthesized and used for polyclonal antibody production. Immunoblot analysis detected a 17-kDa immunoreactive band in the Sertoli cell cytosol. Using Sertoli-germ cell cocultures, sertolin expression was found to be reduced by as much as 5-fold at the time when germ cells attach onto Sertoli cells but preceding the establishment of specialized inter-Sertoli-germ cell junctions. Neither FSH nor 17beta-hydroxy-5alpha-androstan-3-one was able to affect sertolin expression, whereas estradiol-17beta and progesterone induced a significant increase in Sertoli cell sertolin expression in vitro. In addition, interleukin-1alpha, a germ cell-derived cytokine, was also able to elicit a transient but significant increase in Sertoli cell sertolin expression. Sertolin expression was also shown to increase with testicular development and is likely to be associated with the onset of spermatogenesis. In addition, sertolin expression increased in the testis when generalized inflammation was induced in adult rats by injection of fermented yeast. These results show that sertolin will be useful in characterizing cell-cell interactions in the testis.

Obstruction of the vas deferens alters protein secretion by the rat caput epididymidal epithelium in vivo.

Obstruction of epididymal lumen fluid flow alters the intraluminal environment and potentially changes epididymal epithelial cell function when those functions are dependent on intraluminal regulatory molecules. This investigation tested the hypothesis that obstruction of the rat vas deferens alters caput epididymidal protein synthesis and secretion In vivo. Adult male rats were subjected to vasal obstruction or sham operation. Fourteen days later, caput epididymides were subjected to in vivo microperfusion with medium containing a [35S]-amino acid mixture. At the end of a 3-hour perifusion, micropuncture was used to obtain caput lumen fluid (LF). Tubule extract (TE) was obtained as supernatant after homogenization and centrifugation of caput tubules. Tubule extract contained all [35S]-proteins synthesized within the 3-hour experiment, and LF contained the secreted [35S]-proteins. Radioactivity of trichloroacetic acid (TCA)-precipitable proteins in LF and TE was determined, and two-dimensional electrophoresis and autoradiography of each sample were carried out. The resultant autoradiograms were evaluated densitometrically. A protein synthesis index calculated from the TCA-precipitable radioactivity data demonstrated that a significant decline in overall protein synthesis was induced by vasal obstruction. Densitometry of autoradiograms demonstrated that the total number of radiolabeled proteins detected in both the LF and TE of obstructed animals was significantly smaller than the same number in control animals (P < 0.05). Autoradiography revealed seven major, consistently appearing gene products in LF, and these were subjected to amino acid sequence analysis. Cysteine-rich secretory protein (CRISP)-1 proteins were significantly reduced in the LF of obstructed animals, which implies that these proteins are dependent on luminal regulatory molecules for their normal production.