{alpha}-Chlorohydrin Inhibits Glyceraldehyde-3-Phosphate Dehydrogenase in Multiple Organs as Well as in Sperm

Karen Bozak Jelks and Marion G. Miller1,

Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, California 95616

Received December 19, 2000; accepted March 20, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous studies have documented inhibitory effects of {alpha}-chlorohydrin (ACH) on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in spermatozoa. A sperm-specific G3PDH isoform has been described. The possibility that ACH may inhibit G3PDH in cell types other than sperm was investigated in this work. In addition, the onset of ACH-induced epididymal toxicity was described. Changes to epididymal histology occurred 6 h following a single dose of ACH (50 mg/kg po) and were confined to the proximal initial segment. By 24 h, no epithelial cells lined the basement membrane of that region. Three h after ACH administration (50 mg/kg po), G3PDH activity was significantly decreased in sperm (85%) as well as in kidney (31%), liver (49%), and epididymis (35%). Enzyme activity remained inhibited at 6 and 24 h. G3PDH was immunolocalized in the epididymis and staining was highest in the efferent ducts and initial segment as well as in smooth muscle. Since G3PDH is a microtubule-associated protein and microtubule-dependent endocytosis occurs in the epididymis, ß-tubulin was also immunolocalized. ß-tubulin densely stained the apical region of initial segment and caput epithelial cells. Disruption of ß-tubulin immunostaining correlated with the localization and onset of the lesion. Co-localization of G3PDH and ß-tubulin immunostaining was not observed although both antibodies most densely stained the initial segment. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition. Moreover, ACH inhibition of G3PDH is not confined to sperm, although the sperm enzyme is most sensitive to inhibition.

Key Words: {alpha}-chlorohydrin; sperm; epididymal pathology; glyceraldehyde-3-phosphate dehydrogenase activity; ß-tubulin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}-Chlorohydrin (ACH) is an anti-fertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility and sperm intermediary metabolism (Jelks et al., 2001Go; Jones, 1978Go). High doses of ACH produce irreversible effects on the epididymis and induce sterility. A single high dose produces a characteristic pathological lesion in the initial segment of the epididymis (Hoffer et al., 1973Go). Occlusions of the epididymal duct and subsequent degeneration of germinal epithelium in the testis 3–4 days later results in prolonged or permanent sterility (Ericsson, 1970Go). The mechanism underlying the ACH-induced epididymal damage is not known.

Low doses of ACH have been shown to produce a reversible reduction in fertility after 3–6 days with fertility recovering in the animal approximately 5–11 days after cessation of ACH administration (Tsunoda and Chang, 1976Go). In vitro incubation of mature sperm with ACH has been shown to inhibit sperm glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity (Ford and Jones, 1983Go; Jones et al., 1981Go). The inhibitory effect of ACH on metabolic activity of sperm in vitro is thought to occur via ACH oxidation within the sperm to form 3-chlorolactaldehyde (Cooney and Jones, 1988Go). This metabolite has the appropriate chiral conformation to act as an analogue of the G3PDH substrate glyceraldehyde 3-phosphate (Stevenson and Jones, 1985Go). G3PDH inhibition causes a futile cycle in the presence of a glycosable sugar with resultant depletion of sperm ATP (Ford and Harrison, 1985Go). The changes in sperm motion (Slott et al., 1995Go; Toth et al., 1992Go), capacitation (Dravland and Meizel, 1981Go), and fertility seen after ACH are thought to arise because of insufficient sperm ATP.

The present studies investigated the onset and effect of a single, mid-range dose of ACH (50 mg/kg po) on rat epididymal histology and G3PDH activity in rat sperm, epididymis, testis, kidney, liver, and brain within 24 h. G3PDH inhibition has been documented in sperm after exposure to ACH or compounds with similar mechanisms of action (Jones, 1983Go). However, there is conflicting data regarding the effect of ACH on G3PDH activity in other organs. For instance, Kaur and Guraya (1981) reported that all glycolytic enzymes, including G3PDH, were inhibited in testis, epididymis, and sperm of rats treated with ACH in vivo. On the other hand, Ford and Waites (1981) reported that G3PDH was inhibited in spermatozoa but not in testis, liver, brain, and muscle of rats treated in vivo with 6-chloro-6-deoxyglucose. The deoxyglucose is proposed to have a similar mechanism of action to that of ACH (Jones and Dobbie, 1991Go). Since early and direct measurement of G3PDH activity in sperm and multiple organs after in vivo exposure to ACH has not been carried out, we measured G3PDH activity 3, 6, and 24 h after ACH exposure. These time points were selected since decreases in sperm ATP levels collected from the cauda epididymis and vas deferens occur within the first 3 h following in vivo ACH exposure (Jelks et al., 2001Go). In conjunction with the activity assays, G3PDH was immunolocalized in the efferent ducts and the epididymis of the rat.

An additional study was conducted to evaluate the effect of ACH on ß-tubulin. G3PDH has been identified as a microtubule-associated protein involved in endocytosis. Moreover, microtubule disruption leads to inhibition of endocytosis in kidney proximal tubule cells (Elkjaer et al., 1995Go; Robbins et al., 1995Go). Like the kidney, the efferent ducts and initial segment epididymis are responsible for fluid and protein reabsorption through receptor-mediated endocytosis as well as passive diffusion (Ilio and Hess, 1994Go). Since ACH is known to interfere with reabsorption in the epididymis (Wong and Yeung, 1977Go), microtubule dysfunction may compromise these essential processes of endocytotic vesicle transport. To address this, ß-tubulin was immunolocalized within the efferent ducts and epididymis and the pattern of immunostaining in the initial segment was examined over the 24 h after ACH-exposure.

The present study has characterized the onset of the histologic damage in the rat epididymis seen after administration of a mid-range dose of ACH and investigated if ACH inhibits G3PDH activity uniquely in sperm. In addition, G3PDH has been immunolocalized within the epididymis and the effect of ACH on ß-tubulin immunostaining described. A goal of the present work was to explore the possibility that G3PDH inhibition and/or microtubule dysfunction may play a role in the ACH-induced epididymal lesion.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, reagents, and antibodies.
ACH (Lot C-0639, 98.1- 99.2% pure) was purchased from Sigma Chemical Co. (St. Louis, MO) and stored at 4°C. ACH was diluted in distilled water prior to animal treatment. All other chemical reagents were obtained from Sigma and were of the highest available purity unless stated otherwise. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody was obtained from Chemicon International, Inc. (Temecula, CA). Mouse anti-ß-tubulin monoclonal antibody was obtained from Boehringer Mannheim (West Germany). The Vectastain Elite ABC Kit was obtained from Vector Laboratories (Burlingame, CA).

Animals and treatment.
Sprague-Dawley rats were obtained from the Cole Facility Breeding Colony (Department of Animal Sciences, UC Davis). Original stock was purchased from Simonsen (Gilroy, CA) and bred in-house monogamous mating. Rats were maintained on rat chow (Formulab Purina 5008) and water ad libitum, and housed under a 14 h light/10 h dark cycle in humidity- (40–70%) and temperature- (22 ± 2° C) controlled rooms.

Sexually mature adult male Sprague-Dawley rats (100–120 days old, 350–450 g) received a single dose of 50 mg/kg ACH in water by oral gavage (po) since this mid-range dose is the lowest dose required to produce consistent epididymal damage after 5 days (Jelks et al., 2001Go). Controls received water only po at 3, 6, and 24 h after treatment. Rats were killed by asphyxiation (CO2 inhalation) and the liver, kidney, brain, and testes were immediately frozen at –80°C for future analysis. The epididymides were minced, rinsed 3 times in ice-cold buffer followed by filtration through gauze to separate sperm from epididymal tissue. Sperm and epididymal tissue were immediately frozen at –80°C for future analysis.

Histologic assessment of epididymal toxicity.
At various times (3, 6, and 24 h) after ACH administration (50 mg/kg po), testis, efferent ducts, and epididymis were fixed at 25°C in 10% phosphate buffered formalin for a minimum of 72 h (n = 3). Following fixation, testis and whole efferent ducts and caput epididymis were dehydrated in graded alcohol (50–100%), embedded in glycol methacrylate, and longitudinally sectioned at 4.0 µm. Sections were stained with periodic acid-Schiff reagent followed by a hematoxylin counterstain. Examination of the epididymis for ACH-induced changes in epithelial cell histology and luminal contents was determined at each time point.

Assay of G3PDH activity.
Frozen organs (liver, kidney, brain, testes, and epididymis) from control (n = 3) and ACH-treated animals (50 mg/kg po) (n = 3 for each dose) were individually diluted in 4x volume of ice cold Tris–HCl buffer (50 mM, pH 7.4) with phenylmethylsulfonyl fluoride (PMSF) (10 µM) to inhibit proteases. Tissues were disrupted on ice using a PolytronTM set at a low speed for 10 to 15 s. Using protocol 5178 provided by Boehringer-Mannheim (Indianapolis, IN), the homogenate was centrifuged (1000 x g, 8 min, 4°C) and the supernatant sonicated on ice and recentrifuged (12,500 x g, 10 min, 4°C). The resulting supernatant was kept on ice, diluted, and assayed for G3PDH enzyme activity. This assay measures G3PDH activity as the reversible reaction when glyceraldehyde-3-phosphate is formed from 1,3-diphospoglycerate generated from glycerate-3-phosphate catalyzed by 3-phosphoglycerate kinase. Briefly, 50 µl of diluted supernatant (0.01–0.02 mg protein) from each tissue or purified G3PDH with known activity (positive control) was added to 3.11 ml of assay solution consisting of 79.1 mM triethanolamine buffer (pH 7.6), 1.1 mM ATP, 6 mM glycerate 3-phosphate, 0.2 mM NADH, 0.9 mM EDTA, 1.6 mM MgSO4, and 3 U phosphoglycerate kinase. Changes in absorbance at 340 nm recorded NADH disappearance during glyceraldehyde phosphate formation. Activity levels were normalized to protein concentrations as determined by a bicinchoninic acid assay kit (Sigma, St. Louis, MO) and reported as units of activity/mg protein using an extinction coefficient of 6.3 cm–1 µ mole–1. Reagents and chemicals for the assay were purchased from Boehringer-Mannheim (Indianapolis, IN).

Sperm were subjected to 1 cycle of freeze/thaw to disrupt cellular membranes (–80°C), sonicated and then centrifuged (12,500 x g, 10 min, 4°C). The supernatants were diluted (0.2–0.3 mg protein/ml) and were assayed for G3PDH activity.

Immunohistochemical staining for G3PDH.
Testis, efferent ducts, and epididymis were cleanly dissected out and cut so that a given section would include 1 or more of the major regions, i.e., efferent ducts, the initial segment, caput, corpus, and cauda epididymis. The tissues were immersion fixed at 4°C in 1% paraformaldehyde (freshly prepared) for 24 h after which they were dehydrated and embedded in paraffin. Sections (5 µm) were cut and mounted on glass slides, deparaffinized with xylene, and hydrated in graded ethanol solutions (5 min each). Tissue sections were immersed for 10 min in phosphate buffered saline (PBS) containing 3% hydrogen peroxide to inactivate endogenous peroxidase activity.

Prior to incubation with the primary antibody, the tissue sections were blocked for 30 min in PBS containing 10% Blocking Serum (horse serum). Each section was then incubated in a moist chamber with a previously characterized antibody to G3PDH (Kots et al., 1992Go) at a 1:30 dilution (16 h at 4°C). The sections were washed once in PBS (20 min) and incubated for 30 min with a biotinylated secondary antibody (horse anti-mouse) at a dilution of 1:20,000. Following incubation with the secondary antibody, all sections were again washed and then subjected to the Vectastain Elite ABC Kit Protocol using the chromogen diaminobenzidine tetrahydrochloride. After dehydration in a graded series of ethanol solutions (5 min each), sections were immersed in 3 changes of xylene (5 min) and coverslipped with Permount.

Immunohistochemical staining for ß-tubulin.
The procedure for ß-tubulin immunolocalization was the same as the procedure as described in Immunohistochemical Staining for G3PDH. The ß-tubulin primary antibody was used at a dilution of 1:250.

Statistics.
ANOVA followed by Tukey's mean comparison was used (MiniTab+TM) to determine significant differences in G3PDH enzyme activity between the control and the 3, 6, and 24 h time points. Differences between means were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histological Assessment of Epididymal Toxicity
The pathology of the epididymal lesion induced by a mid-range dose of ACH was evaluated at the light level at early time points (3, 6, and 24 h). In our time-course study, which followed the onset and development of the ACH-induced histological lesion, exposure to ACH (50 mg/kg po) resulted in an epididymal lesion after 6 h in the rat. The site of histopathological changes was clearly limited to the proximal initial segment immediately adjacent to the efferent ducts. Our findings, performed at a much lower dose than used in other studies (50 mg/kg vs. 100–140 mg/kg), are consistent with previous reports which also localized epididymal damage to the initial segment of the epididymis (Cooper et al., 1974Go; Hoffer et al., 1973Go).

After 3 h, in control rats (Fig. 1AGo) and the ACH-treated rats (Fig. 1BGo), the initial segment displays characteristic pseudostratified columnar epithelium with long microvilli extending from the principal cells into the lumen. Small empty spaces within the epithelium are routinely seen in control initial segment epididymis and little difference is noted between the initial segment of a control rat and the initial segment of a rat 3 h after ACH treatment. At 6 h following treatment, vacuolization was found both intercellularly and intracellularly throughout the epithelia of the epididymal tubule in the proximal initial segment that lie immediately adjacent to the efferent ducts. An increase in luminal diameter of the tubule can be observed. Some of the cells of the epithelium have lifted off the basement membrane and are sloughing into the lumen. The lumen is filled with debris and few sperm are present (Fig. 1CGo). At 24 h after treatment with ACH, desquamation of epithelial cells occurs and no epithelial cells line the peritubular basement membrane of the initial segment that lies adjacent to the efferent ducts. The distended tubules of the initial segment are filled with sloughed cells and PAS-positive debris and fluid (Fig. 1DGo).



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FIG. 1. Photomicrographs of initial segment epididymal histology in control (A) and ACH-treated rats (50 mg/kg po) (B–D). Animals were killed at 3 h (B), 6 h (C), and 24 h (D) after ACH-administration. The initial segment from control and 3-h rats displays characteristic pseudostratified columnar epithelium with long microvilli extending from the principal cells into the lumen. Six h after ACH, histopathological changes occurred in the proximal initial segment immediately adjacent to the efferent ducts. Intracellular and intercellular vacuolization is present. Luminal diameter is increased and the lumen is filled with sloughed cells and debris, and few sperm are present. At 24 h after treatment with ACH, there is desquamation of epithelial cells and no epithelial cells line the peritubular basement membrane of the initial segment that lies adjacent to the efferent ducts. The distended tubules of the initial segment are filled with sloughed cells and PAS-positive debris and fluid. (periodic-acid Schiff reagent and hematoxylin, magnification: [A] x361, [B] x284, [C] x273, [D] x319)

 
Longitudinal sections of the junction between the efferent ducts and epididymis in control rats show the small efferent ducts coalescing to form the larger columnar, pseudostratified epithelium of the proximal initial segment of the epididymis (Fig. 2aGo). At 6 h after treatment of ACH, the normally columnar, pseudostratified epithelium of the proximal initial segment that lies immediately adjacent to the efferent ducts appears with epithelial vacuolization and an increase in luminal diameter (Fig. 2bGo). Qualitatively, the efferent duct tubules appear slightly distended, although the efferent duct epithelium looks normal and intact.



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FIG. 2. Photomicrographs of longitudinal sections of the junction between the efferent ducts and epididymis in control and ACH-treated rat (50 mg/kg po) 6 h after administration. Longitudinal sections of the junction between the efferent ducts and epididymis in control rats highlight the discrete area of histological damage. In control rats, the small efferent ducts coalesce to form the larger columnar, pseudostratified epithelium of the proximal initial segment of the epididymis (a). Six h after treatment with ACH, the normally columnar, pseudostratified epithelium of the proximal initial segment that lies immediately adjacent to the efferent ducts appears with epithelial vacuolization and an increase in luminal diameter (b). Qualitatively, the efferent duct tubules appear slightly distended, although the efferent duct epithelium looks normal and intact (periodic-acid Schiff reagent and hematoxylin, original magnification x64).

 
Assay of G3PDH Activity
G3PDH activity in sperm, epididymis, brain, liver, kidney, and testis at early time points (3, 6, and 24 h) after ACH treatment (50 mg/kg po) was quantitated. Since sperm ATP concentrations are decreased to approximately 55% of control 3 h following ACH treatment (50 mg/kg po) (Jelks et al., 2001Go), G3PDH activity was determined at the same 3-h time point. At this time following ACH treatment, G3PDH activity was significantly depressed in sperm by 85% (49% in liver, 35% in epididymis, and 31% in kidney) (Fig. 3Go). G3PDH activity was not significantly affected in brain or testicular tissue.



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FIG. 3. Bar graphs of the G3PDH activities in various tissues 3, 6, and 24 h following ACH administration (50 mg/kg po) in the rat. Each bar represents the mean with standard errors; n = 3. An asterisk (*) indicates that the values are significantly different (p < 0.05) from those of control.

 
At 24 h following ACH treatment, G3PDH activity remained significantly depressed in sperm by 77% (71% in kidney, 55% in liver, and 45% in epididymis). The brain and testes appear resistant to ACH treatment with decreases of 12% and 14% respectively that are statistically insignificant at the 24-h time point. Overall, on a per mg of cellular protein basis, sperm were the most sensitive to G3PDH activity inhibition.

Immunohistochemical Staining for G3PDH
In conjunction with activity, G3PDH was immunolocalized in the efferent duct epithelia, the microvilli of the initial segment and the smooth muscle layers surrounding the epididymal tubule along the entire length of the epididymis. Faint background staining was present in an antibody-free control (not shown) but was diffuse and not localized when compared to staining in the presence of the G3PDH antibody. The efferent ducts display moderate immunoreactivity within all cell types although some cells stained more heavily and pocket-like concentration of the enzyme can be seen (Fig. 4AGo). The initial segment displays moderate immunostaining in the microvilli and the thin layer of smooth muscle surrounding the tubule (Fig. 4BGo). Within the caput, corpus, and cauda epididymis, immunostaining for G3PDH was confined to the increasingly layered smooth muscle surrounding the tubule (data not shown). After ACH administration, the pattern of G3PDH immunostaining was disrupted concomitant with the histologic lesion (data not shown).



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FIG. 4. Photomicrographs of control rat efferent ducts and epididymis immunostained with the anti-G3PDH monoclonal antibody. G3PDH appears confined to the efferent duct epithelia, the microvilli of the initial segment, and the smooth muscle layers surrounding the epididymal tubule along the entire length of the epididymis. There is moderate immunoreactivity within all epithelial cell types of the efferent ducts although some cells stain more heavily and pocket like concentration of the enzyme can be seen (A). The initial segment displays moderate immunostaining in the microvilli and the smooth muscle surrounding the tubule (B). Within the caput, corpus and cauda epididymis, immunostaining for G3PDH remains confined to the smooth muscle surrounding the tubule (data not shown). (original magnification x400)

 
Immunohistochemical Staining for ß-Tubulin
The present work evaluated ß-tubulin in the epididymis of the rat over the course of ACH exposure. In control rats, ß-tubulin appeared concentrated in the apical region of epithelial cells within the initial segment and caput region of the epididymis. The dense immunostaining in the apical region of epithelial cells is consistent with the absorptive and secretory function of the region (reviewed in Ilio and Hess, 1994). This immunostaining gradually faded towards the cauda where almost no immunostaining can be detected (data not shown). ACH exposure (50 mg/kg po) altered the ß-tubulin staining pattern in the initial segment epithelia where histological changes were seen to occur.

ß-Tubulin immunostaining is present in the apical region of epithelial cells within the initial segment of a control rat epididymis. It appears to be uniformly distributed throughout all cell types (Figure 5AGo). Three h following a single dose of ACH, ß-tubulin immunostaining within the initial segment was only subtly different from control. At 6 h following ACH treatment, ß-tubulin appears clumped within the epithelia. The overall immunostaining has diminished and lost uniform distribution. At 24 h following ACH treatment, ß-tubulin immunostaining is absent and desquamation of epithelial cells has occurred. No epithelia cells line the basement membrane of portions of the initial segment that lie adjacent to the efferent ducts (Figs. 5B–5DGo).



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FIG. 5. Photomicrographs of control and ACH-treated rat initial segment epididymis immunostained with the anti-ß tubulin monoclonal antibody. ß-Tubulin immunostaining is uniformly present and distributed throughout the apical region of all cell types in the initial segment epithelial cells of control rats (A). At 3 h after ACH exposure (50 mg/kg po), ß-tubulin immunostaining within the initial segment is only subtly different from control (B). At 6 h, ß-tubulin appears clumped in areas between cells. The overall immunostaining has diminished and lost uniform distribution (C). At 24 h following ACH treatment, desquamation of epithelial cells has occurred. No epithelia cells line the basement membrane of portions of the initial segment that lie adjacent to the efferent ducts (D). Original magnification [A] x382, [B] x406, [C] x288, [D] x315.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACH is well known to inhibit G3PDH activity in spermatozoa and to cause epididymal toxicity. Inhibition of this glycolytic enzyme has been implicated as important in the mechanism by which ACH depletes sperm ATP in the presence of a glycosable sugar. A sperm-specific isoform of G3PDH has also been described (Welch et al., 1992Go). Whether this effect of ACH is specific for the sperm G3PDH isoform has not been determined. In the present study, G3PDH activity was inhibited not only in sperm but also in epididymis, liver, and kidney, thus the sperm-specific isoform of G3PDH is not uniquely inhibited by ACH. The first signs of damage observed in the proximal initial segment 6 h after a single, 50 mg/kg dose of ACH was the appearance of vacuoles that were located both inter- and intracellularly. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition.

The region of histological changes in the rat epididymis after ACH administration is in agreement with that described by Hoffer et al., (1973) and Cooper et al., (1974). Our study determined that histologic changes occur within 6 h following ACH exposure. At this time, the proximal initial segment tubule immediately adjacent to the efferent ducts became distended, with epithelial damage and some sloughing of the epithelial cells. Vacuoles or empty looking spaces appear between the cells, the cells and basement membrane, and within cells. By 24 h the lesion progressed with a large proportion of the initial segment tubule epithelium being absent and completely sloughed. No indication of damage in any other region of the efferent ducts or caput epididymis (i.e., distal to the proximal initial segment) was detected except for the appearance of debris within the lumen.

The damage to the proximal initial segment observed after ACH suggests interference with testicular fluid reabsorption since fluid reabsorption is a major function of this region. Intercellular vacuolization could indicate altered fluid secretion/absorption processes and loss of cellular adhesion (Kerr et al., 1993Go). Since Hoffer et al., (1973) detected a striking diminution in the number of coated endocytotic invaginations of the apical plasmalemma of the initial segment following ACH exposure, these data support the theory that early alterations in the absorptive function of the initial segment region is occurring after ACH administration. The initial segment is responsible for absorption of specific proteins and fluid with up to 90% of fluid originating from the testis being absorbed by the efferent ducts and initial segment (Crabo and Appelgren, 1972; reviewed in Ilio and Hess, 1994). ACH did not cause distention of the efferent duct tubules at the 6 h time point when epididymal damage was observed although these vessels are relatively thin-walled. Damage and distention of the efferent ducts ultimately occur at higher ACH dose levels with longer exposure periods (Cooper et al., 1974Go); thus, our data suggest that damage to the efferent ducts occurs subsequent to the damage in the proximal initial segment tubules.

In addition to epididymal toxicity, the sperm is also a target of ACH toxicity. ACH inhibits sperm glycolytic enzymes (primarily glyceraldehyde-3-phosphate dehydrogenase) via a metabolite, 3-chlorolactaldehyde (Cooney and Jones, 1988Go; Stevenson and Jones, 1985Go). Inhibition of G3PDH results in a futile substrate cycle in the glycolytic pathway with a resultant depletion of the spermatozoa's intracellular ATP stores (Ford and Harrison, 1985Go; 1986Go). Insufficient energy for normal sperm function is theorized to be responsible for ACH-induced changes in fertility since sperm are dependent on glycolytic ATP production (Ford and Rees, 1990Go). Sperm motility (Slott et al., 1995Go; Toth et al., 1991Go), sperm capacitation (Dravland and Meizel, 1981Go), and sperm-oocyte penetration (Jelks et al., 2001Go) have been shown to be affected.

It has been theorized that sperm are uniquely susceptible to ACH-induced G3PDH inhibition in the rat (Jones, 1978Go). In support of this is the observation that anti-fertility effects are noted without any other overt signs of toxicity in the rat. Since the response to ACH only occurs in some species and in specific tissues, Jones (1983) theorized that the enzyme responsible for the oxidation of ACH to (S)-3-chlorolactaldehyde, the proposed active inhibitor of G3PDH, may only be localized in mature sperm of some species (Jones, 1983Go). A sperm-specific isoform of G3PDH called GAPD-S has also been identified (Welch et al., 1992Go) and proposed to be the form inhibited by (S)-3-chlorolactaldehyde. However, the selectivity of G3PDH activity inhibition by 3-chlorolactaldehyde is not well documented.

We investigated G3PDH inhibition in multiple organs as well as sperm following in vivo administration of ACH (50 mg/kg po) since G3PDH activity in other tissues following exposure to ACH and precursors of ACH has been contradictory (Ford and Waites, 1981Go; Kaur and Guraya, 1981Go). In our study, G3PDH activity was clearly suppressed 3 h following ACH-exposure in sperm, epididymis, kidney, and liver and remained suppressed for the 24 h of observation. However near-control values were maintained in testis and brain after 24 h. To explain the organ and sperm-specific inhibition by ACH, it is possible that (1) the lactaldehyde metabolite circulates in the blood, reaching multiple tissues, but penetration into brain and testes may be limited or (2) that liver, kidney, and epididymis as well as sperm possess the enzymes capable of metabolizing ACH to the (S)-3-chlorolactaldehyde, and (3) these tissues contain a G3PDH isoform that can be inhibited by the (S)-3-chlorolactaldehyde.

G3PDH inhibition, presumably via on-site metabolism of ACH to the lactaldehyde, is consistent with the presence of metabolic enzymes within the tissues. The liver and kidney are organs rich in metabolizing enzymes and were sensitive to G3PDH inhibition. The testis and brain possess relatively lower metabolic capabilities and appeared insensitive to G3PDH inhibition, thus may not have the required enzyme to metabolically activate ACH to the lactaldehyde. The data are suggestive that the epididymis is an organ with metabolic capabilities. Indeed, recent data has indicated that cytochrome P450 2E1 can be immunolocalized in the efferent ducts and corpus epididymis (Jelks and Miller, 1998Go).

Little is known about the enzyme responsible for converting ACH to (S)-3-chlorolactaldehyde. Stevenson and Jones (1985) identified the enzyme isolated from boar spermatozoa as an NADP+ dependent dehydrogenase, responsible for converting glycerol to glyceraldehyde. Other plausible candidates for ACH metabolism are alcohol dehydrogenase and the cytochrome P450 2E1, which also possesses alcohol dehydrogenase activity. Recently, in vivo administration of the alcohol dehydrogenase inhibitor pyrazole in the rat was shown to increase the urinary excretion of ACH suggesting that alcohol dehydrogenase may be in part responsible for ACH metabolism (de Rooij et al., 1996Go).

In conjunction with G3PDH activity measurements, G3PDH was immunolocalized within the epididymis and found to be localized in the efferent duct epithelium and the initial segment microvilli. Changes in initial segment histopathology were observed where G3PDH is present. Since G3PDH activity is inhibited in the epididymis at 3 h after ACH administration, these data raise the possibility that G3PDH in the initial segment cells may serve a specific function that when disrupted by ACH leads to epididymal toxicity. The functional role of G3PDH in these areas of the epididymis is not known. Moreover the lesion appears confined to only the proximal initial segment in the first 24 h following ACH administration although G3PDH is found associated throughout the initial segment microvilli. Taken together, cell-specific localization of G3PDH does not offer an explanation for site-specific damage.

As previously stated, the efferent ducts and initial segment epididymis play a crucial role in fluid reabsorption via endocytosis as well as passive diffusion (Ilio and Hess, 1994Go). G3PDH has recently been identified to play a role in endocytosis, specifically G3PDH has been identified as a microtubule-associated protein essential for proper endocytotic function (Robbins et al., 1995Go). We investigated changes to tubulin in ACH-mediated epididymal damage since microtubule-based vesicle transport facilitates endocytosis. ß-Tubulin was distributed throughout the epithelium of the efferent ducts and adluminal portions of the initial segment and caput epithelium consistent with a role in secretory and absorptive processes. ß-Tubulin immunostaining appears slightly disrupted 3 h following a single dose of ACH, prior to onset of histological changes, and becomes seriously disrupted and absent in the 24 h following. The early effect on ß-tubulin does support the hypothesis that microtubule-dependent processes may be affected by ACH. Alternatively, microtubule disruption may be secondary to damage of the epithelium and the sequelae of another toxic mechanism induced by ACH. The function of G3PDH with respect to tubulin and endocytosis within the epididymis remains to be determined since strict co-localization of G3PDH and ß-tubulin was not found although both were found to be most abundant in the initial segment.

In summary, histologic damage was observed in a discrete area of the epididymis, the proximal initial segment, within 6 h of ACH treatment. Epididymal G3PDH activity was inhibited prior to the appearance of the epididymal lesion. Whether inhibition of the enzyme is causally related to epididymal toxicity is unknown. The inhibitory effect of ACH on G3PDH activity was observed not only in sperm and the epididymis but also in liver and kidney. Since there is differential sensitivity of sperm and various organs to ACH toxicity, and G3PDH inhibition occurs in sperm as well as the epididymis and other organs, the functional importance of G3PDH could differ between cell types. Overall, these data do not support the hypothesis that a sperm-specific G3PDH isoform is uniquely inhibited by ACH.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (530) 752-3394. E-mail: mgmillersears{at}ucdavis.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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