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B Is Activated in the Rat Testis Following Exposure to Mono-(2-Ethylhexyl) Phthalate
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. Here we show that mono-(2-ethylhexyl) phthalate (MEHP, 1 g/kg) induces translocation of NF-B subunits (p65, p50, and c-Rel) to germ cell nuclei in young rats (Postnatal Day 28) as early as 1 h after exposure. Immunohistochemistry of rat testes exposed to MEHP showed increased p50 and c-Rel presence in spermatocytes and spermatogonia. In addition, there was increased p65 nuclear positivity in Sertoli cells and germ cells after MEHP, while Rel-B localization was unchanged. These alterations correlated with increased nuclear NF-B-binding activity after MEHP exposure, as shown by electrophoretic mobility shift assays of whole-testis nuclear protein extracts. The increased activity of this transcription factor was associated with a transient protection of the seminiferous epithelium manifested as a decreased number of germ cell apoptotic nuclei measured by TUNEL assay 6 h after MEHP exposure. These results suggest that NF-B is involved in the testicular response to MEHP-induced injury.
apoptosis, spermatogenesis, testis, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B has been most extensively studied in the immune system, where it has been identified as a major mediator of the Fas signaling pathway [1, 2]. Extensive reports have shown that NF-B increases expression of numerous antiapoptotic genes, such as Bcl-XL, XIAP, TRAF1&2, c-IAP1&2 [3–6], and proapoptotic genes, such as Fas and FasL [7]. Moreover, the NF-B subunit p65 is critical to survival because mice lacking p65 die embryonically [8]. Despite the extensive research into NF-B, comparatively little is known about the role of this important protein in the testis.
Spermatogenesis is a complex process of germ cell proliferation and maturation. This proliferation is tightly controlled through autocrine-, paracrine-, and endocrine-signaling mechanisms. Particularly important are the somatic cells of the seminiferous tubule, Sertoli cells, which provide physical support, signals, and nutrients to developing populations of germ cells. In the testis, NF-B was first localized to spermatocytes and Sertoli cells in 1998 [9]. Since then, NF-B subunits have been studied in human testis [10] and the inhibitors of NF-B, predominantly IBß in testis, have been identified [11]. NF-B subunit antisera-stained Sertoli cells and germ cells with differential intensity in a stage-specific manner, as determined by immunohistochemistry of adult rat testes [9]. Additional research demonstrated that NF-B induced cAMP-response element binding protein and androgen receptor transcription in Sertoli cells in vitro [12, 13]. Transgenic mice containing the p105/p50 promoter fused to a LacZ marker revealed that induction of p50 transcription is developmentally regulated, with the highest expression demonstrated in pachytene spermatocytes [14]. In addition, testicular NF-B increases postgestation and is maintained from Postnatal Day 18 through adulthood. Although some research has been devoted to characterizing NF-B in rat and mouse testes, few studies have been performed on the NF-B response to testicular injury. Only a preliminary study showed NF-B alterations in mice after mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate, exposure [15]. The role of this important transcription factor in the testicular response to injury and germ cell apoptosis, however, has not been elucidated. Moreover, nuclear NF-B activity in response to apoptotic stimuli has not been described.
Phthalates are testicular toxicants that are ubiquitous environmental pollutants and have been shown to possess endocrine-disrupting activity leading to sexual maldevelopment in male mice exposed in utero [16–19]. In addition, MEHP has been shown to cause Sertoli cell damage and subsequent germ cell apoptosis when administered to 28-day-old rats by oral gavage [20–22]. Paracrine signaling between Sertoli and germ cells is one critical component of the germ cell apoptotic response to MEHP [23]. Interestingly, we have previously shown that the incidence of germ cell apoptosis declines briefly after MEHP exposure [22]. These data suggest that an initial transient antiapoptotic response to MEHP is eventually overwhelmed, resulting in massive germ cell apoptosis. This early protection phenomenon is unexplained but suggests the initial triggering of a germ cell-survival response following Sertoli cell injury.
The Fas system is a key paracrine regulator with Sertoli cells expressing Fas ligand (FasL) and germ cells expressing the Fas receptor. Exposure of gld mice (lacking functional FasL) to MEHP led to significantly less apoptosis than in wild-type counterparts [23]. This demonstrated a role for the Fas system in phthalate-induced germ cell apoptosis. In addition, Fas signaling can induce NF-B transcription [2], while active NF-B dimers can in turn induce Fas transcription [7].
Here we demonstrate an NF-B response during testicular injury following exposure to phthalate. Specifically, NF-B subunits p50, c-Rel, and, to a lesser extent, p65 translocate to the nucleus of germ cells and cause increased DNA-binding activity (by electrophoretic mobility shift assay [EMSA]) in 28-day-old male rats following MEHP exposure. These results show that testicular NF-B responds to MEHP exposure and suggests a role for this important family of transcription factors in modulating germ cell apoptosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats were maintained on a 12L:12D and were housed in 30–70% humidity and 70°F ± 2°F temperature-controlled rooms with access to Purina Rodent Chow 5001 and water ad libitum. All procedures involving animals were performed in accordance with the guidelines of Brown University's Institutional Animal Care and Use Committee in compliance with National Institute of Health guidelines [24, 25]. Newly weaned (21-day-old) Fischer 344 rats were purchased from Charles River Laboratories Inc. (Wilmington, MA) and allowed 6 days to acclimate before experiments were performed. Unless otherwise noted. all chemicals were purchased from Sigma Aldrich (St. Louis, MO)
MEHP Exposure
Male rats (28 days old) were exposed to 1 g/kg MEHP (cat #P1073; TCI America) or corn oil vehicle control by oral gavage at a rate of 4 ml/ kg, as previously described [22]. At the indicated time points (1, 3, 6, 12 h) after MEHP exposure, rats were killed by carbon dioxide asphyxiation and testes were immediately excised and processed.
Immunostaining
Immunostaining was performed based on previous methods [26, 27]. Testes were flash frozen by liquid nitrogen in OCT embedding medium (cat #4583; Sakura Tissue-Tek, Torrance, CA). Testis cross-sections (7 µm) were dried onto polylysine-coated slides and postfixed in –20°C methanol for 3 min. Sections were washed in PBS and blocked (3% BSA in PBS) for 1 h at room temperature. After blocking, samples were probed with p50 (cat #SC-1190), p65 (cat #SC-109), c-Rel (cat #Sc-272), or Rel-B (cat #SC-226) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 (p50, c-Rel) and 1:400 (p65, RelB) dilutions overnight at 4°C. Slides were washed and incubated with secondary antibodies: Alexa Fluor 488 conjugated donkey anti-goat or Alexa Fluor 594 conjugated donkey anti-rabbit (Molecular Probes, Eugene, OR) diluted at 1:500. Negative controls were preabsorption of the antibody with antigen before use or omission of the primary antibody (data not shown). Nuclei were localized by costaining with 50 ng/ml Hoechst 33258 (Sigma-Aldrich) for 5 min. Fluorescent microscopic images were obtained on a Zeiss Axiovert 35 microscope (Carl Zeiss, New York, NY) connected to a Spot RT camera (Diagnostic Instruments Inc., Sterling Heights, MI). Images were downloaded into Photoshop 6.0 imaging software (Adobe Systems Inc., San Jose, CA) for resizing and fluorescent layering. Final figures were assembled using Canvas 8.0 software (Deneba Systems Inc., Miami, FL)
TUNEL
TUNEL assays were performed with the DeadEND Apoptosis Assay kit (cat #G3250; Promega) per manufacturer's instructions with propidium iodide as a counterstain. For quantification, slides were blinded and all tubules in two different sections of testis were counted for the absence or presence of 1–3 or >3 TUNEL-positive germ cells. For every animal, at least 400 tubules were scored for TUNEL positivity.
Electrophoretic Mobility Shift Assay
Nuclear and cytoplasmic protein extraction was performed on detunicated testes based on previously established protocols [28]. Briefly, testes were homogenized with 10 strokes of a dounce homogenizer in 3x volume of 0.1% NP-40 lysis buffer (10 mM HEPES-KOH, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 2 mM MgCl2, 0.5 mM PMSF, 0.5 mM sucrose, 2 µg/ml leupeptin) and placed on ice for 5–10 min. The supernatant, after a 10-min centrifugation at 14 000 x g in 4°C was discarded (cytoplasmic extract). Samples were washed in the above buffer (without NP-40) and incubated for 40 min in 1/16 the volume of high salt buffer (20 mM HEPES-KOH, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 5% glycerol, 0.5 mM PMSF, and 2 µg/ml leupeptin). Samples again underwent 14 000 x g centrifugation for 10 min at 4°C. Supernatant was then resuspended in 1.5x the volume of low-salt buffer (20 mM HEPES-KOH, 50 mM KCl, 0.2 mM EDTA, 1 mM DTT, 20% glycerol, 0.5 mM PMSF, and 2 µg/ml leupeptin). Protein concentrations were determined by Bio-Rad Dc kit (cat #500-0111) and read on a spectrophotometer. For nuclear binding reactions, 10 or 20 µg of nuclear protein was incubated for 15 min at room temperature with binding buffer (cat #E3581; Promega), then for 20 min at room temperature after addition of dsDNA NF-B oligo (cat #SC-2505; Santa Cruz Biotechnology), which was end-labeled with 
-32ATP (cat #Blu002A, Perkin Elmer). For competition experiments, 100-fold excess cold oligo (specific) or mutated NF-B oligo (cat #SC-2511; Santa Cruz Biotechnology) was added to the binding reaction. Supershift experiments were performed by incubating protein with 1 µl of corresponding Santa Cruz Biotechnology antibody p50 (cat #SC-1190X), p65 (cat #SC-109X), or c-Rel (cat #SC-272X) for 30 min on ice before binding reaction. Samples were separated on a 5% polyacrylamide/ Tris borate EDTA gel and subsequently analyzed by autoradiography.
Densitometry
EMSAs of autoradiograms were scanned and band intensity was quantified by using ImageJ software (http://rsb.info.nih.gov/ij/).
Statistics
The mean and standard error of the mean (SEM) were calculated for each data point and represented as mean ± SEM. One-way ANOVA back to the control population was used for all statistical analyses followed by the Bonferroni correction with significance at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B Subunit Localization in Testis
Immunohistochemical staining of Postnatal Day (PND)-28 rat testes with antibodies to NF-B subunits p65, p50, and c-Rel was performed to elucidate cellular localization (Fig. 1). A p65 antibody localized to the somatic Sertoli cells, Leydig cells, spermatogonia, and early round spermatids in all seminiferous tubules (Fig. 1A). Interestingly, p65 was excluded from the nuclei of all PND-28 spermatocytes. In contrast with p65, p50 staining (Fig. 1B) of the same cross-section revealed a markedly different protein localization pattern. A minority of seminiferous tubules contained a group of nuclear p50-positive spermatocytes (Fig. 1, B and D). These p50-positive cells were precisely the populations that were negative for nuclear p65 localization (comparison of Fig. 1, A and B); however, it was clear that only a small percentage of tubules had p50-positive spermatocytes.
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This p50 spermatocyte localization was stage specific; therefore, an individual seminiferous tubule cross-section either had p50 protein expression in all spermatocytes or none. Examination of cross-sections revealed that only early pachytene spermatocytes (those associated with type B and intermediate spermatogonia) localized p50 and 37% of counted tubules had p50 positivity. In addition to these data, an antibody to NF-B subunit c-Rel (Fig. 1C) localized strongly to spermatogonia and occasionally to spermatocytes also in a stage-specific manner. The presence of c-Rel was far more ubiquitous than p50, appearing in all spermatogonia and early spermatocytes (preleptotene, leptotene, and zygotene). Unlike p65 protein expression, c-Rel was neither localized to Sertoli cells nor to round spermatids. Finally, the NF-B subunit Rel-B immunolocalized to all germ cells in all seminiferous tubules (data not shown). Preabsorption of NF-B antibodies with their immunogenic peptides blocked all testis immunostaining (data not shown).
Testicular NF-B Binding Activity after MEHP
To assess NF-B activity, EMSAs were performed on nuclear protein extracts of whole testis after exposure to 1 g/kg MEHP or corn oil vehicle control by oral gavage (Figs. 2 and 3). Nuclear NF-B, bound to a radiolabeled NF-B promoter oligo, migrated as two distinct bands in EMSAs (Fig. 2A, bands labeled A and B) and was selectively competed out by specific (sp) but not mutated (ns) excess cold oligo. Testicular EMSA complexes A and B increased in intensity by 1 h (n = 3) after MEHP exposure, compared with control (n = 4) and subsequently decreased to control and subcontrol levels by 3 (n = 3) and 6 (n = 3) h after exposure, indicating increased testicular NF-B activity shortly after toxicant exposure. Quantification of complex A and B by image densitometry revealed a significant four- and twofold increase in NF-B DNA binding, respectively, 1 h after MEHP exposure (Fig. 2B).
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To assess which NF-B subunits were involved in these EMSA complexes, antibody supershift experiments were performed on pooled nuclear testis protein extracts (Fig. 3A). Nuclear NF-B promoter binding again increased 1 h after MEHP exposure compared with control and subsequently decreased at 3 and 12 h postexposure (pooled samples; n = 3 for all time points). Addition of an antibody to p50 caused complex B to supershift. DNA protein complex specificity was again determined by loss of bands with sp but not ns mutated NF-B unlabeled excess oligo. An additional supershift EMSA with pooled testis nuclear protein extracts from a third set of PND-28 rat testes was performed to verify these results and analyze p65, p50, and c-Rel supershifted complexes (Fig. 3B). Again, an increase in NF-B-binding activity was observed 1 h after MEHP exposure compared with two control groups (pooled samples; each n = 3). The addition of a p65 or c-Rel antibody decreased the intensity of complex B and A (especially after toxicant exposure), indicating a supershift and presence of those proteins in the EMSA complex. Supershifts of p50 lanes were similar to previous results, with a shifted B complex. This shifted B complex appeared to migrate to the A complex region of the gel (most evident in the Fig. 3A control lane).
NF-B Protein Localization Following Toxicant Exposure
To characterize further the testicular NF-B response to MEHP, immunohistochemistry and TUNEL staining were performed on control testes and testes following MEHP exposure at 1, 3, 6, and 12 h. Figures 4 and 5 are representative of controls 1 and 6 h after exposure, while the 3- and 12-h time points are not shown. Testes from control rats gavaged with corn oil demonstrated a p65 and p50 (Fig. 4A; p65 = red, p50 = green) and c-Rel and p50 (Fig. 5A; c-Rel = red; p50 = green) staining pattern similar to untreated rats (Fig. 1). Although the majority of seminiferous tubules exhibited no p50 staining, some tubules did demonstrate a ring of p50-positive spermatocytes in a stage-specific manner (Figs. 4A and 5A). The p65 subunit was absent in spermatocytes and present in Sertoli cells, Leydig cells, spermatogonia, and round spermatids (Fig. 4A). Sertoli cell localization was based on its characteristic spoke-like pattern in the seminiferous tubule and touch preps (data not shown). As shown in untreated rats (Fig. 1), c-Rel intensely stained spermatogonia in all stages and less intensely stained spermatocytes in a stage-specific manner (Fig. 5A). One hour following MEHP exposure, most seminiferous tubules had a ring of spermatocytes with p50 staining (Figs. 4B and 5B) while c-Rel and p65 protein localization remained unchanged. Moreover, p50 staining appeared in c-Rel-positive spermatogonia (Fig. 5B) as early as 1 h after toxicant exposure.
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Testes from rats 3 h after MEHP exposure (data not shown) exhibited strong p65 Sertoli cell localization and less intense staining in some spermatocytes, while p50 protein staining involved almost all spermatocytes and many spermatogonia, where it colocalized with nuclear c-Rel protein (data not shown). By 6 h after toxicant exposure, testis sections revealed strong Sertoli cell p65 immunolocalization, while all germ cell populations were positive for c-Rel, p65, and p50 (Figs. 4C and 5C). Spermatogonia, p50 negative in controls, were intensely positive and spermatocytes, which were infrequently positive for c-Rel, colocalized c-Rel with p50 (Fig. 5C). This nuclear colocalization of the c-Rel and p50 subunits persisted at 12 h after MEHP exposure (data not shown). Once again, p50 was present in the nuclei of all spermatocytes while p65 was perinuclear in spermatocytes but present in nuclei of spermatogonia and intensely in Sertoli cells. Moreover, strong interstitial p50 and c-Rel immunostaining also was observed at this time point (data not shown). In contrast with these changing immunolocalization patterns throughout MEHP toxicant exposure, the Rel-B subunit of NF-B staining remained unchanged, present in all germ cell types before and after injury (data not shown).
In addition to NF-B immunohistochemistry, TUNEL assays were performed at each MEHP postexposure timepoint (Fig. 6). In the seminiferous tubules, there was a basal level of TUNEL-positive apoptotic germ cells in the vehicle corn oil control group that persisted at 1 and 3 h after MEHP exposure. Interestingly, fewer TUNEL-positive cells were observed at 6 h after MEHP; however, a large number of apoptotic TUNEL-positive germ cells were apparent by 12 h after MEHP. In control testes (n = 5), 26.1% of the seminiferous tubules (>400 counted in all cases) had between one and three TUNEL-positive germ cells and 4.1% of the seminiferous tubules had greater than three TUNEL-positive germ cells. These numbers remained unchanged at 1 (n = 3; 29.9% and 4.2%) or 3 (n = 3; 27.7% and 5.2%) h after toxicant exposure. Interestingly, 6 h (n = 4) after MEHP gavage, there were fewer seminiferous tubules with 1–3 (17.0%) or 
3 (1.7%) TUNEL-positive germ cells, suggesting a transient protective phenomenon. As expected, 12 h after MEHP exposure, there was a significant increase in the number of seminiferous tubules with >3 TUNEL-positive cells (37.4%; n = 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B upregulates many genes related to apoptosis in numerous cell types in response to a multitude of stimuli. Despite the extensive study of NF-B over the past several years, very little is known about this important gene family in testis. Here we have shown a testicular NF-B response to MEHP-induced injury, both by EMSA and immunohistochemical localization. Nuclear NF-B binding activity, measured by EMSA, increased significantly by 1 h after MEHP exposure when compared with corn oil vehicle-treated control rats. NF-B subunits p50 and c-Rel were identified as the main subunits activated, causing NF-B promoter gel shift by supershift experiments.
To identify cell types responsible for increased NF-B activity, immunohistochemistry was performed. A stage-specific expression of p50 protein was observed in control 28-day-old rats. This pattern is different from adult rats, where pachytene spermatocytes in stages VII–XI were most intense for NF-B staining [9]. Here, using p50 antibodies on pubertal rats, we observed strong protein localization to spermatocytes in those stages in which they are associated with type B and intermediate spermatogonia. In response to MEHP-induced injury, however, spermatocytes not normally expressing the p50 subunit of NF-B began to exhibit nuclear localization of this protein. Interestingly, spermatogonia and spermatids, which were found to be negative for p50 protein expression in control rats, expressed p50 after MEHP exposure, indicating not only nuclear translocation of p50 in response to injury but also induction of p50 protein expression. This differential protein pattern before and after injury was also seen with anti-c-Rel antibodies and to a lesser extent with anti-p65 staining. Antibodies to c-Rel localized strongly to all spermatogonia and weakly to some spermatocytes in control testes and then exhibited intense nuclear localization in spermatocytes, spermatogonia, and mild upregulation in spermatids after phthalate exposure. While germ cell immunolocalization was observed with p50 and c-Rel, p65 was present in Sertoli cells as well as spermatogonia and spermatids before toxicant-induced injury. This staining pattern is diagrammed in Figure 7. After MEHP exposure, p65 was present in all cell types. The fact that Rel-B immunolocalization was present in all germ cells but not altered after MEHP exposure suggests that the other NF-B subunit responses are not an artifact of MEHP injury of the testes. The differential protein expression and localization of p50 and c-Rel in spermatocytes after toxicant exposure is particularly interesting given that these meiotic germ cells are the population most sensitive to MEHP-mediated damage (Fig. 7).
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What has been well documented in the literature is the NF-B response to injury, particularly in the immune system [29]. These data include NF-B activation and its subsequent role as an anti- or proapoptotic factor in hepatocytes, endothelium, and the immune system. In hepatocytes, NF-B upregulates antiapoptotic genes following partial hepatectomy [30, 31]. Conversely, NF-B has proapoptotic roles in the Fas-mediated endothelial cell response to homocysteine [32]. In the immune system, NF-B can have both pro- and antiapoptotic roles during silica-induced macrophage injury [33] and in T cells where NF-B is proapoptotic with phorbol myristate acetate and ionomycin stimulus but antiapoptotic when treated with glucocorticoids [1]. The increased transcription of antiapoptotic genes following NF-B activation has been well documented. Tumor necrosis factor-
(TNF-)-induced NF-B activation leads to increased expression of c-IAPs and TRAF1 and 2 [3]. The c-IAPs inhibit apoptosis by binding to effector caspases 3 and 7. TRAF2 in association with c-IAPs may be recruited to the TRAF1 complex to also block caspase 8, thereby inhibiting downstream effector caspases. Through these functions, NF-B has been shown to be critically important both to the immune system and to normal liver homeostasis. Knockouts of either p65 or IKKß (part of the NF-B activating complex) are embryolethal due to massive liver hemorrhage [8, 34]. The involvement of NF-B responses in many different cellular injuries and its role in protective phenomenon prompted us to characterize MEHP-induced NF-B alterations in the testis.
The Sertoli cell response to MEHP exposure has been characterized both in vivo and in vitro, although a proximal MEHP target has yet to be discovered. Interestingly, studies have shown that germ cell apoptosis, detected by the TUNEL assay, transiently decreases below control levels after MEHP exposure [22]. By 12 and 24 h after MEHP exposure, this protection phenomenon is overwhelmed and a large number of TUNEL-positive cells are observed. Here we showed that with the 1 g/kg dose of MEHP in PND-28 rats, the protection phenomenon (fewer seminiferous tubules containing TUNEL-positive germ cells) was observed at 6 h. The initial experiments referenced showed the protection phenomenon at 3 h instead of 6 h, a difference that we attribute to the twofold greater dose of MEHP used previously.
The eventual germ cell apoptosis following MEHP exposure is dependent, at least in part, on a functional Fas system [23, 35, 36]. A potent downstream target of Fas signaling is the activation of NF-B [2]. Therefore, it is possible that, through Fas signaling, NF-B is being activated. Given this Fas and NF-B connection and the initial antiapoptotic wave seen during this injury, we hypothesized that NF-B activity may play a protective role in testicular injury.
Indeed, an increase in whole-testis NF-B activity was seen at 1 h after MEHP gavage, which is quite rapid, given the time required for toxicant distribution after gavage. Of particular interest is the timing of germ cell apoptosis following MEHP. MEHP exposure causes Sertoli cell damage, which leads to germ cell apoptosis. The germ cell response to Sertoli cell injury has been observed, in large part, many hours after MEHP exposure [22, 25, 37–39]. If NF-B is involved in the protective phenomenon observed with this injury, then its activation would have to occur soon after exposure. By EMSA and immunostaining, the data presented here show increased NF-B activity soon after MEHP administration. If the subsequent upregulation of antiapoptotic genes by NF-B occurred in spermatocytes soon after exposure, then the decreased TUNEL positivity at 6 h (5 h after NF-B activation) correlates nicely, given the fact that the DNA fragmentation (assessed by TUNEL) is a relatively late stage of apoptosis.
What was surprising, however, was the dramatic increase in p50 staining after MEHP exposure. This increased staining could result from either epitope unmasking or de novo synthesis of new p50. The proprietary p50 peptide used to generate the anti-p50 antibody is from its carboxy terminus (personal communication, Santa Cruz Biotechnology). Translocation of NF-B dimers from the cytoplasm to the nucleus occurs after the nuclear localization signal (NLS) is revealed [40]. Although direct binding of the NLS of p50 and IB does not occur, there is a NLS extension from amino acids 363–376 at the carboxy terminus [41]. This NLS extension does bind IB and is in the region of the anti-p50 antibody epitope; therefore, IB bound to p50 in the cytoplasm could result in epitope masking [41]. Alternatively, an actual increase in the p50 protein expression after MEHP injury could occur; the latter would be an interesting finding, given that increased NF-B protein expression is not usually observed after NF-B activation [42].
A question beyond the scope of these experiments is if NF-B has a pro- or antiapoptotic function in this system. There is a preponderance of literature reporting NF-B antiapoptotic effects in other cells types. It is important to note, however, that NF-B upregulates numerous proapoptotic genes, including death receptors (4, 5, and 6) and Fas itself. Fas is expressed on spermatocytes and is a key component of MEHP-induced germ cell apoptosis [23, 35, 36]. In addition, round spermatids and pachytene spermatocytes express the potent activator of NF-B, TNF- and Sertoli cells express the TNF receptor [43, 44]. It is possible that germ cell NF-B activation results from Fas or TNF- signaling. Although it is possible for NF-B to have proapoptotic functions in certain cell types, the confounding factor disputing this idea is the early period of NF-B activation (1 h) in contrast with the late time frame (12 h) in which apoptosis is observed. Expression of Fas, however, increases 3 and 6 h after MEHP exposure in the testes [35]. Because NF-B can promote Fas transcriptional activity, it is possible that the early activation of NF-B could lead to later sensitization of germ cells. In summation, the data presented here suggests a role for NF-B in the testicular response to MEHP injury. These associations are not necessarily causal and critical questions regarding the signaling pathway involved in this response and roles of NF-B during normal testicular homeostasis remain to be elucidated by future studies.
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FOOTNOTES |
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Received: 13 July 2004.
First decision: 18 August 2004.
Accepted: 1 October 2004.
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REFERENCES |
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