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Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.
It is well known that Sertoli cells provide structural and functional support to the development of testicular germ cells. Apoptosis of germ cells serves as a mechanism to match their numbers to the finite “supportive capacity” of Sertoli cells [1, 2]. Previous studies [3–5] have demonstrated that FAS/FASL paracrine signaling has a key role in mediating germ cell apoptosis in the testis after phthalate-induced Sertoli cell injury. In this model, Sertoli cell-expressed FASL acts on FAS-expressing germ cells to initiate their elimination by apoptosis.
Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate, is an established model for decreasing Sertoli cell supportive capacity that ultimately leads to an increase in the incidence of testicular germ cell apoptosis. The functional participation of FAS/FASL signaling in directly triggering germ cell apoptosis after MEHP-induced Sertoli cell injury has been described [3, 6]. Our previous study  described a direct effect of MEHP on Sertoli cells that enhanced the transcriptional regulation of FASL through the activation of transcription factors SP1 and NFKB1. We also demonstrated an indirect effect triggered by the increased production and release of soluble tumor necrosis factor α (sTNFA) from germ cells, resulting in the further activation of NFKB1 in Sertoli cells through the activation of the TNFRSF1A (TNFR1) signaling pathway. Therefore, knowledge of the mechanisms that specifically control the induced expression of FASL and TNFA over the basal levels is required for a complete understanding of the regulation of germ cell apoptosis for the establishment of functional spermatogenesis.
Tumor necrosis factor α has previously been reported to regulate spermatogenesis , modulate Leydig cell steroidogenesis [9–11], and influence the expression of cell-cell adhesion molecules [12, 13]. The objectives of this study were to determine the functional significance of TNFA in modulating the sensitivity of germ cells to undergo apoptosis and to evaluate the mechanism of TNFA processing in the seminiferous epithelium. Metalloproteinases (MPs) are essential for processing the TNFA precursor [14–17], and several MPs in the testis have been described [18–21]. Most MPs are extracellular enzymes that are released as an inactive latent form and are further activated through proteolytic processing. Metalloproteinase activity can be controlled by transcriptional regulation, by zymogen activation, and by tissue inhibitors of MPs (TIMPs) . In this study, in vivo and in vitro experimental approaches were used to examine the relationship between sTNFA sensitivity and testicular germ cell apoptosis after toxicant-induced Sertoli cell injury. The results of these studies provide insights into the cross talk that occurs between germ cells and Sertoli cells and that controls germ cell survival in response to decreased Sertoli cell supportive capacity.
Twenty-eight-day-old male wild-type C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The climate of the animal room was kept at a constant temperature (mean ± SEM, 22 ± 0.5°C) at 35%–70% humidity with a 12L:12D photoperiod. Animals were given standard laboratory chow and water ad libitum. All procedures involving animals were performed in accord with the guidelines of the Institutional Animal Care and Use Committee of the University of Texas at Austin in compliance with guidelines established by the National Institutes of Health. Twenty-eight-day-old mice were given a single dose of MEHP (1 g/kg, in corn oil; TCI America, Portland, OR) by oral gavage, a standard procedure for the investigation of MEHP-induced testicular toxicity . Control animals received a similar volume of corn oil vehicle. Control and MEHP-treated animals were killed by CO2 inhalation, and testes were either immediately frozen in liquid N2 for protein analysis or fixed in Bouin solution (Polysciences, Inc., Warrington, PA) and paraffin embedded for histology.
Primary cocultures of rat Sertoli cells and germ cells were prepared as previously described . Briefly, the testes of young (21-day-old) Fisher rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were detunicated and subjected to a series of enzymatic digestions. Primary coculture cells (2 × 106) were plated on 35-mm laminin-coated culture dishes containing Dulbecco modified Eagle medium/Ham F12 media (Invitrogen, Gaithersburg, MD) with epidermal growth factor (1 ng/ml; Sigma, St. Louis, MO), ITS+ premix (insulin, transferrin, selenious acid, bovine serum albumin, and linoleic acid, 10 μg/ml; BD Biosciences, San Jose, CA), gentamicin (50 μg/ml; Invitrogen), and 1% penicillin-streptomycin (Invitrogen) and then incubated at 37°C. After 48 h, primary cocultures were treated with 200 μM MEHP diluted in dimethyl sulfoxide (DMSO). After treatment, the conditioned media were replaced with 20 mM Tris-HCl (pH 7.4) to cause germ cells to separate from Sertoli cells .
A detailed description of total protein preparation from rat coculture cells and mice tissue, as well as Western blot analysis, has been published previously . Total cellular proteins (30 μg) were tested using primary antibodies against TIMP1, TIMP2, TIMP3, and TIMP4 (1:1000; Chemicon, Temecula, CA); matrix MP 2 (MMP2) and MMP9 (1:1000; Abcam Inc., Cambridge, MA); FASL (1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA); TNFA (1:500; R&D Systems Inc., Minneapolis, MN); a distintegrin and MP 10 (ADAM10) (1:500; R&D Systems Inc.), ADAM17 (1:500; Abcam Inc.); and β-actin (ACTB) (1:500; Santa Cruz Biotechnology Inc.) coupled with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology Inc.). The ECL chemiluminescent substrate (Amersham Bioscience, Piscataway, NJ) was used as the detection reagent, and ACTB was used as a loading control. Densitometry for bands on Western blots was determined using ImageJ software (National Institute of Mental Health, Bethesda, MD).
The MMP2 and MMP9 gelatinolytic activity in primary coculture media was assayed by gelatin zymography with minor modifications . Briefly, 12 h after the addition of MEHP (200 μM) or vehicle (0.04% DMSO) to the cocultures, conditioned media were collected and directly applied to a 10% polyacrylamide gel containing 2 mg/ml of gelatin (Sigma). After electrophoresis, the gels were washed twice for 15 min with 2.5% Triton X-100 in 50 mM Tris-HCl (pH 7.5) to remove the SDS and restore enzyme activity and were incubated overnight at 37°C in activation buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2, and 0.02% sodium azide). The gels were then stained with 30% methanol/10% acetic acid containing 0.5% (w/v) Coomassie brilliant blue G-250 for 30 min and were destained in the same solution without dye. Clear bands shown on the blue background indicate enzyme activity.
Bouin-fixed testes were washed in 70% ethyl alcohol-Li2CO3 saturated solution and embedded in paraffin for histological analysis. Cross sections (5 μm) of paraffin-embedded tissues were evaluated by periodic acid-Schiff-hematoxylin (PAS-H) staining  and then imaged using a Nikon E800 microscope (Natick, MA) and captured with a Canon-5D digital camera (Canon Inc., Tokyo, Japan). At least three mice in each treatment group were examined. The average diameter of the lumen was determined by measuring the diameters of 20 seminiferous tubules per testicular cross section, and two sections of each mouse were examined.
Apoptotic fragmentation of DNA in mouse paraffin-embedded testis cross sections was determined by TUNEL analysis using the ApopTag kit (Chemicon). The apoptotic index was calculated as the percentage of seminiferous tubules containing more than three TUNEL-positive germ cells in each cross section. At least two testicular cross sections per mouse and at least three mice in each treatment group were analyzed.
A total of 2 × 106 primary Sertoli cell-germ cell cocultures were exposed to 200 μM MEHP for 12 h. The culture media were collected and centrifuged to remove cellular debris. The supernatant was rapidly frozen at −80°C until assayed for MMP2 and TNFA by ELISA (R&D Systems Inc.). To determine the effect of MMP2 on TNFA processing, recombinant MMP2 protein (0, 5, 10, and 50 ng/ml) was added to primary coculture cells for 12 h, and the media were collected for ELISA. The cell number was counted to normalize the protein expression assayed by ELISA. All experiments were performed in triplicate.
SB-3CT is a gelatinase-specific inactivator that inhibits MMP2 and MMP9 by binding the active site of the enzymes . In vitro, SB-3CT (Chemicon) (0, 5, 10, and 20 μM) was added to primary Sertoli cell-germ cell cocultures for 12 h in the presence of MEHP. Primary coculture cells were harvested for protein analysis, and conditioned media were collected to determine TNFA levels by ELISA. In vivo, SB-3CT (5 mg/kg) and vehicle solution (10% DMSO in normal saline) were injected i.p. into 28-day-old C57BL/6J wild-type mice. After 6 h, MEHP (1 g/kg) was administrated to SB-3CT-pretreated C57BL/6J mice by oral gavage for another 12 h. One testis was collected and prepared for paraffin embedding, and the other testis was immediately frozen at −80°C for protein analysis.
All experiments were performed in triplicate and repeated at least three times. The results are presented as the individual means ± SEM. The data were subjected to Student t-test or parametric one-way ANOVA followed by Tukey test for post hoc comparisons. Statistical significance was considered to be achieved when P < 0.05.
To determine whether the expression of specific MPs in the testis was influenced by MEHP exposure, 28-day-old C57BL/6J mice were treated with MEHP (1g/kg), and testes were collected at various time points after exposure. Western blot analysis shows that TIMP1 and TIMP2 expression in whole-testis homogenates decrease in a time-dependent manner after MEHP exposure, while no significant changes are observed in TIMP3 and TIMP4 expression (Fig. 1A). MMP2 protein expression levels in whole-testis homogenates are increased after MEHP exposure, but no differences in MMP9 protein levels are observed (Fig. 1B). An increase in the ADAM10 processed form (65 kDa) is detected early after MEHP exposure, but its expression is time dependently decreased afterward. In primary cocultures of rat Sertoli cells and germ cells, TIMP2 is expressed only in Sertoli cells and is significantly reduced after MEHP exposure, while MMP2 is detected in both primary rat Sertoli cells and primary rat germ cells, and its expression is slightly increased in primary germ cells in response to MEHP challenge (Fig. 1C). To determine the activity of MMP2 and MMP9, conditioned media from primary rat coculture cells were collected and analyzed by gelatin zymography. Only MMP2 is strongly detected in the conditioned media, and its activity is increased in response to MEHP exposure (Fig. 2A). In addition, sMMP2 is significantly increased in a time-dependent manner in response to MEHP exposure (Fig. 2B).
When the specific gelatinase inhibitor SB-3CT is added to primary rat coculture cells, MEHP-induced endogenous MMP2 activity is suppressed in a dose-dependent manner by gelatin zymography (Fig. 3A). Also, sTNFA is decreased in a dose-dependent manner by ELISA (Fig. 3B, right group).
To understand the participation of MMP2 in the production of sTNFA in the testis, recombinant MMP2 is added to primary rat Sertoli cell-germ cell cocultures, and after 12 h, the amount of sTNFA expression is measured by ELISA. Soluble TNFA is increased in response to the addition of MMP2 in a dose-dependent manner (~5.4-fold at the highest dose of recombinant MMP2) (Fig. 3B, left group). However, MEHP treatment alone triggers a greater increase in sTNFA (~9.46-fold, black bar).
SB-3CT is injected i.p. into 28-day-old C57BL/6J mice for 6 h, followed by MEHP exposure for another 12 h. MMP2 protein expression in whole-testis homogenates is slightly decreased in SB-3CT-pretreated mice compared with MEHP treatment alone. Moreover, MEHP-enhanced processing of TNFA is suppressed by SB-3CT injection (Fig. 4A), and MEHP-induced germ cell apoptosis is decreased as well (7.36% compared with 23.33% after MEHP treatment) (Fig. 4B). Testicular histology shows that an obvious germ cell detachment in seminiferous tubules occurs after MEHP exposure (~4.5% of tubules per cross section), but fewer germ cell detachment events are observed in the SB-3CT-pretreated mice (~0.2%) (Fig. 5A). The average diameter of the lumen in seminiferous tubules is significantly increased after MEHP exposure for 12 h (84.49 ± 6.04 and 112.24 ± 4.86 μm in the absence and presence of MEHP, respectively). However, despite these differences in lumen diameter, the average outside diameter of the seminiferous tubules in MEHP-treated mice testes is the same as that in control mice testes (175.41 ± 1.32 and 180.08 ± 2.14 mm), suggesting that the increase in the lumen dilation was not due to the increase in the luminal fluid. TUNEL-positive germ cells are increased after MEHP exposure, and SB-3CT pretreatment is able to prevent germ cells from MEHP-induced apoptosis, especially in spermatocytes (Fig. 5B). These observations indicate that the suppression of MMP2 leads to protection of germ cells from MEHP-induced apoptosis and to preservation of the interaction between Sertoli cells and germ cells.
In C57BL/6J mice, MEHP induces significant MMP2 protein expression at 1 h, while MMP2 expression is slightly decreased after 3 h of exposure (Fig. 6). An increase in the production of sTNFA is also detected at 1, 3, and 12 h. FASL expression is increased in a time-dependent manner from 1 to 6 h after MEHP exposure. A dramatic increase in MEHP-induced germ cell apoptosis is observed at 12 h but is reduced to basal level by 24 h (Fig. 7A). After 24 h of exposure, the diameter of lumen appeared to increase due to the retraction of Sertoli cell cytoplasm and the loss of germ cells (84.49 ± 6.04 and 167.34 ± 7.45 μm 0 and 24 h after MEHP treatment, respectively) (Fig. 7B).
Within the seminiferous epithelium, the dynamic interaction between Sertoli cells and germ cells is critical in controlling spermatogenesis. Our previous findings demonstrated that exposure of young rodents to the Sertoli cell toxicant MEHP results in the enhanced production of sTNFA by germ cells and its consequent initiation of germ cell apoptosis through the activation of the FASL/FAS signaling pathway . Herein we further explore the mechanisms responsible for the generation of sTNFA in the seminiferous epithelium after MEHP exposure. We found that TIMP2 expression is decreased in MEHP-injured Sertoli cells, allowing for MMP2-mediated TNFA processing and the consequent increase in germ cell apoptosis during the peripubertal period of testis development. To our knowledge, this is the first evidence showing the possible mechanism by which MPs involve the regulation of programmed cell death in the testis.
Four members of the TIMP family, TIMP1, TIMP2, TIMP3, and TIMP4, interact with active sites of distinct MPs with 1:1 stoichiometry and regulate the activity of MPs and their associated physiological and pathological processes . In the mouse testis, the RNA levels of all four TIMPs are constitutively expressed during development . Our observations of C57BL/6J mouse testis homogenates indicate that a decrease in the protein levels of TIMP1 and TIMP2 occurs after MEHP exposure, while no significant changes are observed in TIMP3 or TIMP4 levels (Fig. 1A). Only TIMP2 and TIMP4 were detectable in conditioned media of primary Sertoli cell-germ cell cocultures (data not shown), further narrowing the likely participation of TIMPs in the seminiferous epithelium to these two TIMP family members. Previous studies [19, 31, 32] indicate that both TIMP1 and TIMP2 are expressed by Sertoli cells, that TIMP3 is found within interstitial cells such as Leydig cells, and that TIMP4 is present in both interstitial cells and Sertoli cells. Our results suggest that TIMP2 is predominantly expressed in Sertoli cells and show that, after MEHP exposure, its protein levels are specifically reduced in vitro (Fig. 1C). The down-regulation of TIMP2 has also been observed in human lung cells in response to cobalt exposure  and in hypoxic human endothelial cells , implying that decreases in TIMP2 levels maybe a general stress-induced response. It has been shown that TIMP1 and TIMP2 levels are up-regulated by follicle-stimulating hormone (FSH) through a cAMP-dependent signaling pathway [35, 36]. This signaling pathway has previously been shown to be inhibited in the rodent testis after MEHP exposure . Follicle-stimulating hormone influences Sertoli cell proliferation and the synthesis of Sertoli cell-derived products that are essential for germ cell survival. A decrease in FSH corresponds to an increase in germ cell apoptosis in rats ; therefore, MEHP-decreased Sertoli cell FSH signaling may be responsible in part for changes in Sertoli cell TIMP2.
The ADAM family of proteins is widely reported to have a role in the proteolytic shedding of membrane-bound proteins . ADAM10 and ADAM17 both have TNFA-converting enzyme activity  and have been previously described as cleaving membrane-bound TNFA . In the testis, the processed active form of ADAM10 (65 kDa) is slightly increased shortly after MEHP exposure, while ADAM17 expression remains low throughout the time course (Fig. 1B), suggesting that other protein(s) besides these two ADAM family members are involved in TNFA processing in response to MEHP exposure.
MMP2 and MMP9 are components of extracellular matrix between Sertoli cells and peritubular cells [19, 41]. A recent study  shows that MMP2 is mainly localized in apical ectoplasmic specializations associated with the heads of elongate spermatids. The present data indicate that total MMP9 expression remains steady after MEHP exposure (Fig. 1B) and that no significant induction of its activity is detectable in conditioned media from primary cocultures (Fig. 2A). However, the mRNA level of MMP9 is increased after 12 h of MEHP exposure (data not shown), suggesting that posttranscriptional regulation may occur. MMP2 is found in both Sertoli cells and germ cells (Fig. 1C). Total testis protein levels of MMP2 are rapidly increased 1 h after MEHP treatment but then decrease by 3 h (Fig. 1B). It is well recognized that TIMP2 is involved in the activation of pro-MMP2 , which is also important for testicular development . Time-independent increases in the expression and activity of sMMP2 after MEHP exposure were detected by zymography and ELISA (Fig. 2, A and B) and indicate that MEHP induces the activation of MMP2 in the testis. Therefore, a decrease in Sertoli cell-derived TIMP2 expression after MEHP treatment might contribute to the activation of MMP2 by decreasing the TIMP:MP ratio in the testis.
To address the correlation between MEHP-induced activation of MMP2 and MEHP-induced TNFA processing in the testis, in vitro and in vivo experiments were performed using a specific gelatinase inhibitor, SB-3CT. This compound is commonly used in animal models to determine the effect of MMP2/9 inhibition on metastatic activity, proteolysis, and apoptosis . The use of SB-3CT in primary Sertoli cell-germ cell cocultures suppressed MEHP-enhanced MMP2 activity in a dose-dependent manner (Fig. 3A), as well as MEHP-induced sTNFA production (Fig. 3B). These observations point to a key role of MMP2 in the processing of TNFA in vitro. In the absence of MEHP, recombinant MMP2 induces only ~50% of the sTNFA that is produced after MEHP exposure (Fig. 3B), demonstrating that MEHP-induced TNFA activation may be only partially regulated by MMP2. In vivo, a low dose of SB-3CT (5 mg/kg) inhibits TNFA processing (Fig. 4A) and consequently results in a significant decrease in MEHP-induced germ cell apoptosis (Figs. 4B and and5B).5B). Testicular histology shows obvious germ cell detachment in MEHP-treated C57BL/6J mice but not in SB-3CT-pretreated mice (Fig. 5A). Therefore, both in vitro and in vivo results demonstrate that a low dose of SB-3CT can provide protection from MEHP-induced germ cell apoptosis and point to the significant role of MMP2 in TNFA processing and germ cell apoptosis in the testis.
The complex and dynamic communication between Sertoli cells-Sertoli cells and Sertoli cells-germ cells is highly regulated by cytokines such as TNFA and transforming growth factor β . The inhibition of TNFA decreases the permeability of lung tight junctions , and the MMP inhibitor GM-6001 decreases TNFA-induced breakdown of the blood-cerebrospinal fluid barrier . In vitro, MEHP disrupts tight junctions between Sertoli cells  and causes germ cell detachment . It has been recently suggested that the activation of MMP2 disrupts the laminin-integrin complex, resulting in restructuring of the blood-testis barrier . Therefore, MEHP-induced TNFA signaling may be in part responsible for disrupting the junctional connections in the seminiferous epithelium, as well as facilitating germ cell detachment and loss from the testis.
The present findings, together with those of our previous studies, have led to the development of our working model shown in Figure 8. This model predicts that TIMP/MMP-regulated TNFA processing has a key role in the mechanism instigating testicular germ cell apoptosis after MEHP-induced Sertoli cell injury. MEHP exposure decreases Sertoli cell secretion of TIMP2 into the adluminal space and allows for the activation of MMP2. The consequent processing of TNFA in germ cells allows for its ability to activate Sertoli cell TNFRSF1A and its triggering of the NFKB1 signaling pathway to produce an increase in FASL. The increased FASL protein levels instigate apoptosis of FAS-positive germ cells by the classic death receptor pathway (Figs. 6 and and7A).7A). Taken together, these findings demonstrate that change in the TIMP:MMP ratio in the testis is a critical mechanism to account for the sensitivity of germ cells to undergo apoptosis in response to decreases in the Sertoli cell supportive capacity during the critical period of peripubertal testis development.
We appreciate the assistance of Jessica Cobarrubia in performing some experiments and her expert editorial assistance in the preparation of the manuscript.
1Supported in part by grants from the National Institute of Environmental Health Sciences (NIEHS)/ NIH ( ES009145 & ES007784) and the Center for Molecular and Cellular Toxicology.