Disappearance of Vimentin in Sertoli Cells: A Mono(2-ethylhexyl) Phthalate Effect

Abstract

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V–FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V–FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

Di(2-ethylhexyl) phthalate (DEHP) is one of the most common plasticizer used in consumer products, food packaging materials (Blount et al. 2000), and biomedical devices (Cole et al. 1981). Studies on DEHP have shown reduced fertility and testicular atrophy in laboratory animals (Teirlynck et al. 1988). When administered orally to rodents, DEHP is rapidly hydrolyzed in the gut and other tissues by nonspecific esterases to produce the monoester metabolite, mono(2-ethylhexyl) phthalate (MEHP) and 2-ethylhexanol (Albro and Thomas 1973). MEHP has been found to be the active toxic metabolite of DEHP (Oishi and Hiraga 1980). Previous researches have shown that young animals are more sensitive to MEHP exposure (Poon et al. 1997) and that the Sertoli cells are the primary target of MEHP in the testis (Boekelheide 1993). Loss of spermatogenic cells attached to the seminiferous epithelium and appearance of spermatogenic cells in the lumen of the seminiferous tubule are the phenomena frequently observed with several Sertoli cell toxicants, including phthalates (Creasy et al. 1987; Gray and Beamand 1984).

Sertoli cells are the supportive cells of the seminiferous epithelium, and provide the appropriate hormonal and nutritional environment necessary for the differentiation of immature spermatogenic cells into spermatozoa (Allard, Johnson, and Boekelheide 1993). These Sertoli cells possess a highly organized and quite active cytoskeleton. In most cells, the cytoskeleton consists of three major components, namely, microfilaments, microtubules, and intermediate filaments. In Sertoli cells, intermediate filaments are of the vimentin type, with a molecular weight of about 55 to 58 kDa (Frank, Grund, and Schmid 1979). These vimentin filaments are reported to concentrate around the basally positioned nucleus and radiate towards the periphery of the cells. They are also thought to play a role in anchoring spermatogenic cells to Sertoli cells (Amlani and Vogl 1988).

Because spermatogenic cells are fully dependent on Sertoli cells for maturation, differentiation, and survival, we can also presume that chemicals that affect Sertoli cells would also have an indirect effect on spermatogenic cells. Unfortunately, none of the major Sertoli cell toxicants of environmental and occupational significance have a known mechanism of action. So far, from our preliminary findings, we have observed that MEHP exposure causes an increase in spermatogenic cell death. However, the actual mechanism still remains ambiguous. Therefore, the current study was undertaken to shed some light on what happens to Sertoli cells during MEHP exposure.

MATERIALS AND METHODS

Animals and Dosing

Twenty-one-day-old C57Bl/6N male mice (Charles River, Japan) were given a single dose of 800 mg/kg MEHP in corn oil by oral gavage. Mice received MEHP in corn oil at a volume equal to 4 ml/kg. Control animals received the same volume of corn oil. MEHP was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). At 0, 3, 6, 9, and 24 h after administration, mice were sacrificed. Testes from 0-, 3-, 6-, and 9-h sampling were used for Western blot analysis. Testes from the 24-h sampling were fixed in 4% PFA (paraformaldehyde) at 4°C in phosphate-buffered saline (PBS). Thereafter, they were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Paraffin blocks were cut at 5 μm in thickness.

Sertoli Cell Culture

Mixed cultures of Sertoli and spermatogenic cells were prepared from 21-day-old C57Bl/6N mice (Charles River) as previously described by Worrell et al. (1989) with adaptations, and were maintained at 32°C in 25-cm² tissue culture flasks. In brief, testes were excised, decapsulated, cut into small pieces, and treated with 0.1% collagenase (Sigma, St. Louis, USA). Single cells were obtained by gravity sedimentation and usage of 50% typsin-EDTA (Gibco, NY, USA). Cells were finally suspended at a concentration of 1 × 10⁸ cells/ml and maintained for 24 h in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), and 10% fetal bovine serum (Sigma). Thereafter, cells were maintained in serum-free medium, which was changed every 24 h. At 72 h, spermatogenic cells were removed by treatment with hypotonic Tris buffer (20 mM Tris-HCl, pH 7.4) for 5 min as described by Galdieri et al. (1981). The Sertoli cell–enriched cultures obtained were then maintained in serum-free medium for 24 h before being reseeded onto chamber slides. The samples were then cultured for a further 24 h. Subsequently, they were exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. Some slides were stained with 0.5% toluidine blue for light microscopic observation, whereas the rest were used for immunohistochemical analysis.

Vimentin Immunohistochemistry

For vimentin immunohistochemistry, a monoclonal antivimentin antibody (mouse immunoglobulin M [IgM] isotype, clone LN-6; Sigma) at a dilution 1:100 was used for staining vimentin in Sertoli cells. Slides were fixed in 4% PFA for 10 min, rinsed in PBS, blocked with a TNB (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% Blocking Reagent, pH 7.5) blocking buffer, supplied in TSA (Tyramide Signal Amplification) Biotin System kit (Perkin Elmer Life Sciences, Boston, USA), and incubated overnight at 4°C with primary antibody. Thereafter, the sections were incubated with biotinylated goat anti-mouse IgG secondary antibody for 1 h at room temperature, followed by the ABC kit (Funakoshi, Tokyo, Japan). Immunoreaction was visualized with 0.05% diaminobenzidine tetrahydrochloride (DAB) (Wako, Tokyo, Japan) with H₂O₂ in PBS. The sections were lightly counterstained with hematoxylin. For a negative control, the incubation step with the primary antibody was omitted. Meanwhile, for paraffin sections, they were first deparaffinized, rehydrated, and later heated in 0.01 M citrate buffer (pH 6.0) for 10 min in a microwave to facilitate antigen retrieval. Then they were treated with 3% peroxidase for 30 min to eliminate endogenous peroxidase, blocked with a TNB blocking buffer, and incubated overnight at 4°C with primary antibody. Thereafter, protocols were similar as described above.

Annexin V–FITC

An annexin V–FITC (fluorescein isothiocyanate) apoptosis detection kit (BioVision, USA) was used. In brief, 5 μl of annexin V–FITC and 1 μl of TOPRO-3 were added to 500 μl of 1× binding buffer. Slides containing Sertoli cells were later incubated with this solution at room temperature for 20 min in the dark. Henceforth, they were observed using a LSM510 confocal laser scanning system (Carl Zeiss, Jena, Germany) with a dual filter set for FITC and TOPRO-3.

TUNEL

The apoptosis of Sertoli and spermatogenic cells was examined using the TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) method according to the protocol of the In Situ Apoptosis Detection Kit (TaKaRa, Tokyo, Japan). For Sertoli cell cultures, slides were fixed in 4% PFA for 10 min, and then washed in PBS. The paraffin-embedded sections were deparaffinized, rehydrated, and then predigested with 10 μg/ml protease K for 15 min. Thereafter, detection protocols were similar for both types of samples, whereby they were incubated in PBS containing 3% H₂O₂ for 15 to 30 min to block endogenous peroxidase activity. Then they were incubated with a fluorescein isothiocyanate (FITC)-labeled TdT enzyme in a humidified chamber at 37°C for 90 min. After washing, the slides were incubated with anti-FITC HRP (horseradish peroxidase) conjugate for 30 min at 37°C. Finally, TUNEL-positive cells were detected by DAB substrate development, counterstained with methyl green dye, mounted, and observed using optical microscopy for counting apoptotic cell number per each seminiferous epithelium (an average of 5 to 10 seminiferous tubules were counted per mice, n = 6). To check for the nonspecific reaction, the sections were incubated with PBS buffer alone instead of FITC-labeled TdT enzyme.

Western Blot

Testes were harvested at 0, 3, 6, and 9 h after initial MEHP treatment. Each tissue was washed with ice-cold PBS, homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 nM phenylmethylsulfonyl fluoride [PMSF], 1% Triton X, and 1× protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)), and centrifuged at 40,000 rpm at 4°C for 1 h. After the protein concentration of each supernatant was determined by the BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL, USA), 10 μg of each sample was boiled with nonreduced sodium dodecyl sulfate (SDS) sample buffer, and electrophoresed in a 10% to 20% gradient gel. After the gel was transferred to nylon membrane, the blot was incubated with monoclonal antivimentin antibody (mouse IgM isotype, clone LN-6; Sigma) (1:1000). The labeled protein was then visualized with HRP-labeled goat anti-mouse IgG antibody in combination with the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). In addition, approximately equal amounts of protein sample per lane were confirmed by silver staining of the replica gel.

Statistical Analysis

Data are expressed as means ± SE. Statistical analysis was conducted using the Student’s t test. p values of less than .05 were considered statistically significant.

RESULTS

Fig. 1 shows the vimentin staining and TUNEL analysis for both control and treated mice, respectively. In control mice, vimentin could be seen as projections extending from the basal region into the lumen (Fig. 1A ). Meanwhile, in mice treated with a single oral dose of 800 mg/kg MEHP, vimentin showed shorter projections and concentration near the basal region (Fig. 1B ). For TUNEL analysis, mice treated with MEHP showed an increase in the number of TUNEL-positive cells (Fig. 1D ) compared to the control mice (Fig. 1C ). The number of TUNEL-positive cells per all spermatogenic cells in the seminiferous tubule was calculated and the results are shown in Fig. 1E . In control mice, the normal cell death percentage was less than 1% (0.6% ± 0.26%). On the other hand, mice treated with a single oral dose of MEHP showed an astonishing 30-fold increase (30.6% ± 1.56%) in TUNEL-positive cells after 24 h, which was quite significant (p < .001).

Sertoli cells in culture treated with MEHP, and later stained with toluidine blue, showed the presence of vacuoles in the cytoplasm. This incident was found to be increased in time-and dose-dependent manners (Fig. 2). Morphological features of a nontreated Sertoli cell in culture are shown in Fig. 2A and were used as a control for comparison. Sertoli cells treated with 1nmol/ml MEHP for 3 h showed no abnormal changes (Fig. 2B ). The presence of vacuoles could be observed as early as 3 h after MEHP exposure in Sertoli cells treated with 100 nmol/ml MEHP (Fig. 2C ). Meanwhile, Sertoli cells treated with 1 nmol/ml MEHP, a lower concentration of MEHP, only began to show the signs of vacuolation at 6 h after initial exposure (Fig. 2D ). Thereafter, the number and size of vacuoles increased in both treated groups (Fig. 2E –H), with the most severe vacuolation seen in Sertoli cells treated with 100 nmol/ml MEHP for 24 h (Fig. 2I ).

The results from the vimentin immunohistochemistry are shown in Fig. 3. Positive vimentin decreased as time and concentration of MEHP increased (Fig. 3). Sertoli cells treated with 1 nmol/ml MEHP for 3 and 6 h (Fig. 3B, D ) showed no signs of vimentin disruption (Fig. 3A ). However, different results were obtained from the Sertoli cells treated with 100 nmol/ml MEHP. Even at 3 h after initial exposure, significant disruption in vimentin could be observed (Fig. 3C ). Vimentin was found to be more concentrated around the perinuclear region and undetectable at the apical region of the cell. Henceforth, significantly less vimentin was observed, as exposure time to MEHP increased for both concentrations (Fig. 3E–H ). Vimentin almost disappeared in Sertoli cells treated with MEHP of the highest concentration and longest time (Fig. 3I ).

The decrease in vimentin expression can also be detected in the in vivo system (Fig. 4). Western blot analysis showed the gradual decrease of vimentin expression as the exposure to MEHP increased.

An annexin V–FITC apoptosis detection kit was used to observe the translocation of phospholipid phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the surface of cells undergoing apoptosis. This detection was done by staining the cells with a fluorescent conjugate of annexin V, a protein that has a high affinity for PS. As a result, detection of annexin V–FITC in Sertoli cells showed that some were preapoptotic, seen as green spots (data not shown). However, these results could not be correlated with the findings from TUNEL staining, as no TUNEL-positive cells were detected (data not shown).

DISCUSSION

According to Teirlynck and Belpaire (1985), MEHP has a half-life of approximately 3 to 5 h, so that within 24 h more than 90% of the compound is eliminated. However, Teirlynck et al. (1988) noted that the rats treated with a single dose of 800 mg/kg MEHP showed significant testicular atrophy even at 7 days after administration. Histological examination of the testes revealed a dose-dependent increase in the number of atrophic seminiferous tubules, with loss of spermatocytes (Teirlynck et al. 1988). Interestingly, this event supports the view that after single oral administration, MEHP induces a sequence of events within the testes, even after MEHP has been eliminated. In the present study, we focused only on documenting the effects of MEHP on Sertoli cells within a 24-h period.

Mice were used in this experiment because there are insufficient reports on the effects of phthalates on them and pre-pubertal mice were chosen because they are more sensitive to phthalate esters compared to adults (Gray and Gangolli 1986; Sjöberg et al. 1986). The differences in absorption, distribution, and metabolism of phthalate esters are recognized between the immature and adult individuals (Sjöberg et al. 1985). Moreover, spermatogenic cell apoptosis normally occurs in young mice to limit the clonal expansion of spermatocytes. Therefore, young mice provide a convenient model to evaluate alterations in the process of apoptosis.

In the current in vivo experiment, the 800 mg/kg MEHP dosage, a threshold dose, was chosen because our preliminary study revealed that higher doses would cause the sloughing of spermatogenic cells. The dose chosen was suitable for a 24-h exposure treatment and would generate the highest number of apoptosis. This dose was low compared to the 2000 mg/kg MEHP used by Awal et al. (2005) on prepubertal guinea pigs.

Apoptosis of selected spermatogenic cells occurs normally in testes, and is essential for maintenance of spermatogenesis (Print and Loveland 2000). In our in vivo findings, normal mice have a TUNEL-positive percentage of about 1%, whereas mice treated with a single oral dose of MEHP showed a sudden and quite significant increase (about 30%) in TUNEL-positive cells within 24 h after exposure. According to Richburg (2000), such an increase in apoptotic (TUNEL-positive) spermatogenic cells was often observed in testes injured by various physical or chemical stimuli, which proved that the testes of mice in this experiment were indeed injured in some way by the MEHP treatment. Furthermore, Richburg and Boekelheide (1996) also found that the primary consequence of MEHP exposure to rodents showed a large increase in spermatogenic cell apoptosis.

The Sertoli cell has been identified as the primary target for phthalate-induced injury by 91)—(1) early histopathological changes in this cell type by in vivo or in vitro exposure; (2) early alterations in Sertoli cell function and biochemistry; and (3) rapid disruption of the Sertoli-spermatogenic cell physical interaction (Boekelheide 1993). This prompted us to focus our attention to the primary effects of MEHP on Sertoli cells. Because determining a suitable MEHP concentration to use in the in vitro system and finding a logical correlation with the in vivo system were difficult, we concluded to use a 1 nmol/ml MEHP (low-dose) and 100 nmol/ml MEHP (high-dose) concentration based on findings from other researchers (Andriana et al. 2004a, 2004b), who found significant damage to the Sertoli cells of rats and goats after MEHP exposure in vitro.

Toluidine blue–stained Sertoli cell cultures in the current experiment showed the presence of vacuoles. Vacuolation is the earliest morphological sign of testicular injury and the cardinal response seen with many of the Sertoli cell toxicants. At the light microscope level, two rather distinct forms of basal Sertoli cell vacuolation could be observed: presence of either multiple, small vacuoles or a single, large vacuole (Boekelheide 1993). In our findings, we observed the presence of either multiple, small vacuoles or multiple, large vacuoles. The presence of multiple, small vacuoles in the basal Sertoli cell cytoplasm is a prominent feature of the early response to phthalate exposure in young rodents (Creasy, Foster, and Foster 1983). Although the occurrence of single, large vacuoles has been described in normal adult rat seminiferous epithelia (Chapin, Morgan, and Bus 1983), the incidence and characteristic of vacuolation appears to be altered by exposure to certain Sertoli cell toxicants. For example, following exposure to 2,5-hexanedione, both the number and size of Sertoli cell vacuoles observed in a testicular cross section increased many fold (Chapin, Morgan, and Bus 1983; Boekelheide 1987, 1988). In our study, the presence of multiple, small and large vacuoles was observed in the Sertoli cell cultures.

Intermediate filaments of the vimentin type are an important component of the Sertoli cell cytoskeleton and are thought to play a role in anchoring spermatogenic cells to the Sertoli cell. Vimentin filaments have been reported to occur in the perinuclear region of the cell and in the apical cytoplasm, which was also observed in our current findings. In this study, we found that vimentin became less visible as the exposure time and dose of MEHP increased. Vimentin began to decrease from the apical region and recede towards the nuclei, until vimentin filaments were no longer detectable by immunohistochemistry. Interestingly, this decrease of vimentin did not change the Sertoli cell physical appearance. The Sertoli cell remained intact and spread out, whereas only the vimentin faded. This decrease was also seen in the in vivo sections, whereby the vimentin apical extensions became shorter. But, this does not necessarily mean that the Sertoli cell is no longer projecting into the lumen, which our data on Sertoli cell culture clearly show. Some studies have found that this vimentin disruption correlates with the loss of structural integrity of seminiferous epithelia along with spermatogenic cell apoptosis (Richburg and Boekelheide 1996). The vimentin disruption seen in our in vivo experiment correlates with the sudden increase in TUNEL-positive cells, thereby confirming the previous observation. However, the Sertoli cell could still be physically supporting the spermatogenic cells, even though they might have lost their connection with vimentin. We can only hypothesize that MEHP somehow affects the Sertoli cells, causing an immediate damage to the vimentin filaments. This disruption signifies the loss of some sort of support mechanism for spermatogenic cells and could be the cause of the increased TUNEL-positive cells. Without a healthy Sertoli cell to hold and nurture spermatogenic cells, the vimentin-detached cells would eventually die. The exact pathway of this effect still remains ambiguous and requires further research.

At concentrations used in this experiment, a time-based correlation can be drawn up by comparing the appearance of vacuoles and the fading of vimentin in the Sertoli cells. As time and dose of MEHP treatment increased, the appearance of vacuoles also increased, both in number and in size. At the same time, the gradual disappearance of vimentin was observed. At this point in time, the cause-and-effect relationship between the appearance of vacuoles and the disruption of vimentin is unknown and other mechanisms may also exist. However, both events are believed to be connected in one way or other.

Exposure to MEHP does not cause Sertoli cell death. Although some of the Sertoli cell cultures were annexin V–FITC positive, a preapoptotic indicator, no TUNEL-positive Sertoli cells were observed. Two possible reasons for this are, first, there may exist a time difference between being annexin V–FITC positive and being TUNEL-positive. The TUNEL assay detects only DNA strand breaks, which is a relatively late event in the cellular process of apoptosis (Richburg, Nanez, and Gao 1999). On the other hand, the annexin V–FITC apoptosis system detects PS on the surface of cells that are about to undergo apoptosis. The direct comparison of the TUNEL assay and annexin V affinity assay indicates that PS externalizations can be measured prior to the detection of DNA strand breaks (O’Brien, Reutelingsperger, and Holdaway 1997). All experimental evidence obtained thus far indicate that loss of membrane asymmetry is a quite early phenomenon during apoptosis, initiated at a time following the caspase proteolytic cascade but possibly preceeding nuclear condensation and breakdown of intracellular cytoskeletal and nuclear matrix constituents (van Engeland et al. 1998). This means that the annexin V–FITC–positive cells in this experiment would eventually be TUNEL positive. Second, the Sertoli cell possesses a recovery mechanism that allows the cell to repair physical damage and return to normal conditions after a certain period of time. The annexin V–FITC–positive cells are in the process of recovery and would eventually be undetected. However, the possibility is not supported by experimental results as PS exposure seems to last from the early execution phase of apoptosis until the final stage, at which the cell has broken up into apoptotic bodies (Verhoven, Schlegel, and Williamson 1995). We know that Sertoli cells are the nurse cells of spermatogenic cells and play an important role in spermatogenesis and as such probably have evolved a recovery or defense mechanism to protect themselves from trauma.

We also believe that the vimentin disruption seen in Sertoli cell cultures is not permanent, and would eventually recover if the causative agent was removed. We found that mice treated with a single dose of MEHP showed significant spermatogenic cell loss and cell death. But, in time, the cell-death count returned to normal and the seminiferous epithelium showed no significant loss of spermatogenic cells (data not shown). Because the loss of vimentin correlates with the increased apoptotic spermatogenic cell numbers, return to normal rate of apoptosis would indicate that the vimentin also recovered. However, this speculation is yet to be proven and it would be interesting to know if Sertoli cells possess a recovery mechanism.