Mono-(2-ethylhexyl) Phthalate Rapidly Alters both Sertoli Cell Vimentin Filaments and Germ Cell Apoptosis in Young Rat Testes

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

137, 42–50 (1996)

0055

Mono-(2-ethylhexyl) Phthalate Rapidly Alters both Sertoli Cell Vimentin Filaments and Germ Cell Apoptosis in Young Rat Testes JOHN H. RICHBURG AND KIM BOEKELHEIDE1 Department of Pathology and Laboratory Medicine, Brown University, Box G-B518, Providence, Rhode Island 02912 Received July 27, 1995; accepted November 14, 1995

tyl, dipentyl, and dihexyl phthalate. It has been demonstrated that these phthalates, when administered orally, are rapidly hydrolyzed in the gut and other tissues by nonspecific esterases to produce the corresponding monoesters (Albro, 1987; Thomas and Thomas, 1984). The monoesters, of which mono-(2-ethylhexyl)phthalate (MEHP) is one of the most potent, are recognized as the ultimate testicular toxic metabolites. Both in vivo and in vitro experiments have demonstrated that the Sertoli cell is the primary site of phthalate-induced testicular toxicity (for review, see Boekelheide, 1993). Sertoli cell alterations include vacuolization, inhibition of seminiferous tubule fluid formation, loss of mitochondrial succinate dehydrogenase activity, inhibition of transferrin secretion, and abnormalities in the glycolytic pathway. In addition, MEHP has been reported to specifically disrupt follicle stimulating hormone-induced increases in cyclic AMP in cultured rat Sertoli cells (Grasso et al., 1993). Germ cell enzymatic activities are not directly affected (Fukuoka et al., 1993; Oishi, 1986; Zhou et al., 1990). These Sertoli cell alterations lead to a progressive degeneration of spermatocytes and spermatids which ultimately slough off into the tubular lumen. In addition to the biochemical changes in Sertoli cells after phthalate exposure there is also a rapid disruption of the Sertoli–germ cell physical interaction, leading to germ cell sloughing from the seminiferous epithelium (Albro, 1987; Thomas and Thomas, 1984). Experiments using Sertoli–germ cell cocultures show that MEHP induces a concentration-dependent increase in the rate of germ cell detachment (Creasy et al., 1988). Since the MEHP-induced germ cell detachment from cocultures occurs at lower exposure levels than those which cause general cytotoxicity, a specific mechanism is likely to be responsible for this effect. In order to study the consequences of phthalate-induced Sertoli cell toxicity, we investigated the influence of MEHP on germ cell apoptosis in young (28-day-old) rat testes. Sertoli cells are responsible for directing germ cell differentiation and survival by providing a nutritional and hormonal microenvironment. Apoptosis has been described to occur in cells after growth factor or hormone deficiency (Bardon

Mono-(2-ethylhexyl) Phthalate Rapidly Alters both Sertoli Cell Vimentin Filaments and Germ Cell Apoptosis in Young Rat Testes. RICHBURG, J. H., AND BOEKELHEIDE, K. (1996). Toxicol. Appl. Pharmacol. 137, 42–50. Mono-(2-ethylhexyl) phthalate (MEHP) is a widely studied Sertoli cell toxicant. Here we describe alterations in Sertoli cell vimentin filament distribution and the incidence of testicular germ cell apoptosis in young (28-day-old) Fischer rats that were treated with MEHP (2 g/kg, po) and killed 0, 3, 6, or 12 hr after exposure. A collapse in vimentin filaments was observed 3 hr after MEHP exposure without accompanying changes in the pattern of Sertoli cell tubulin or actin. A progressive increase in the perinuclear condensation of the vimentin filaments was observed from 6 to 12 hr after exposure. To evaluate the consequences of these Sertoli cell changes on germ cells, the role of apoptosis in MEHP-induced testicular toxicity was examined. DNA isolated from testis of rats 6 and 12 hr after MEHP exposure showed a marked increase in low-molecular-weight DNA resulting from internucleosomal cleavage. In addition, DNA fragmentation visualized in frozen testis cross sections by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end label (TUNEL) staining demonstrated a progressive increase in germ cell apoptosis from 6 to 12 hr after MEHP exposure. However, 3 hr after MEHP exposure, the incidence of TUNEL-positive germ cells was significantly decreased compared to that seen in controls. Taken together, the early collapse in Sertoli cell vimentin filaments and the concurrent decrease in germ cell apoptosis suggests that MEHP engenders Sertoli cell dysfunction resulting in the disruption of the physiological mechanism of germ cell apoptosis. q 1996 Academic Press, Inc.

Phthalic acid esters are found widespread in the environment due to their use as plasticizers in food packaging and biomedical devices. Phthalic acid esters have been shown to reduce fertility and induce testicular atrophy in laboratory animals (for reviews see Albro, 1987; Thomas and Thomas, 1984). Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely studied male reproductive toxicants in rats. Other male reproductive toxic phthalic acid diesters include dibu1 To whom reprint requests and correspondence should be addressed. Fax: (401) 863-9008. E-mail: [email protected].

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0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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et al., 1987; Kyprianou and Isaacs, 1988, 1989; Tapanainen et al., 1993). Therefore, in an analogous manner, altered secretion of Sertoli cell factors as a result of MEHP exposure may trigger germ cell apoptosis. Here we describe the novel observation of an MEHP-induced inhibition of germ cell apoptosis, a process that normally occurs in testis of young rats to limit the clonal expansion of spermatocytes in the immature seminiferous epithelium. The present study was initiated based on preliminary experiments which suggested that phthalates cause alterations in the rat Sertoli cell cytoskeleton, particularly the intermediate filament vimentin. Vimentin filaments surround the Sertoli cell nucleus and extend long apical filaments which radiate toward the periphery of the cell where they associate with the plasma membrane in the region of Sertoli–germ cell attachments. In the past, intermediate filaments were thought to play a simple structural role in cellular support. However, more recently, it has been suggested that vimentin filaments play a role in positioning the Sertoli cell nucleus, maintaining Sertoli–germ cell attachment sites, or act as mediators of cell signal transduction between the plasma membrane and the nucleus (for reviews, see Aumuller et al., 1992; Klymkowsky et al., 1989). The function of Sertoli cell vimentin filaments, as with that of other intermediate filaments, has not yet been clearly defined. Recently, a mouse model was reported that contained a null mutation in the vimentin gene (Colucci-Guyon et al., 1994). These mice underwent normal development and were able to reproduce, suggesting that vimentin filaments do not play a vital role in cells. This finding seems at odds with the fact that intermediate filament gene structure, protein sequence, and patterns of cell type expression are highly conserved (Klymkowsky et al., 1989), implying that intermediate filament proteins perform important functions. Defining the function(s) of intermediate filaments has been difficult due to the absence of membrane-permeable drugs that specifically disrupt intermediate filament organization. In the present study, we describe a specific and early MEHPinduced collapse of Sertoli cell vimentin filaments that occurs coincident with a decrease in normal testicular germ cell apoptosis. We speculate that a detachment of Sertoli– germ cell contact disrupts an apoptotic signal transduction pathway between these cells. MATERIALS AND METHODS General. Male Fischer (28-day-old) rats (Charles River Laboratories, Wilmington, MA) were given water and chow (Pro-Lab Rat, Mouse, and Hamster chow 3000) ad libitum. The animal room climate was kept at a constant temperature (70 { 27F) at 35–70% humidity with a 12-hr alternating light–dark cycle. All rats were acclimatized 1 week prior to treatment. Experimental protocol. MEHP was a gift from the National Institute of Environmental Health Sciences and was determined to be 95% pure by the Midwest Research Institute (Kansas City, MO). Rats (28-day-old) re-

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ceived a single 2 g/kg dose of MEHP in corn oil by gavage at a volume equal to 4 ml/kg. Control rats received a similar volume of corn oil vehicle. Vehicle and MEHP-treated rats were killed at 3, 6, and 12 hr after treatment and both testes were removed. From each rat, one testis was rapidly frozen in OCT compound (Miles Inc., Elkhart, IN) in liquid nitrogen and the other testis was cut in half for immersion fixation in Bouin’s fixative (kept at 47C) and in neutral buffered formalin. For the Bouin’s fixed testis, the tissue was removed after 24 hr and immersed in 30% sucrose/phosphate-buffered saline solution for at least 48 hr prior to rapid freezing in OCT compound and storage at 0807C. Histopathology. To examine the morphological appearance of seminiferous tubules following MEHP treatment, testes were fixed in 10% neutral buffered formalin and embedded in glycol methacrylate using a Historesin embedding kit (Reichert-Jung, Heidelberg, Germany). Sections (2 mm) were stained with periodic acid–Schiff’s reagent and hematoxylin (PAS/H). Cytoskeletal staining. Cryosections (8 mm) of testis that were fresh frozen (vimentin) or Bouin’s fixed (tubulin) were mounted on poly-L-lysine coated slides and post fixed for 10 min in acetone (0207C). A monoclonal antibody to bovine vimentin (clone V9, Sigma Chemical Co., St. Louis, MO) was used at a dilution of 1:40, while a monoclonal antibody to atubulin (clone 5a6, D. Brown, U. Ottawa) was used at a dilution of 1:200. Sections were blocked for endogenous avidin–biotin using a blocking kit (Vector Laboratories, Burlingame, CA) and for endogenous peroxidase activity using 1% hydrogen peroxide in methanol for 15 min. Nonspecific binding was blocked using 5% normal goat serum and 1% bovine serum albumin in phosphate-buffered saline (PBS/). Primary antibody was detected using a biotin-conjugated goat anti-mouse IgG secondary antibody (Calbiochem, San Diego, CA) and the ABC-Elite kit (Vector Laboratories) with 0.09% H2O2 , 0.04% NiCl2 , and 0.25 mg 3,3*-diaminobenzidine (DAB)/ml phosphate-buffered saline. The distribution of filamentous actin was examined by fluorescent labeling of actin using tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin. Cryosections (8 mm) of fresh frozen testis were mounted on poly-L-lysine coated slides and postfixed in acetone (0207C) for 10 min and then allowed to air dry. After incubating for 10 min in PBS/, the slides were incubated with 0.3 mg/ml TRITC-conjugated phalloidin for 20 min followed by three rinses in PBS. Low-molecular-weight DNA isolation. The presence of a low-molecular-weight DNA ladder was determined as described by Strauss (1994) with modifications. Testes were decapsulated, washed in modified Eagle’s media, and frozen in liquid nitrogen. Frozen tissue was homogenized for 5 sec in Tris buffer (5 mM Tris–HCl, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) and incubated with diethyl pyrocarbonate (DEPC) for 60 min. The homogenate was centrifuged at 27,000g for 20 min to remove cellular debris and high-molecular-weight DNA. The supernatant was digested with proteinase K (0.5 mg/ml, Boehringer Mannheim, Mannheim, Germany) for 60 min and extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). The aqueous phase was ethanol-precipitated overnight, pelleted, and resuspended in TE buffer (pH 8.0) containing 10 mM Tris–HCl and 1 mM EDTA. The resulting DNA was digested with RNase free of DNase followed by extraction. The aqueous phase was ethanol precipitated overnight, pelleted, and resuspended in TE buffer. A total of 30 mg of DNA was loaded on a 2% agarose gel and separated by electrophoresis (30 V, 3 hr). DNA was stained with ethidium bromide and visualized with an ultraviolet transilluminator at 302 nm (Spectronics Corporation, Westbury, NY). The sizes of resulting DNA bands were estimated by comparison with a standard 100-bp DNA ladder. In situ TUNEL staining and quantitation. Terminal deoxy-nucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) was performed on formalin-fixed 8-mm cryosections of testis using the ApopTag kit

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(Oncor, Gaithersburg, MD.). Briefly, digoxigenin-dUTP end labeled DNA was detected with anti-digoxigenin-peroxidase antibody followed by peroxidase detection with 0.05% diaminobenzidine (DAB) and 0.02% H2O2 . Tissue was counterstained with methyl green. Only intact, essentially round seminiferous tubules were counted. The number of apoptotic germ cells was counted in 100 randomly selected seminiferous tubules of testis cross sections from each of four different rats. The incidence of apoptosis was then categorized into either of five groups defined as 0, 1–3, 4–6, 7–9, or §10 positive cells per seminiferous tubule cross section. The data, calculated as a percentage of total, were

expressed as the mean { SEM. Significance between groups (p õ 0.05) was determined by single factor analysis of variance (ANOVA) with a Fisher’s least significant differences test comparison using StatView software (Abacus Concepts Inc., Berkeley, CA).

RESULTS

Prepubertal rats (28-day-old) were chosen as the animal model since they are more sensitive to the reproductive ef-

FIG. 1. Progressive stages of MEHP-induced testicular histopathology. Fischer rats (28-day-old) were given a single dose of MEHP (2 g/kg, po) or corn oil vehicle. Rats were killed 0, 3, 6, or 12 hr after MEHP treatment and the testes removed, fixed, embedded in glycol methacrylate, and sectioned. (A) Typical seminiferous tubule morphology of control testis. (B) Displacement of spermatogonia away from the basement membrane of seminiferous tubule occurred within 3 hr after MEHP administration (asterisk). Beginning at 6 hr (C), and continuing to 12 hr (D) a progressive sloughing of germ cells (asterisks) from the seminiferous epithelium was apparent (PAS&H, 1871).

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fects of phthalate esters (Gray and Butterworth, 1980; Gray and Gangolli, 1986; Sjoberg et al., 1986a). The sensitivity of young rats to phthalate esters is attributed, in part, to differences in absorption, distribution, and metabolism between young and old rats (Heindel and Powell, 1992; Sjoberg et al., 1986b). In addition, in young rat testes there is normally a significant incidence of germ cell apoptosis that

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occurs to limit the clonal expansion of spermatocyes. Therefore, young rats provide a convenient model to evaluate alterations in the process of apoptosis. Histopathology Histopathology was evaluated using cross sections of plastic embedded testis stained with PAS/H. Within 3 hr after MEHP

FIG. 2. MEHP-induced collapse of Sertoli cell vimentin filaments. Immunohistochemical distribution of vimentin was visualized using a monoclonal antibody to bovine vimentin. (A) Typical seminiferous tubule expression of vimentin filaments in normal 28-day-old rat testis. Vimentin staining is seen radiating from the Sertoli cell perinuclear region with apical ‘‘spoke-like’’ extensions (arrows). (B) Many of the Sertoli cell vimentin apical extensions have collapsed 3 hr after MEHP administration. A dense vimentin filament staining is observed surrounding the nucleus (short arrows). During the 6-hr (C) and 12-hr (D) period after MEHP treatment, a progressive increase in sloughing of the seminiferous epithelium is evident by detached germ cells (thick arrows) (1871).

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treatment, there was a displacement of spermatogonia away from the basement membrane (Fig. 1B) as compared to control (Fig. 1A). From 6 to 12 hr after MEHP exposure there was progressive displacement of spermatogonia away from the basement membrane, sloughing of germ cells into the lumen, and basal Sertoli cell vacuolization (Figs. 1C, 1D). Cytoskeletal Staining Vimentin was visualized by immunohistochemical staining. In Sertoli cells from control rats, vimentin was seen surrounding the nucleus and extending apical extensions in a ‘‘spoke-like’’ pattern (Fig. 2A). Three hours after MEHP exposure there was a dramatic loss in the staining of the apical vimentin extensions with a concomitant increase in the perinuclear staining intensity (Fig. 2B). Six and 12 hr after MEHP exposure, intense vimentin staining was seen surrounding the nucleus (Figs. 2C, 2D), suggesting that vimentin filaments had collapsed toward the Sertoli cell nucleus in MEHP-exposed animals. To determine if the effects of MEHP were specific for Sertoli cell vimentin filaments, the cytoskeletal staining patterns of actin and a-tubulin were examined. No difference in the atubulin staining pattern, which was characterized by long defined tracts extending along the long axis of the Sertoli cell, were observed between control and MEHP-treated rats (data not shown). The observed a-tubulin staining pattern, which reflects Sertoli cell microtubule filaments, demonstrated that MEHP did not disrupt Sertoli cell microtubule networks. Actin staining in control testes cross sections occurred at a basal location in Sertoli cell tight junctions and was found displaced toward the lumen after 6 and 12 hr of MEHP exposure (data not shown). These changes in actin staining occurred coincident with displacement of germ cells toward the lumen which likely reflects an elevation of Sertoli cell tight junctions produced by basal Sertoli cell vacuolization. Apoptosis The role of apoptosis in the mechanism of phthalateinduced germ cell loss was investigated by two different approaches. Apoptosis was detected by the presence of lowmolecular-weight DNA fragmentation, resulting in a characteristic DNA ‘‘ladder’’ pattern on agarose gels and quantitated by the in situ TUNEL technique. Testes from control 28-day-old rats showed a faint 180-bp DNA ladder pattern that is typical of ongoing apoptosis occurring at this age (Fig. 3, lane 0). A similar faint DNA staining pattern was observed 3 hr after MEHP exposure (Fig. 3, lane 3). However, a marked increase in the intensity of DNA staining was observed in samples isolated from testes 6 and 12 hr after MEHP treatment (Fig. 3, lanes 6 and 12). In order to evaluate the incidence of apoptosis after phthalate exposure quantitatively, the in situ TUNEL technique

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FIG. 3. Increased low-molecular-weight DNA fragmentation follows MEHP exposure. Low-molecular-weight DNA isolated from rat testes at various times after MEHP exposure was separated by agarose (2%) gel electrophoresis. Each lane represents DNA isolated from an individual rat. A faint DNA ladder pattern is observed at both the 0 and the 3-hr time points. Beginning 6 hr after MEHP exposure and progressing to 12 hr, an obvious increase in the intensity of the DNA ladder pattern is apparent. STD lane represents a 100-bp DNA standard ladder.

was used to label the 3* ends of fragmented DNA in apoptotic cells. This method provides a very sensitive indicator to detect breaks in DNA and often labels cell DNA early in the process of apoptosis before morphologic evidence of cell death can be seen. In agreement with the DNA ladder technique, apoptotic germ cells were observed in control rat testes (Fig. 4A). The incidence in apoptosis was markedly increased in germ cells 6 and 12 hr after MEHP exposure (Figs. 4C, 4D). However, unexpectedly, 3 hr after MEHP exposure, the incidence of apoptotic events was decreased compared to that seen in controls (Fig. 4B). The incidence of apoptotic events observed in 100 seminiferous tubule cross sections of testes from each of four rats was counted and tabulated into categories. The categories were defined as 0, 1 – 3, 4 – 6, 7 – 9, or §10 incidences of apoptotic germ cells per tubule cross section. In control testes, 44.5% of the seminiferous tubule cross sections did not contain any apoptotic cells (Fig. 5). However, 3 hr after MEHP treatment, the number of tubule cross sections with no incidence of apoptosis significantly increased to 63.3%. This shift was reflected by a significant decrease in the incidence of tubules containing 1 – 3 apoptotic cells per cross section at 3 hr. Seminiferous tubule cross sections from the 6- and 12-hr MEHP-exposure groups had a dramatic increase in the number apoptotic events as is evident by the increased incidence of seminiferous tubules which contained 4 – 6, 7 – 9, and §10 apoptotic germ cells (Fig. 5) and a decrease in the incidence of seminiferous tubule cross sections that contained no apoptosis (35.0 and 27.8%, respectively).

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FIG. 4. MEHP-induced alterations in germ-cell apoptosis. DNA fragmentation, a characteristic feature of apoptosis, was detected in germ cells by TUNEL staining of testis cross sections. Arrows mark germ cells undergoing apoptosis. (A) Characteristic TUNEL staining in germ cells from control rat testis cross sections. (B) The number of TUNEL positive events per seminiferous tubule was markedly decreased 3 hr after MEHP exposure. By 6 hr (C) and continuing to 12 hr (D), the number of TUNEL staining positive events markedly increased over that of control (1771).

DISCUSSION

One of the major changes seen after phthalate exposure in animal models is the sloughing of testicular germ cells. The underlying mechanism for this detachment of germ cells from the seminiferous epithelium has not yet been determined. The Sertoli cell of the testicular seminiferous epithelium is the primary target of the phthalates based upon Ser-

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toli cell specific biochemical and morphological alterations. The Sertoli cell is responsible for orchestrating the differentiation of testicular germ cells as well as providing nutritional and physical support. In further characterizing the effects of the biologically active and toxic phthalate metabolite, MEHP, on the Sertoli cell, we have made two novel observations: an early and specific collapse of Sertoli cell vimentin filaments and a decrease (followed by a later increase) in

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FIG. 5. Quantitation of TUNEL-stained testis cross sections. The incidence of apoptosis at different times after MEHP exposure was determined by counting the number of TUNEL-positive cells for each seminiferous tubule categorized groups defined as 0, 1–3, 4–6, 7–9, or §10 positive cells per seminiferous tubule cross section. A total of 100 randomly selected seminiferous tubule cross sections were analyzed for each of four rats at each time point with data expressed as a percentage of total. Bars represent the means { SEM. Significant differences (p õ 0.05) between times of MEHP exposure among groups of apoptotic cell counts are indicated by different letters.

the normal physiological incidence of germ cell apoptosis seen in young rats. Both of these effects occurred as early as 3 hr after MEHP exposure. To investigate the consequences of Sertoli cell dysfunction on germ cell viability, the role of apoptosis in MEHPinduced germ cell death was studied by two methods: identification of low-molecular-weight DNA fragmentation patterns on agarose gels and the in situ TUNEL technique to stain cells undergoing apoptosis. DNA isolated from whole testis of rats showed a characteristic apoptotic DNA ‘‘ladder’’ pattern that progressively increased in intensity from 6 to 12 hr after MEHP exposure. These results indicated that the germ cells were dying at these time points by an increase in apoptosis rather than necrosis. This conclusion was confirmed by immunohistochemistry using in situ TUNEL stained cryosections. However, in contrast, the novel finding of a significant decrease in the physiological incidence of germ cell

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apoptosis 3 hr after toxicant exposure via the TUNEL method indicate that MEHP inhibited the physiologic mechanism by which germ cells undergo apoptosis. Coincidentally, both the phthalate-induced collapse of Sertoli cell vimentin filaments and the inhibition of germ cell apoptosis occurred 3 hr after MEHP exposure. Taken together, these observations imply that either (1) the collapse in Sertoli cell vimentin filaments and the inhibition of germ cell apoptosis are casually related or (2) the process of apoptosis and vimentin filament collapse share similar biochemical components and mechanisms affected by MEHP. The contribution of the collapse of Sertoli cell vimentin filaments to MEHP-induced germ cell apoptosis needs to be addressed. Vimentin filaments in the Sertoli cell are found both surrounding the nucleus and as apical extensions which radiate to the periphery of the cell where they associate with the plasma membrane in the region of attachment to germ cells. It has been proposed that these filament extensions are involved in maintaining the integrity of Sertoli cell–germ cell contacts (Aumuller et al., 1992). Therefore, the MEHPinduced collapse in vimentin filaments may lead to alterations in germ cell apoptosis by a disruption in contactmediated communication between the Sertoli cells and germ cells. Vimentin filaments may also be envisioned to have a more direct role in coupling Sertoli–germ cell apoptotic signaling. Though vimentin intermediate filaments are prominent elements of the cytoskeleton of many eukaryotic cells, these filaments have yet to be ascribed any vital cellular function. However, there is strong structural, biochemical, and direct experimental evidence that cytoplasmic intermediate filaments interact with the plasma membrane. Vimentin binds indirectly to the avian erythocyte plasma membrane proteins ankyrin and spectrin and other unidentified proteins (for review, see Green and Jones (1990)). A specific association of a receptor and G-protein-regulated phospholipase C with a component of the detergent-insoluble cytoskeleton was reported in turkey erythrocyte preparations (Vaziri and Downes, 1992). Therefore, it is conceivable that vimentin could be associated with the apoptotic cellular signal transduction pathway. MEHP could also be envisioned to interact directly with the apoptotic signaling pathway. The cellular mechanisms and signal transduction events which regulate apoptosis are poorly defined and currently the subject of intensive research. However, the trigger for apoptosis might be integrated with other normal cellular signal transduction systems (Corcoran et al., 1994). MEHP disrupts the follicle stimulating hormone-linked signal transduction pathway in primary Sertoli cell cultures by a mechanism located at the cell membrane (Grasso et al., 1993). These findings suggest the possibility that MEHP interacts directly with components of the apoptotic signal transduction pathway.

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Sertoli cells provide both physical support as well as secreted factors that are required for germ cell survival. In addition, Sertoli cells may also influence germ cell function by direct ‘‘communication’’ between these two cell types. Sertoli and germ cells are in close contact and communications may be mediated though transport of messengers through gap junctions or by contact-mediated mechanisms (for review, see Enders (1993)). To explain the differences in germ cell apoptosis seen 3 and 6 hr after MEHP exposure, we hypothesize that the Sertoli cell is responsible for directing germ cell apoptosis and that the signaling mechanism between these two cells requires that they remain in close physical contact. Therefore, disruption of Sertoli cell–germ cell contacts by MEHP-induced detachment of germ cells would uncouple the signal transduction mechanism between these cells leading to an initial (3 hr) decrease in germ cell apoptosis. Later in the MEHP exposure period (6–12 hr), Sertoli cells may increase the secretion of a factor to mediate apoptosis in detached germ cells or, alternatively, detachment of germ cells may initiate their apoptotic pathway by an autocrine mechanism. The design of our future experiments is guided by analogy to the mechanism by which cytotoxic T lymphocytes (CTLs) kill infected target cells (for review, see Nagata and Golstein (1995)). This mechanism is currently the best understood process by which one cell type causes the death of another by apoptosis. The interaction between CTLs and their target cells is mediated by the Fas-associated apoptotic pathway. Fas is a well characterized apoptosis-related cell surface molecule that acts as a receptor protein for Fas ligand which triggers an intrinsic suicide program in the target cell (Itoh et al., 1991). Indeed, abundant Fas ligand has been reported to be expressed in rat testis (Suda et al., 1993). Future experiments will address the cell type specificity of Fas and Fas ligand expression in the testis and the influence of MEHP on this cell death system. ACKNOWLEDGMENTS This publication was made possible by Grants RO1 ES05033 (K.B.) and T32 ES 07272 (J.R.) from the National Institute of Environmental Health Sciences, NIH. Kim Boekelheide is a Burroughs Wellcome Fund Scholar in Toxicology and this work was supported in part by a grant from the Burroughs Wellcome Fund.

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