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BOR - Papers in Press, published online ahead of print January 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.038596
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BIOLOGY OF REPRODUCTION 72, 1232–1240 (2005)
DOI: 10.1095/biolreprod.104.038596
© 2005 by the Society for the Study of Reproduction, Inc.

Nonylphenol Alters Connexin 43 Levels and Connexin 43 Phosphorylation Via an Inhibition of the p38-Mitogen-Activated Protein Kinase Pathway1

Jayaprakash Aravindakshan , and Daniel G. Cyr 2 

INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Quebec, Canada H9R 1G6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocrine-disrupting chemicals are exogenous compounds that mimic or inhibit the action of estrogens or other hormones. Nonylphenol, an environmental contaminant distributed along the St. Lawrence River, has been reported to act as a weak estrogen. Previous studies from our laboratory have shown that rats that were fed fish taken from nonylphenol contaminated sites have altered spermatogenesis and decreased sperm count. The mechanism responsible for this effect is unknown. Gap junctional intercellular communication (GJIC) in the testis is critical for coordinating spermatogenesis. The objectives of the study were to determine the effects of nonylphenol on GJIC and connexin 43 (Cx43) in a murine Sertoli cell line, TM4. Cells were exposed for 24 h to different concentrations (1 to 50 µM) of either nonylphenol or 17ß-estradiol. GJIC was determined using a microinjection approach in which Lucifer yellow was injected directly into a single cell, and GJIC was assessed 3 min postinjection. Nonylphenol exposure decreased GJIC between adjacent cells by almost 80% relative to controls. A significant concentration-dependent reduction in GJIC was observed at nonylphenol concentrations between 1 and 50 µM. Cx43 immunofluorescent staining was reduced at both 10 and 50 µM doses of nonylphenol. Cx43 phosphorylation, as determined by Western blot analysis, was reduced at both 10 and 50 µM concentrations, which may explain, at least in part, the inhibition of GJIC. In contrast, no effect on GJIC or Cx43 protein was observed in cells exposed to 17ß-estradiol at these concentrations. Cx43 has been reported to be phosphorylated via the p38-mitogen-activated protein kinase (MAPK) pathway. P38-MAPK activity was assessed in both control and nonylphenol-exposed cells. A dose-dependent decrease in p38-MAPK activity was observed in nonylphenol-exposed Sertoli cells. Protein kinase C activity was also measured and was not influenced by nonylphenol. These results suggest that nonylphenol inhibits GJIC between Sertoli cells and that this is modulated via nonestrogenic pathways.

environment, Sertoli cells, signal transduction, testis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several reports suggest a deterioration in the quality of male human reproductive function over the last 50 yr [1, 2]. Male reproductive parameters that have been reported as altered include a decrease in sperm count, increased incidences of congenital malformations of the male reproductive tract, and testicular cancer among young men [3, 4]. While it is not clear what factors or changes in lifestyle may be responsible for these changes, it has been suggested that exposure to certain environmental contaminants may be contributing to these effects [5]. Among the different classes of reproductive toxicants present in the environment, those that act as endocrine-disrupting chemicals have been singled out as contributing to male reproductive dysfunction [2, 6, 7]. We have previously shown that spermatogenesis is delayed in fish exposed to xenoestrogens in the St. Lawrence River [8]. Furthermore, if lactating rats are fed fish from xenoestrogen contaminated sites, the male pups will produce fewer sperm and sperm with decreased motility when they reach adulthood. As well, spermatogenesis appears to be altered, and a decrease in the expression of the gap junctional protein connexin 43 (Cx43) in the testis has been shown [9].

Studies have reported that alkylphenol ethoxylates from municipal sewage effluent are estrogenic chemicals that may contribute to the xenoestrogenic effects observed in fish from the St. Lawrence River [8, 10]. There has been speculation that certain phenolic plasticizing agents, such as p-nonylphenol, which are now prevalent in the environment, may affect Sertoli cell development and function, because males have much lower levels of estradiol than females [3, 11]. Studies have reported that severe testicular abnormalities, including poor germ cell differentiation and reduced sperm counts, are observed following gestational, lactational, or direct exposure of male rats to moderate levels of several alkylphenols [12–14]. Similar adverse effects on testicular structure and function have been observed following exposure to artificial estrogens, such as diethylstilbestrol or ethinylestradiol in rodents [15]. It has been suggested that the toxic effects of alkylphenols are mediated via estrogen receptors [16]. Of interest, alkylphenols have also been shown to induce apoptosis in a wide variety of cells [17], including rat primary germ and Sertoli cell cultures, while 17ß-estradiol was without effect [18]. Another report has shown that hCG-stimulated steroidogenesis in cultured mouse Leydig tumor cells was inhibited by octylphenol in an estrogen receptor-independent manner [19], suggesting that the biological effects of alkylphenols may not be mediated entirely through direct interaction with estrogen receptors.

Spermatogenesis requires direct intercellular communication between Sertoli cells, which is mediated by gap junctions [20]. An important role of gap junctions is to regulate cell growth and differentiation by controlling the passage of small molecules, including secondary messengers, between adjacent cells [21]. Gap junctions are composed of intercellular pores that allow the passage of small molecules between adjacent cells (<1 kDa). These pores are composed of hexomeric connexins from each cell, which are themselves formed by the oligomerization of connexins. Cx43 is present in many tissues, including the testis, and is localized between adjacent Sertoli cells, Sertoli cells and germ cells, and between Leydig cells [22–25]. Several lines of evidence indicate that Cx43 is essential for normal testicular function [20, 24, 26–28].

The objective of the present study was to determine whether nonylphenol could alter intercellular communication in Sertoli cells and to determine if this effect was mediated via an estrogenic pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

TM4 mouse Sertoli cells were purchased from the American Type Culture Collection (Manassas, VA). TM4 cells were grown in medium containing a mixture of Ham F12 medium and Dulbecco modified Eagle medium (1:1) with 1.2 g/L sodium bicarbonate and 15 mM Hepes (92.5%), horse serum (5%), and fetal bovine serum (2.5%) supplemented with 100 U/ml penicillin and 100 U/ml streptomycin. For subculturing, the medium was removed, and the cells were rinsed with a solution of 0.25% trypsin and 0.03% EDTA. The solution was removed and an additional 1 to 2 ml of trypsin-EDTA solution was added. The flask was incubated at 37°C until the cells detached. The cells were then washed with fresh culture medium and subsequently aspirated and pelleted by centrifugation. The supernatant was discarded and the cells were then resuspended in fresh culture medium and dispensed into new culture flasks.

Treatment and Cell Viability

To determine whether nonylphenol affects Sertoli cells via an estrogenic pathway, we treated TM4 Sertoli cells with either nonylphenol or 17ß-estradiol. Nonylphenol and 17ß-estradiol were dissolved in ethanol (0.05%), which was used as the vehicle. TM4 cells were passaged at a concentration of 1 x 106 cells/ ml and incubated overnight. The cells were then washed and left untreated (control), exposed to 0.05% ethanol (vehicle), exposed to varying concentrations of nonylphenol (0.1 nM to 50 µM; Schenectady International, Schenectady, NY), or exposed to varying concentrations of 17ß-estradiol (0.1 nM to 1 µM). After 24 h, cells were harvested and cell viability was assessed by trypan blue exclusion.

Immunolocalization of Cx43

Cells were plated on glass coverslips and fixed in ice-cold methanol for 30 min at –20°C. Cells were washed in PBS and blocked with 2% BSA in PBS for 20 min at room temperature. This was followed by three 5-min washes in PBS. Immunocytochemical localization of Cx43 was performed using rabbit polyclonal anti-Cx43 antisera (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Cells were incubated for 90 min in a hydrated chamber with the primary antibody at room temperature. The cells were then washed in PBS and incubated for 45 min with a fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (1: 2000; Jackson Immunoresearch, West Grove, PA). The cells were subsequently washed three times in PBS and mounted with Vectashield containing propidium iodide (Vectastain Laboratories, Burlington, ON). The cells were viewed and photographed under a Leica fluorescent microscope.

Estimation of Gap Junctional Intercellular Communication by Dye-Transfer Assay

TM4 Sertoli cells were cultured with 17ß estradiol, nonylphenol, or vehicle alone for 24 h. To determine the functionality of gap junctions, gap junctional intercellular communication (GJIC) was assayed by scoring the number of dye-coupled cells after microinjection of a single cell with Lucifer yellow (Sigma Chemical Co., St Louis, MO) using an Eppendorf pressure injection system. The extent of GJIC was determined by the number of neighboring fluorescent cells, scored with the aid of a fluorescence/ phase-contrast microscope 3 min postinjection. For each experimental point, 10–20 independent injections were carried out.

Western Blot Analysis

Culture medium was removed and the cells were rinsed with PBS at room temperature. The following steps were performed on ice using fresh, ice-cold buffers. A 1-ml aliquot of RIPA buffer (1x Tris-buffered saline [TBS], 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide, and 100 µM sodium orthovanadate) was added to the cell culture plate and adherent cells were scraped with a cell scraper. The scraped lysate was transferred to a clean microcentrifuge tube. The plate was washed once again with 0.3 ml of RIPA buffer and then combined with the first lysate. The lysate was passed through a 21-gauge needle to shear the DNA and incubated for 60 min on ice. The lysates were centrifuged at 10 000 x g for 10 min at 4°C. The supernatant was collected and protein concentration determined using the Bradford method (Protein Assay Kit; Bio-Rad Laboratories, Mississauga, ON). An aliquot of 40 µg of the whole cell lysate was mixed with an equal volume of electrophoresis sample buffer (5% v/v glycerol, 2.5% v/v 2-mercaptoethanol, 1% SDS, 125 mM Tris-HCl pH 6.7, and 0.4% w/v bromophenol blue) and loaded on a 10% polyacrylamide gel with a 4% stacking gel. Electrophoresis was carried out at 90 V until the dye front reached the bottom of the gel. The proteins were transferred from the gel onto a polyvinylidene difluoride membrane using an electroblotting apparatus according to the manufacturer's protocols (Bio-Rad Laboratories). To block nonspecific binding, the membranes were incubated in 5% Blotto for 60 min at room temperature. The blocked membrane was incubated with anti-Cx43 antisera (1:200 in Blotto; Santa Cruz Biotechnology) for 1 h at room temperature. The membranes were washed three times for 5 min each with TBST (TBS with Tween-20, pH 7.4). The membrane was then incubated for 45 min at room temperature with an alkaline phosphatase-conjugated secondary antibody (1:2000 in Blotto; Sigma-Aldrich, Mississauga, ON). Following the incubation, the membranes were once again washed three times for 5 min with TBST and once for 5 min with TBS (pH 7.4). Quantification of Cx43 was performed by scanning the immunoblots using ImageQuant software (Bio-Rad Laboratories).

p38-Mitogen-Activated Protein Kinase Assay

The activity of p38-mitogen-activated protein kinase (MAPK) was analyzed by using a p38 MAPK assay kit (Cell Signaling Technology Inc, Beverly, MA) according to the manufacturer's instructions. Briefly, phosphorylated p38-MAPK was immunoprecipitated from 200 µg of cell lysate with an anti-p38-MAPK phospho-specific antibody. The immunoprecipitated protein pellets were washed thoroughly and resuspended in kinase buffer containing ATP and 1 µg of recombinant activating transcription factor-2 (ATF-2) as a p38-MAPK substrate. The reaction was incubated at 30°C for 45 min and terminated by the addition of 3x SDS sample buffer (187.5 mM Tris-HCl pH 6.8 at 25°C, 6% w/v SDS, 30% glycerol, 150 mM dithiothreitol, and 0.03% w/v bromophenol blue). The kinase reaction (ATF-2 phosphorylation) was detected using anti-phospho-ATF-2 (Thr71) antisera by Western blotting and chemiluminescent detection.

Protein Kinase C Activity Assay

The protein kinase C (PKC) activity of TM4 Sertoli cells was determined using the PepTag PKC assay kit (Promega, Ottawa, ON), according to the manufacturer's instructions. This assay utilizes a florescent peptide substrate that is specific to PKC. Phosphorylation by PKC changes the net charge of the substrate from +1 to –1, allowing the phosphorylated and nonphosphorylated forms of the substrate to be separated on an agarose gel (1%), because the phosphorylated species migrates toward the anode, while the nonphosphorylated substrate migrates toward the cathode. The samples were separated on an agarose gel at 100 V for 15 min, and the bands were visualized under UV light. The negatively charged phosphorylated bands were excised using a razor blade, placed in a graduated microcentrifuge tube, and heated at 95°C until the gel slice melted. The volume of the solution was adjusted to 250 µl with water. The hot agarose solution (175 µl) was added to a separate tube containing 75 µl of gel solubilization solution, 100 µl of glacial acetic acid, and 150 µl of distilled water. The mixture was vortexed and then transferred to a spectrophotometric cuvette, and absorbency was read at 570 nm. One unit of kinase activity is defined as the number of nanomoles of phosphate transferred to a substrate in 1 ml/min. One phosphorylation site exists on each peptide; therefore, the number of moles of peptide present in the negatively charged, phosphorylated bands is equivalent to the number of moles of phosphate transferred. The number of moles of phosphorylated peptide ({epsilon}) was determined by calculating the number of units of kinase activity in each slice of agarose.

Statistical Analysis

Data are expressed as mean ± SEM unless otherwise indicated, of at least three independent experiments performed at different time points. The data were tested for normality and homogeneity of variance using Kolmogorov-Smirnov test. All data were analyzed using one-way analysis of variance (ANOVA) (SigmaStat for Windows, Version 2.0, Jandel Corporation, San Rafael, CA). If the differences were significant, a Student-Newman-Keuls test was used for post-ANOVA multiple comparisons (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Nonylphenol on Cell Viability

To establish whether or not nonylphenol or estradiol altered cell viability, Sertoli cells were exposed for 24 h to different concentrations of nonylphenol (0.1 nM to 50 µM) and viability was assessed by trypan blue exclusion. Concentrations of nonylphenol ranging between 0.1 nM and 25 µM did not exert any significant effects on Sertoli cell viability (Table 1). In contrast, when cells were exposed to a 50 µM concentration of nonylphenol, the number of viable cells was decreased by 28% (P < 0.05; Table 1). Because nonylphenol is at least 1000 times less estrogenic than 17ß-estradiol, we treated the Sertoli cells with doses ranging between 0.1 nM to 1 µM. 17ß-Estradiol did not alter cell viability at any of these doses (data not shown). Nonylphenol exposure at the 10 µM dose did not appear to alter either apoptosis or cell proliferation as determined either by the general morphology of the cells or in the expression of cell cycle or apoptosis genes (data not shown).


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  		TABLE 1. TM4 cell viability after 24 h treatment with nonylphenol.*

Effects of Nonylphenol on GJIC

To assess the effects of nonylphenol on functional GJIC, Sertoli cells were microinjected with Lucifer yellow and GJIC was assessed by examining the transfer of dye between cells. In control cells, baseline coupling varied from 4 to 6 cells, with dye passing to a mean of 5.56 cells (Fig. 1Aa). 17ß-Estradiol treatment did not affect GJIC between adjacent cells and these cells were not different from controls (Fig. 1Ab). A significant reduction in GJIC was observed at 10 µM nonylphenol (Fig. 1Ac). Following exposure to 10 µM nonylphenol, the number of coupled cells decreased between 0 and 1 cells, an almost 80% reduction relative to controls. This indicated that the functionality of gap junctions was reduced (Fig. 1B).



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  		FIG. 1. Functional analysis of gap junctions in TM4 Sertoli cells. Confluent cell cultures of TM4 Sertoli cells were treated for 24 h with either vehicle (0.05% ethanol), 1 µM 17ß-estradiol, or 10 µM nonylphenol. Cells were microinjected with Lucifer yellow dye and examined 3 min postinjection for fluorescent dye transfer. Several cells from different cultures were microinjected to establish the effect of treatment. A) Each longitudinal series of panels show the cells before microinjection, the single cell microinjection with Lucifer yellow under fluorescent light, and the transfer of Lucifer yellow between cells after 3 min. Vertical panels indicate (a) control, (b) 1 µM 17ß-estradiol treated cells, and (c) 10 µM nonylphenol treated cells. Arrows indicate the cell injected with Lucifer yellow. Magnification x400. B) Quantitative analysis of GJIC in TM4 cells treated with either vehicle (control), 17ß-estradiol, or nonylphenol (10 µM). Asterisk indicates a significant difference from both control and estradiol-treated groups (ANOVA, P < 0.05)

Immunolocalization of Cx43

To assess whether or not the decrease in gap junctional communication was associated with a decrease or alteration in the cellular localization of Cx43, immunolocalization was performed following 24 h of treatment. In control cells (Fig. 2a), a punctate immunostaining for Cx43 along the plasma membrane of adjacent cells was observed. Sertoli cells treated with different doses of 17ß-estradiol did not affect Cx43 localization, and the intensity of the immunostaining appeared to be similar to that of control cells (Fig. 2b). However, in cells treated with nonylphenol (1–50 µM), the intensity of the immunostaining was markedly decreased at all doses used. The localization of Cx43 in the nonylphenol-treated cells did not appear to be affected by treatment, and the immunostaining was restricted to the plasma membrane of the cells at areas of contact between neighboring cells. However, in cells treated with 10 µM nonylphenol, the intensity of the immunoreaction was substantially decreased (Fig. 2c) and was almost completely absent in cells treated with 50 µM (Fig. 2d).



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  		FIG. 2. Photomicrographs of TM4 Sertoli cells showing the immunolocalization of Cx43. a) In the untreated TM4 Sertoli cells, punctate Cx43 immunofluorescence (arrows) is prominent in the appositional plasma membranes between adjacent cells. b) 17ß-Estradiol treated cells did not show significant changes in Cx43 staining. In cells incubated with either 10 µM (c) or 50 µM (d) of nonylphenol for 24 h, Cx43 immunostaining (green) is dramatically decreased (arrows). Nuclei are stained with propidium iodide (red). Magnification x600

The effects of nonylphenol appeared to be both dose- and time-dependent. The decrease in Cx43 immunostaining induced by nonylphenol was evident as early as 6 h after the addition of nonylphenol and decreased gradually until 24 h (data not shown).

Effects of Nonylphenol on Cx43 Levels in Sertoli Cells

Cx43 levels in Sertoli cells were determined by Western blot analysis. Cx43 is a phosphorylated protein, and its phosphorylation status has been shown to be critical for its function. Western blots of enriched membrane fractions of Sertoli cells revealed two major bands, representing the phosphorylated and nonphosphorylated forms of Cx43 (Fig. 3A).



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  		FIG. 3. Western blot analysis of Cx43 levels in nonylphenol treated TM4 Sertoli cells. Cells were incubated with either medium alone (C), vehicle (0.05% ethanol), or varying concentrations of nonylphenol (1, 10, 25, or 50 µM) for 24 h. Total proteins (40 µg) were extracted from TM4 cell monolayers and proteins were separated by SDS-PAGE. Cx43 was detected using an alkaline phosphatase-conjugated secondary antibody and revealed by chemiluminescence (A). Protein loading was standardized using actin as an internal control (A). Unphosphorylated (B) and phosphorylated (C) Cx43 levels were determined by densitometry and corrected for protein loading using actin levels. The ratio of phosphorylated to nonphosphorylated Cx43 was also assessed (C). Data are expressed as the mean + SEM of four different experiments. Asterisks indicate significant differences from control and vehicle group (ANOVA; P < 0.05)

Sertoli cells incubated with nonylphenol expressed significantly less Cx43 (Fig. 3B). Furthermore, both the phosphorylated and nonphosphorylated Cx43 levels were significantly lower in nonylphenol-exposed cells (Fig. 3, B and C). However, when the levels of phosphorylated Cx43 were compared with those in either nonexposed or vehicle-treated cells, there was a significant reduction in the ratio of phosphorylated to nonphosphorylated Cx43 in the nonylphenol-treated cells (Fig. 3D). The decrease in Cx43 phosphorylation occurred as early as 6 h after the start of treatment and continued to decrease until 24 h (Fig. 4, A–D). There were no significant differences in the phosphorylation status of Cx43 in the 17ß-estradiol treated cells (Fig. 5, A–D). This suggested that nonylphenol not only decreased Cx43 expression levels but also its phosphorylation status.



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  		FIG. 4. Western blot analysis of Cx43 levels in nonylphenol treated TM4 Sertoli cells at different times following the start of treatment. Cells were incubated with either medium alone (C), vehicle (0.05% ethanol), or nonylphenol (10 µM). Total proteins (40 µg) were extracted from TM4 cell monolayers and proteins were separated by SDS-PAGE at 0.5, 1, 6, 12, and 24 h following the addition of nonylphenol. Cx43 was detected using an alkaline phosphatase conjugated secondary antibody and revealed by chemiluminescence (A). Protein loading was standardized using actin as an internal control (A). Unphosphorylated (B) and phosphorylated (C) Cx43 levels were determined by densitometry and corrected for protein loading using actin levels. The ratio of phosphorylated to nonphosphorylated Cx43 was also assessed (D). Data are expressed as the mean + SEM of four different experiments. Asterisks indicate significant differences from control and vehicle group (ANOVA; P < 0.05)



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  		FIG. 5. Western blot analysis of Cx43 levels in 17ß-estradiol treated TM4 Sertoli cells. Cells were incubated with either medium alone (C), vehicle (0.05% ethanol), or varying concentrations of 17ß-estradiol (10–9 to 10–6 M) for 24 h. Total proteins (40 µg) were extracted from TM4 cell monolayers and proteins were separated by SDS-PAGE. Cx43 was detected using an alkaline phosphatase-conjugated secondary antibody and revealed by chemiluminescence (A). Protein loading was standardized using actin as an internal control (A). Unphosphorylated (B) and phosphorylated (C) Cx43 levels were determined by densitometry and corrected for protein loading using actin levels. The ratio of phosphorylated to nonphosphorylated Cx43 was also assessed (D). Data are expressed as the mean + SEM of four different experiments. There were no significant differences between the control and vehicle exposed groups

p38-MAPK and PKC Activity

To understand the mechanisms by which Cx43 phosphorylation was altered by nonylphenol, both p38-MAPK and PKC activity were measured, because these are known to phosphorylate Cx43 in other cell types [29–31].

We first determined whether or not p38-MAPK represented a biologically active kinase in TM4 Sertoli cells. This was done by testing whether phosphorylated p38-MAPK found in TM4 Sertoli cells could phosphorylate its downstream substrate, ATF-2. Using an in vitro kinase assay we were able to demonstrate that p38-MAPK can phosphorylate ATF-2 and that it is therefore active in TM4 cells (Fig. 6A).



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  		FIG. 6. Effects of nonylphenol on p38-MAPK activity in TM4 Sertoli cells. Using a commercial assay kit, p38-MAPK activity was determined by measuring the phosphorylation levels of the ATF-2 peptide. Protein extracts from TM4 cells were analyzed by immunoprecipitation followed by kinase activity assay. Cell extracts were immunoprecipitated with immobilized phospho-p38 MAPK (Thr180/Try182) monoclonal antibody. In vitro p389-MAPK assays were performed using the ATF-2 fusion protein as a substrate. Phosphorylation of ATF-2 at Thr71 was measured by Western blotting using a phospho-ATF-2 (Thr71) antibody and quantified by densitometry. A) Cells were incubated with either medium alone (C), vehicle (V, 0.05% ethanol), or varying concentrations of nonylphenol (1, 10, 25, or 50 µM) for 24 h. Data are expressed as the mean + SEM from four separate experiments (B). To assess whether p38-MAK was altered by estradiol, cells were incubated with either medium alone (C), vehicle (0.05% ethanol), or varying concentrations of 17ß-estradiol (10–9 to 10–6 M) for 24 h (C). Data from these experiments are expressed as the mean + SEM from four separate experiments (D). Asterisks indicate a significant difference from either control or vehicle exposed cells (ANOVA; P < 0.05)

Once it had been established that TM4 Sertoli cells possessed p38-MAPK activity we assessed whether or not nonylphenol could alter its activity. Western blot analysis, with ATF-2-phospho-specific antibody, revealed that exposure to nonylphenol resulted in a dose-dependent decrease of ATF-2 phosphorylation by as much as 80%, indicating an inhibition of p38-MAPK activity (Fig. 6B). There was no significant change in p38-MAPK activity in cells treated with 17ß-estradiol (Fig. 6, C and D).

Because PKC has also been shown to phosphorylate Cx43, we examined the level of PKC activation in TM4 Sertoli cells exposed to either nonylphenol or 17ß estradiol. PKC activity was not altered in cells exposed to either nonylphenol or 17ß-estradiol, compared with that of untreated control cells (Fig. 7, A and B).



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  		FIG. 7. Activation of PKC in TM4 Sertoli cells treated with either nonylphenol or 17ß-estradiol for 24 h. Total cell lysates were prepared and subjected to the PepTag assay. The phosphorylated peptide bands (A) were excised, and PKC activity was estimated by spectrophotometry (B). Cells were incubated with either medium alone (C), vehicle (V, 0.05% ethanol), 17ß-estradiol (A; 1 µM), or varying concentrations of nonylphenol (1 µM, B; 10 µM, C; 25 µM, D; 50 µM, E) for 24 h (A). Data are expressed as the mean + SEM from four separate experiments (B). There were no significant differences between any experimental group (ANOVA)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nonylphenol is the final biodegradation product of nonylphenol polyethoxylates, a major contaminant in the St. Lawrence River [32, 33]. Rats whose mothers are fed fish from these sites during lactation have reduced testicular Cx43 expression [9]. In the present study we have shown that TM4 Sertoli cells form functional intercellular gap junctions between adjacent cells by allowing the passage of microinjected Lucifer yellow between cells. Using both immunocytochemistry and Western blot analyses, we demonstrated that TM4 Sertoli cell gap junctions are composed of Cx43. These junctions appear as punctate structures localized to cell-cell contacts between adjacent cells. Gap junctions were shown to be present between adjacent Sertoli cells of the testis [34]. McGinley et al. [35] also reported that gap junctions were present between Sertoli cells and developing germ cells. Tan et al. [23] first reported that Cx43 was localized between adjacent Sertoli cells, as well as between developing germ cells and Sertoli cells in the adult rat. Other studies have reported the presence of connexins 26, 31, 32, 33, 37, 40, and 45 in the seminiferous tubules of the rat [36].

While exposure of the cells to estradiol did not alter intercellular communication, exposure to nonylphenol dramatically reduced intercellular communication at all doses tested. This effect suggests that while nonylphenol is considered an estrogenic substance [37], its effects on intercellular communication are not mediated via the estradiol receptor pathway. This is despite the fact that estrogen receptors have been shown to be expressed in TM4 Sertoli cells [38, 39].

While there have not been any studies to our knowledge regarding the effects of nonylphenol on intercellular communication, there is a growing list of environmental toxicants that appear to alter intercellular communication. Using the microinjection technique, we were able to demonstrate that nonylphenol almost completely inhibited GJIC at higher exposure doses. Studies in nonreproductive tissues have also been shown to reduce GJIC. Hexachlorobenzene, an organochlorine, can promote gender-specific tumor promotion by blocking intercellular communication between hepatocytes in the rat [40]. Other toxicants such as dioxins, polychlorinated aromatic hydrocarbons, and cadmium can also decrease gap junctional communication in cultured hepatocytes [41, 42]. Studies by Defamie et al., [43] have shown that lindane can abolish GJIC between rat Sertoli cells at the 50 µM dose. The importance of Cx43 gap junctions for spermatogenesis is indicated by the severe depletion of germ cells in prenatal male and female mice lacking the Cx43 gene [26]. Postnatal proliferation of spermatogonia is also impaired in Cx43 null mutants [20]. Insertion of Cx32 or Cx40 coding regions into the Cx43 coding region of Cx43–/– mice restored oogenesis and other deficiencies caused by Cx43 deletion, but spermatogonial amplification and spermatogenesis remained defective [27]. The importance of Cx43 gap junctions to the regulation of spermatogenesis in adults is supported by the fact that Cx43 immunoreactivity is reduced in spermatogenesis-deficient mutants [24, 28]. Thus, Cx43 is an essential component of GJIC pathways, which support the early phases of spermatogenesis. Previous studies have shown that nonylphenol can affect spermatogenesis [44–47].

The effects of nonylphenol on intercellular communication and Cx43 expression were both dose- and time-dependant. Time-response studies in which cells were exposed to 10 µM of nonylphenol indicated that there was a decrease in Cx43 over time, which peaked 24 h after the start of the exposure. These results suggest that nonylphenol does not appear to act at the level of the gap junction itself, but rather exerts a progressive effect either on the level of renewal of the connexins, or on their synthesis, or both. Further studies will be necessary to clearly establish whether or not this effect is at the transcriptional or post-transcriptional level.

Cx43, like most connexins, is a phosphoprotein with multiple electrophoretic isoforms when analyzed by SDS-PAGE. Phosphorylation has been implicated in the regulation of a broad variety of connexin processes, such as trafficking, assembly/disassembly, degradation, and gating of gap junction channels. Alterations in the phosphorylation status of Cx43 appear to influence gap junctional communication, whether it is hyperphosphorylated or hypophosphorylated [48–53]. Exposure of Sertoli cells to nonylphenol resulted not only in a decrease in the levels of Cx43, but also in its phosphorylation. Our results suggest that nonylphenol inhibited GJIC between Sertoli cells and that aberrant phosphorylation may have contributed to this effect. These effects were not observed when the cells were treated with 17ß-estradiol. These observations are consistent with other studies that show that Cx43 dephosphorylation is correlated with a reduction in communication via gap junctions [54–59].

The one or more protein kinases responsible for phosphorylating Cx43 or the site or sites targeted in unstimulated cells are unknown. Moreover, the exact number of phosphorylated sites per Cx43 is not known; however, at least five different Cx43 phosphopeptides have been observed, suggesting that Cx43 is phosphorylated at multiple sites [60], on serine and threonine residues [49, 61]. Cx43 has been shown to be phosphorylated by PKC [29, 31] and MAPK [30].

To understand the mechanism by which nonylphenol decreased the phosphorylation of Cx43, we studied whether or not the p38-MAPK and PKC pathways were altered in nonylphenol-treated Sertoli cells. When cells were treated with nonylphenol, p38-MAPK activity was significantly reduced in a dose-dependent manner. Of interest, there was no change in the PKC activity. The decrease in p38-MAPK suggests that nonylphenol may be selectively inhibiting this pathway, thereby resulting in a decrease in Cx43 phosphorylation. This is in contrast to another environmental contaminant, lindane, which increases Cx43 phosphorylation in Sertoli cells by stimulating the extracellular signal-regulated kinases (ERK) without altering either the JNK or p38-MAPK pathways [62]. The hyperphosphorylation of Cx43 by lindane also resulted in an increase in the cytoplasmic localization of Cx43, which is absent in nonylphenol-treated Sertoli cells in which Cx43 is hypophosphorylated. This suggests that hyperphosphorylation and altered ERK pathways are implicated in the localization of Cx43 in Sertoli cells [62].

Clearly, the effects observed in the present study indicate that nonylphenol alters intercellular communication via an estrogen-independent pathway, despite the fact that nonylphenol is considered an estrogenic chemical [11, 63]. Studies have reported that nonylphenol can alter the production of reactive species (ROS) in neuronal cells [64]. Furthermore, it has been shown that Cx32 expression is sensitive to increased ROS production in cultured hepatocytes. Therefore, it is tempting to speculate that nonylphenol action on intercellular communication in Sertoli cells may be mediated by an increased production of ROS. Furthermore, the possibility that nonylphenol is acting on other aspects of cell-cell interactions, such as cell adhesion, and that these affect GJIC cannot be discounted.

In rats exposed to nonylphenol, there is a decrease in circulating levels of serum testosterone and altered spermatogenesis [65, 66]. Our results indicate that nonylphenol can decrease intercellular communication and Cx43 levels, which have been shown to be important regulators of both Leydig cell function and spermatogenesis. These results suggest that the in vivo effects of nonylphenol on the testis may be, in part, the result of a decrease in intercellular communication. Furthermore, it has been reported that the activation of p38-MAPK by transforming growth factor-ß is essential for the formation and maintenance of the blood-testis barrier, which is formed by tight junctions between adjacent Sertoli cells [67, 68]. The results from this study would therefore suggest that as a result of decreased p38-MAPK activity, nonylphenol may also be targeting the blood-testis barrier. It has been reported that Cx43 colocalizes with tight junction proteins in the blood-testis barrier [69, 70]; hence, the complex association of both tight and gap junctions in the blood-testis barrier may be altered by nonylphenol. Further studies will be necessary to establish whether or not this is the case, and whether these effects all contribute to nonylphenol-induced testicular dysfunction.

In conclusion, the present study provides the first evidence that nonylphenol impairs gap junctional intercellular communication between Sertoli cells. This effect is, in part, the result of a decrease in the expression and phosphorylation of Cx43. The effects of nopnylphenol on testicular GJIC are mediated via an estrogen receptor-independent mechanism and an inhibition of the p38-MAPK pathway.


   ACKNOWLEDGMENTS
 
We thank M. Gregory and J. Dufresne (INRS) for their suggestions and assistance.


   FOOTNOTES
 
1 The Armand-Frappier Foundation provided postgraduate support for J.A. This study was supported by the Canadian Network of Toxicology Centres and the Natural Sciences and Engineering Research Council of Canada. Back

2 Correspondence: Dr. Daniel G. Cyr, INRS-Institut Armand Frappier, Université du Québec, 245 Hymus Boulevard, Pointe-Claire, Québec, H9R 1G6. FAX: 514 630 8850; daniel.cyr{at}iaf.inrs.ca Back

Received: 29 November 2004.

First decision: 17 December 2004.

Accepted: 11 January 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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