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Published-Ahead-of-Print March 21, 2007, DOI:10.2164/jandrol.107.002550
Journal of Andrology, Vol. 28, No. 4, July/August 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.107.002550

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Chronic Exposure to Low Levels of Dibromoacetic Acid, a Water Disinfection By-product, Adversely Affects Reproductive Function in Male Rabbits

D. N. R. VEERAMACHANENI*, J. S. PALMER* AND G. R. KLINEFELTER{dagger}

From the * Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; and the {dagger} Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratories, Office of Research and Development, Environmental Protection Agency, Research Triangle Park, North Carolina.

Correspondence to: Dr Veeramachaneni, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683 (e-mail: D_N_Rao.Veeramachaneni{at}colostate.edu).
Received for publication January 19, 2007; accepted for publication March 5, 2007.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Four groups (minimum of 10/dose group) of male Dutch-belted rabbits were treated daily with dibromoacetic acid (DBA) via drinking water beginning in utero from gestation day 15 to adulthood; target dosages were 1, 5, and 50 mg DBA/kg body weight. Developmental, prepubertal as well as postpubertal reproductive sequelae were evaluated. One (out of 22), 2 (out of 32), and 1 (out of 21) male offspring in the 1, 5, and 50 mg DBA/kg groups were unilaterally cryptorchid. There were no significant differences in serum follicle-stimulating hormone, luteinizing hormone, and testosterone (basal concentrations or in response to exogenous gonadotropin-releasing hormone) in both prepubertal and adult rabbits. Chronic exposure to DBA adversely affected the mating abilities of some rabbits. The number of sperm produced was not affected, but spermiogenesis was disrupted, resulting in unique sperm acrosomal-nuclear malformations even at the 1-mg dose level. Concentrations of SP22, a specific sperm membrane fertility protein, in detergent extracts of ejaculated sperm were significantly lower (P < .05) in all DBA-treated groups compared with controls. The conception rates following artificial insemination of a constant number of sperm for 1, 5, and 50 mg DBA/kg groups were 55% (10/18), 65% (13/20), and 55% (9/16), respectively, vs 85% (17/20) for control group. Histologic lesions in testes characterized by spermatogenic arrest predominantly at the round spermatid stage, pyknosis of differentiating germ cells, and ultimate degeneration and desquamation leaving focal vacuolation in seminiferous epithelium were evident in DBA-treated groups. Thus, male rabbits exhibit reproductive toxicity with exposure to DBA during reproductive development at dosages as low as 1 mg/kg body weight.

     Key words: Testis, acrosomal dysgenesis, sexual dysfunction, infertility, SP22


Haloacetic acids are formed when disinfectants such as chlorine react with organics and bromide in source water. By weight, haloacetic acids are among the most abundant water disinfection by-products and are a concern from health and regulatory standpoints (Uden and Miller, 1983). Haloacetic acids, including dibromoacetic acid (DBA), have been shown to cause adverse effects on development and reproductive function in some laboratory animal models. Testicular toxicity following acute exposure to DBA (10–1250 mg/kg/d via oral gavage for 1–14 days) has been demonstrated in adult male rats with marked effects on sperm motility and morphologic features (Linder et al, 1994a,b). In a subsequent 70-day study, a dose-related decrease in the ability of males to sire multiple litters following oral gavage with 10, 50, or 250 mg/kg DBA was observed (Linder et al, 1995). The observed qualitative and quantitative alterations in sperm were later associated with defects in spermatogenesis, including delayed spermiation and the formation of atypical residual bodies (Linder et al, 1997, Linder et al, 1997). These histologic alterations were also observed in P0 and F1 males exposed to 250 and 650 ppm DBA (ie, 56 and 132 mg DBA/kg, respectively) in a 2-generation study (Christian et al, 2002).

A 2-week exposure of rats to the structurally related haloacid bromochloroacetic acid (BCA) resulted in significantly reduced fertility of proximal cauda epididymal sperm upon in utero insemination (Klinefelter et al, 2002). The reductions in fertility were significant at all doses; 8 mg/kg was the lowest dose tested. In this study, fertility was highly correlated with the levels of a specific isoform of a sperm membrane protein, previously identified as SP22, in detergent extracts of cauda epididymal sperm (Klinefelter et al, 1997; Welch et al, 1998). More recently DBA and BCA were shown to be dose and effect additive with respect to decreasing both fertilizing ability and the putative SP22 biomarker (Kaydos et al, 2004); low effect levels of 2 and 1.6 mg/kg were observed for DBA and BCA, respectively. Finally, exposure was shown to delay puberty in both male and female offspring (Klinefelter et al, 2004). This delay was attributed to continuous exposure throughout reproductive development rather than a specific window of susceptibility.

A study in mice demonstrated no significant effects in gametogenic potential following exposure to 5 or 50 mg DBA/kg/d from gestation day 15 throughout life (Weber et al, 2006), but effects have been observed at higher doses in the mouse (Tully et al, 2005), suggesting that the mouse is less sensitive to a haloacid insult than the rat. While the observed differences in sensitivity may be due to species-related differences in haloacid metabolism/disposition, additional research in a nonrodent species is indicated.

In this study, we sought to determine the effects of chronic exposure via drinking water (to simulate a natural situation) to relatively low doses of DBA in the rabbit. We selected a rabbit model because rabbits have a long infantile period before initiation of changes culminating in puberty, mimicking human development, whereas in rodents these changes start at birth (Amann, 1982; Veeramachaneni et al, 2001). Furthermore, use of rabbits (in contrast to rodents) facilitates multiple evaluations of seminal quality and sexual capacity including the first quantitative assessment of the sperm biomarker SP22 in an ejaculate. Herein, we report reproductive sequelae, measured before puberty and also after sexual maturity, in rabbits following chronic prenatal plus postnatal exposure to DBA.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Rabbit Husbandry

Six-month-old, specific pathogen-free, Dutch-belted rabbits were obtained from Myrtle's Rabbitry (Thompson Station, Tenn) and individually housed in standard stainless steel cages in the university animal care facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animals were treated according to the guidelines stipulated by Colorado State University's Institutional Animal Care and Use Committee. The rooms were maintained on a 12:12-hr light:dark cycle at approximately 19°C to 21°C and ~40% humidity. Rabbits were fed certified rabbit ration (#7009; Harlan Teklad, Madison, Wis).

After acclimatization for 4 to 5 weeks, 61 rabbit does were artificially inseminated using procedures previously described (Veeramachaneni et al, 2001, 2006). Each rabbit doe was given an intramuscular injection of 10 µg of gonadotropin-releasing hormone (GnRH; Calbiochem, La Jolla, Calif) to induce ovulation and was inseminated with 20 million spermatozoa pooled from semen collected from 11 bucks to increase biologic heterogeneity of the experimental subjects. Rabbits were palpated for pregnancy 14 days after insemination, and resulting pregnant does (n = 45) were randomly assigned to treatment groups for appropriate exposures (see below). On gestation day 28 nesting boxes were placed in the cages of pregnant does.

Dosing Regimen

Four groups (n = minimum of 10/dose group) of pregnant rabbits were treated daily with 0 (deionized water; control) or target dosages of 1, 5, or 50 mg DBA/kg body weight via drinking water. Individual water bottles were filled daily. Amber, borosilicate glass water bottles fitted with fluorocarbon septums containing stainless steel sipper tubes, equipped with balls to minimize water dripping were used. Bottle systems were steam-cleaned twice weekly.

DBA (CAS #631-64-1, lot #03807JS, purity 97%) was purchased from Aldrich Chemical Co (Milwaukee, Wis). Stock solutions were prepared by adding DBA to deionized water, bringing the pH to 6.8 to 7.4 using 1 N NaOH, and refrigerating. To minimize loss of chemical due to volatility, stock solutions were prepared twice weekly and dosing solutions were prepared daily. DBA concentrations in dosing solutions were computed as follows. Water consumption during a 24-hour period was recorded daily from gestation day 15 (when dosing began) until 6 weeks postpartum (when pups were weaned) in dams and twice weekly from 6 weeks to 12 weeks and once weekly from 12 weeks to 24 weeks in offspring. Body weights were recorded weekly. Dosing solutions were prepared based on the average body weight and average daily water consumption in each treatment group during the previous week such that the animals received 1, 5, or 50 mg DBA/kg body weight/d. This strategy ensured continuous delivery of doses very close to those intended.

Dosing of dams began on gestation day 15 and continued through parturition and weaning at 6 weeks postpartum. After weaning, rabbit pups were housed individually and DBA treatments continued until necropsy at 12 weeks (prepuberty; n = 10–22/dose group) or 24 weeks (postpuberty; n = 9–10/dose group). Thus, the offspring (experimental units) were exposed to DBA in utero beginning from gestation day 15, throughout nursing via dam's milk and then in drinking water. Rabbits in each dose group represented at least 6 different litters.

Evaluation of Mating Ability, Semen Characteristics, and Fertility

Beginning at 20 weeks, rabbits were trained for semen collection using an artificial vagina and one of several female rabbits as a teaser. Between 22 and 24 weeks, mating ability and semen characteristics were evaluated by attempting to collect 6 seminal ejaculates from each rabbit, 1 ejaculate every third day. All of these procedures are detailed in Veeramachaneni et al (2006).

Briefly, one of several female teasers was selected randomly for each attempt at semen collection. For each episode, mating ability was evaluated by monitoring the outcome and recording 1) sexual interest, 2) status of penile erection, and 3) time from introduction of teaser to ejaculation (reaction time). Once the teaser was introduced, evaluation continued until ejaculation or a maximum period of 180 seconds.

For each sample, seminal volume (after removing the gel) was recorded (to the nearest 0.05 mL), semen was mixed, and a 50-µL aliquot of semen was fixed in 950 µL of phosphate-buffered formal saline (93 mM NaCl, 35 mM Na2HPO4, 19 mM KH2PO4; 12.5% v/v formalin; Hancock, 1957) and stored at 4°C until evaluation. Sperm concentration (x 106/mL) was determined by hemocytometer (4 chambers/sample) and total sperm per ejaculate (x 106) calculated by multiplying volume times sperm concentration. Morphologic features of sperm were evaluated in a treatment-blinded manner using a light microscope equipped with differential interference contrast optics. Two hundred sperm were evaluated in wet smears of each ejaculate for abnormalities of the acrosome, head, midpiece, and principal pieces; retention of cytoplasmic droplet; and presence of residual cytoplasm using criteria previously established for rabbits (Veeramachaneni et al, 2001, 2006).

At the end of the seminal evaluation period at 24 weeks 2 additional seminal ejaculates were collected from each rabbit; one was used for quantification of SP22 (see below) and the other for fertility testing. Rabbit does of proven fertility were artificially inseminated with 20 million spermatozoa from each male, 2 does per male. Pregnancy outcome was documented as an indicator of fertility potential.

Quantification of SP22 on Ejaculated Sperm by Enzyme-Linked Immunosorbent Assay

Sperm were prepared for protein analysis as described by Klinefelter et al (2004). For each semen sample, seminal volume was measured after removal of the gel fraction and the semen was transferred to a 15-ml centrifuge tube and diluted with 10 mL of sperm isolation buffer (95 mL/L 10x Hanks balanced salts solution, 0.35 g/L NaHCO3, 4.2 g/L HEPES, 0.9 g/L glucose, and 10 mL/L 100x sodium pyruvate [pH 7.4]). Following centrifugation (3000 x g for 10 minutes), the pelleted sperm were washed twice more and then extracted for 1 hour at room temperature with 1 mL of 80 mM n-octyl-ß-D-glucopyranoside in 10 mM Tris (pH 7.2) containing freshly added phenylmethyl sulfonyl fluoride. Following a final centrifugation (10 000 x g for 5 minutes), the supernatant was removed and frozen (–70°C). Prior to SP22 enzyme-linked immunosorbent assay (ELISA), sperm extracts were thawed and each extract concentrated with 1 mM Tris buffer (pH 7.2) by 2 centrifugations (3000 x g for 45 minutes at 4°C) in Ultrafree-4 centrifugation filter units (Millipore Corp, Billerica, Mass). Protein concentrations were determined using a protein assay kit from Pierce (Rockford, Ill). Sample volumes equivalent to 10 µg of protein were lyophilized.

The SP22 ELISA was performed as described recently by Kaydos et al (2004). For this, 96-well tissue culture plates (Costar; Corning Inc, Corning, NY) were used. A standard curve was generated using serial dilutions of antigen (i.e., full-length rat recombinant SP22 [rSP22]; Klinefelter et al, 2002): 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 ng in 50 µL/well; all dilutions were in BupH phosphate-buffered saline (PBS; pH 7.2) (Pierce). For each sperm extract, 10 µg of lyophilized protein was diluted with BupH PBS and plated at 50 µL/well. Duplicate wells were used for both the SP22 standards and each sperm extract. The plates were stored overnight at 4°C to maximize antigen absorption. The following day unbound antigen was removed by inverting the plate and shaking gently. A blocking step consisted of adding milk protein (caseinate or dry milk powder) in Dulbecco PBS (DPBS; 150 µg/well), followed by incubation for 1 hour at 37°C. Sheep anti-rSP22 diluted 1:1000 in DPBS + 1% bovine serum albumin (BSA) was added (50 µL/well) and allowed to bind during incubation for 1 hour at 37°C. After 3 washes with DPBS + 1% BSA (200 µL/well), peroxidase-conjugated rabbit anti-sheep antibody (Pierce) diluted 1:500 in DPBS + 1% BSA was added (50 µL/well) and allowed to incubate for 1 hour at 37°C. After 4 washes with DPBS + 1% BSA, the peroxidase substrate 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (Pierce) was added (100 µL/well). The reaction was allowed to develop over a 15- to 20-minute period. Absorbance was read using FLUOstar Galaxy software (BMG Labtechnologies Inc, Durham, NC) at 405 nm excitation, and no emission, and values were expressed as the absorbance or optical density. Sample values were found to always fall within the range of the standard curve.

Tissue Collection and Processing

During the morning hours of the day before termination of the experiments (at 12 and 24 weeks), a GnRH challenge test was performed to determine the response of anterior pituitary gland to hypothalamic stimuli. After a blood sample was taken by jugular venipuncture, 10 µg of GnRH was injected intramuscularly. Two additional blood samples were collected 30 and 120 minute later. Serum was separated and samples stored at –20°C until they were assayed for gonadotropins and testosterone.

A day after the GnRH challenge tests rabbits were euthanized by CO2. Anogenital distance was measured and visceral and reproductive organs examined. Liver, kidneys, testes, epididymides, and accessory sex glands (prostate, vesicular, and bulbourethral glands) were evaluated for any gross abnormalities, removed, and weighed. Testes and epididymides were weighed individually. Each left testis and epididymis were processed for light and transmission electron microscopy (Veeramachaneni et al, 1986, 1993). Each testis was sliced into 2 pieces; one piece was fixed in Bouin fixative and the other in 4% (v/v) glutaraldehyde in 0.1 M sodium cacodylate. Each epididymis was fixed in Bouin fluid. Tissues fixed in Bouin fluid were embedded in paraffin, and those fixed in glutaraldehyde were postfixed in osmium tetroxide and embedded in Poly/Bed 812 (Polysciences Inc, Warrington, Pa). Thick sections (5 µm) were cut from paraffin-embedded tissues and stained with hematoxylin and eosin (light microscopy), and thin sections (60–80 nm) were cut from Poly/Bed-embedded tissues and stained with uranyl acetate and lead citrate (transmission electron microscopy).

Each right testis and epididymis were processed for quantification of daily sperm production and epididymal sperm reserves. The epididymis was dissected from the testis, cut into 2 segments (caput-corpus and cauda), weighed, frozen, and stored at –80°C. The testis was decapsulated, weighed, frozen, and stored at –80°C.

Daily Sperm Production and Epididymal Sperm Reserves

Daily sperm production and epididymal sperm reserves were determined by procedures described for rabbits (Amann and Lambiase, 1969) with minor modifications. Briefly, testicular parenchyma was thawed, minced on a watch glass, and homogenized for 1 minute in a semimicro Waring blender using 50 mL of buffer (0.145 M NaCl containing 4 mM NaN3 and 0.05% [v/v] Triton X-100). The number of homogenization-resistant elongated spermatid nuclei was determined without further dilution using a hemocytometer (4 chambers/sample). Daily sperm production was calculated using a time divisor of 5.35 days (Amann and Lambiase, 1969) and expressed per gram of testicular parenchyma. Caput-corpus and cauda epididymidis were thawed, minced on a watch glass, and homogenized for 3 minutes using 50 or 125 mL of buffer, respectively. Without further dilution, sperm nuclei were counted using a hemocytometer (4 chambers/sample). Epididymal sperm reserve was expressed per epididymal segment.

Histopathology of the Testis

Seminiferous epithelium and interstitium were evaluated using a Nikon Microphot-FXA light microscope and a JEOL-1200EX transmission electron microscope. For light microscopy, 100 randomly selected, essentially round (major diameter <1.5x minor diameter) cross sections of seminiferous tubules from each animal were classified into 1 of 8 different grades indicating relative severity of seminiferous epithelial abnormalities (Veeramachaneni et al, 1986, 2006). Briefly, grade 0 = normal, intact seminiferous epithelium; grade 1 = seminiferous epithelium with pyknotic germ cells and desquamation or focal vacuolation; grade 2 = seminiferous epithelium intermediate between grades 1 and 3; grade 3 = seminiferous epithelium with premeiotic germ cells and Sertoli cells; grade 4 = Sertoli cells only; grade 5 = no seminiferous epithelium, leaving only the basement membrane; grade 6 = seminiferous tubule with sperm stasis, sperm granuloma, or mineralization; grade 7 = fibrosis of the seminiferous tubule. A weight between 0 and 1, to reflect relative absence of germ cells, was assigned to each grade: 0, 1/4, 2/4, 3/4, and 4/4 to grades 0, 1, 2, 3, and 4 through 7, respectively. The degree of germinal epithelial lesions/loss (DGEL) was calculated by multiplying the percentage of tubules in each grade by the respective assigned weight and summing products. For electron microscopy, corresponding tissue sections were evaluated for any subtle lesions or abnormal cells.

Hormone Assays

Serum concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined by validated radioimmunoassay (Pau et al, 1986; Bodensteiner et al, 2004) using homologous rabbit reagents provided by Dr A. Parlow and the Pituitary Hormone Program, National Institutes of Health. The final dilutions of antisera for FSH and LH were 1:72 000 and 1:2.16 x 106, respectively. For a given hormone, all samples were assayed in a single assay and the intra-assay coefficients of variation were 11.4% for FSH and 4.9% for LH. Testosterone was extracted from each serum sample and assayed using a validated radioimmunoassay (Berndtson et al, 1974). All samples were assayed concurrently, and the intra-assay coefficient of variation was 4%.

Statistical Analyses

Differences in parameters were analyzed by one-way analysis of variance using the GLM procedure of SAS (Statview version 5.0; SAS Institute Inc, Cary, NC). If a significant difference (P < .05) was indicated, a Tukey-Kramer post hoc test was used as an indicator of differences among means. For fertility and morphologic data, the percentage values were transformed using arcsine of the square root of the percentage/100 to minimize inequality in variance and Fisher's protected least significant difference post hoc test was used to detect differences among means.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Delivered Doses

The calculated average (± SE) daily delivered doses for the postweaning rabbits were 0, 1.02 ± 0.03, 5.8 ± 0.55, and 61.08 ± 4.64 for the 12-week group and 0, 0.99 ± 0.02, 5.2 ± 0.23, and 55.57 ± 1.95 mg/kg body weight for the 24-week group (Table 1). These doses were very close to the targeted dosages (ie, 0, 1, 5, and 50 mg DBA/kg body weight). Water consumption in the high-dose exposure (50 mg/kg) was tested in a preliminary study and did not differ significantly from that of control in the definitive study (Table 1).


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  		Table 1. Average daily consumption of water and doses of DBA delivered*{dagger}
 

General Parameters

DBA did not cause any overt toxic effects in the pregnant dams. The average body weights for 0, 1, 5, and 50 mg/kg dose groups on gestation days 22 and 29 were 2.50 and 2.46 kg, 2.56 and 2.50 kg, 2.45 and 2.39 kg, and 2.54 and 2.42 kg, respectively.

DBA had no effect on growth rate or total body weight in either age group (Table 2). Testicular descent was impaired in DBA-treated rabbits: 1 (out of 22), 2 (out of 32), and 1 (out of 21) in 1, 5, and 50 mg DBA/kg groups, respectively, were unilaterally cryptorchid; in each instance, the retained testis was abdominal and was associated with a poorly developed gubernaculum. No other gross abnormalities of the reproductive tract were observed. Anogenital distances were similar between control and treated rabbits in both age groups. Except for an increased (P < .05) liver weight in the 50 mg/kg group of adult (24 weeks) rabbits, none of the organ weights differed from those of controls. No gross abnormalities of the viscera were observed.


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  		Table 2. Incidence of cryptorchidism, anogenital distances, and organ weights at necropsy in 12-week-old and 24-week-old rabbits exposed to 0, 1, 5, or 50 mg DBA/kg/d*{dagger}
 

Hypothalamo-Pituitary-Testicular Axis

In both prepubertal and adult animals, serum FSH and LH peaked at 30 minutes and declined by 120 minutes post-GnRH; there were no significant differences in basal concentrations or in response to exogenous GnRH (Table 3). Similarly, except for a significant increase (P < .05) in testosterone by 30 minutes post-GnRH in the 50 mg DBA/kg group, there were no differences in basal or post-GnRH concentrations of testosterone.


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  		Table 3. Reproductive hormone levels in 12-week-old and 24-week-old male rabbits exposed to 0, 1, 5, or 50 mg DBA/kg/d*{dagger}
 

Mating Ability

Chronic exposure to DBA adversely affected the mating ability of some rabbits. On at least 1 occasion out of the 6 episodes, 1 rabbit in each of the 1 and 5 mg DBA/kg groups and 2 rabbits in the 50 mg DBA/kg group failed to accomplish ejaculation (Table 4). One rabbit in the 1 mg DBA/kg group never evinced sexual interest in a female teaser and failed to achieve penile erection or ejaculation in all 6 episodes. The serum concentration of testosterone in this rabbit was not substantially different than the values of other rabbits in this group. Excluding episodes culminating in failure, the interval between placement of a teaser into the male's cage and ejaculation averaged 15 seconds for control males but averaged 29 seconds for the 50 mg DBA/kg males (Table 1); the averages for the 1 mg DBA/kg and 5 mg DBA/kg groups were not significantly different (P > .1) than that of the control group (Table 4).


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  		Table 4. Mating ability and fertility of 24-week-old rabbits exposed to 0, 1, 5, or 50 mg DBA/kg/d*{dagger}
 

Seminal Parameters and Spermatogenesis

Chronic exposure to DBA at the dose levels tested did not affect the number of sperm produced but impaired spermiogenesis, resulting in morphologically defective sperm (Table 5). Daily sperm production per gram of testis, sperm reserves in the epididymis, and total number of sperm per ejaculate for DBA-treated rabbits were not different (P > .1) than those of controls. However, the volume of the ejaculate in each of the DBA groups was higher (P < .05) than that of the control, resulting in lower (P < .05) sperm concentrations in the DBA treatment groups.


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  		Table 5. Semen parameters measured in 24-week-old rabbits exposed to 0, 1, 5, or 50 mg DBA/kg/d*{dagger}
 

The percentage of morphologically normal spermatozoa was reduced (P < .05) in all DBA groups compared with the control group (Table 5). Predominant sperm morphologic defects in DBA-treated rabbits involved the acrosome and nucleus. Nine percent to 18% of sperm were affected by one or both defects in DBA-treated rabbits vs ~2% in control rabbits.


Figure 1
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  		Figure 1. Acrosomal dysgenesis: shared acrosome. (A) Differential interference contrast micrograph of ejaculated sperm of a 24-week-old rabbit exposed to 5 mg of dibromoacetic acid. Note the dysplastic acrosome (arrow) shared between 2 sperm heads resulting in conjoined sperm. Inset light micrograph shows 2 fused spermatid nuclei sharing a common acrosomic vesicle (arrow). Scale bar = 10 µm. (B) Transmission electron micrograph of a testicular section depicting morphogenesis of conjoined sperm sharing a common vesiculated acrosome. Scale bar = 1 µm.

 
These defects included abnormal acrosomes characterized by cystic or knobbed appearance (Figures 1A and 2A and B). These dysplastic acrosomes occasionally spread around 2 or more spermatids in a cohort (Figure 1B), resulting in conjoined heads sharing a common acrosome (Figure 1B) and causing nuclear aberrations (Figure 2C). Occasionally clusters of such abnormal sperm were exfoliated along with Sertoli cell remnants (Figure 3A). At an electron microscopic level, it was evident that these cells were from a cohort of spermatids in which nuclear division was impaired during spermiogenesis (Figure 3B).


Figure 2
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  		Figure 2. Acrosomal dysgenesis: vesiculation and dysplasia. (A) Differential interference contrast micrograph of ejaculated sperm of a 24-week-old rabbit exposed to 5 mg of dibromoacetic acid. Note the presence of knobbed/cystic acrosomes (arrows). Dysplastic acrosomal membranes manifest as craters and protrusions on the sperm head (left). Scale bar = 10 µm. (B) Transmission electron micrograph of a testicular section depicting acrosomes enveloping elongating spermatid nuclei. Note excessive acrosomal matrix and vesiculation of the spermatid on the left. The cytoplasmic inclusions of the acrosomal cyst (arrow) resemble that of Sertoli cells. Scale bar = 0.5 µm. (C) Transmission electron micrograph of a testicular section depicting acrosomal/nuclear dysplasia. Note aberrant membranes (arrows) and irregular, fragmented nuclear chromatin (asterisks). Scale bar = 1 µm.

 

Figure 3
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  		Figure 3. Nuclear dysgenesis in spermatid cohorts: incomplete nuclear division. (A) Differential interference contrast micrograph of ejaculated sperm of a 24-week-old rabbit exposed to 5 mg of dibromoacetic acid. Note a cluster of sperm (arrow) embedded in a denuded seminiferous epithelial fragment. That these sperm are a cohort that failed to segregate during spermiogenesis is evident upon ultrastructural examination. Scale bar = 10 µm. (B) Light (inset) and transmission electron micrographs of a testicular section depicting impaired spermiogenesis. Note the connection (arrow) between 2 spermatid nuclei extending through the intracellular bridge of a cohort. As they condense and spermiate, these sperm cells appear to exfoliate still embedded in fragments of Sertoli cells, as seen in Panel A. Scale bar = 1 µm.

 


Figure 4
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  		Figure 4. Histogram depicting concentrations of SP22 measured by enzyme-linked immunosorbent assay in detergent extracts of ejaculated rabbit spermatozoa. Values are expressed as the mean integrated optical density (IOD). IOD is decreased significantly in each dibromoacetic acid treatment group; asterisk denotes significant difference from control (P < .05). n = 8 samples/group.

 
SP22 and Fertility

Concentrations of SP22 in detergent extracts of ejaculated sperm of DBA-treated groups were significantly lower (P < .05) than that in the control group (Figure 4). The conception rates for 1, 5, and 50 mg DBA/kg groups were 55% (10/18), 65% (13/20), and 55% (9/16), respectively, vs 85% (17/20) for the control group (Table 4). One male from the 5 mg DBA/kg group and 2 males from the 50 mg DBA/kg group failed to impregnate either test female.

Histopathology of Testes

Histopathologic changes in the testes representing grades 1 and 2 were found in all groups of rabbits (Table 6), including controls; the incidence was higher (P < .05) for grade 1 lesions in the 5 and 50 mg DBA/kg groups and for grade 2 lesions in the 1 and 50 mg DBA/kg groups. The higher incidence of these lesions in DBA-treated rabbits was mirrored by fewer (P <.05) normal (grade 0) seminiferous tubules (Figure 5) and higher DGEL (Table 6). Lesions in seminiferous epithelium were characterized by pyknosis of differentiating germ cells, formation of "multinucleated giant cells" (a syncytium of spherical spermatids), and ultimate degeneration and desquamation leaving focal vacuolation in seminiferous epithelium (Figure 5B). Exfoliated premature germ cells were evident in the excurrent ducts (Figure 5D and E) and seminal ejaculates. Spermiogenesis was arrested largely at the round spermatid stage in 1 of the 50 mg DBA/kg rabbits (Figure 5C and F).


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  		Table 6. Histopathologic changes in the seminiferous epithelium of 24-week-old rabbits exposed to 0, 1, 5, or 50 mg DBA/kg/d*{dagger}
 

Figure 5
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  		Figure 5. Photomicrographs of testicular and epididymal sections from 24-week-old control and dibromoacetic acid (DBA)-treated rabbits. (A) Seminiferous tubules in a control testis showing normal progression of spermatogenesis. (B) Seminiferous tubules in a 50-mg DBA rabbit showing extensive vacuolation in seminiferous epithelium resulting from exfoliation of germ cells. Multinucleated giant cells (spherical spermatid syncytia; arrows), which eventually get denuded, leaving vacuoles in seminiferous epithelium, are shown. (C) Seminiferous tubules in a 50-mg DBA rabbit showing impaired spermiogenesis. Spermiogenesis is largely arrested at the round spermatid stage. Exfoliation of syncytia of round spermatids (arrows) is seen. (D) Caput epididymidis of a control rabbit showing normal luminal contents. (E) Caput epididymidis of the 50-mg DBA rabbit (same as in Panel B) with luminal sperm containing aggregates of exfoliated immature germ cells. (F) Caput epididymidis of the 50-mg DBA rabbit (same as in Panel C) showing exfoliated premature germ cells in the lumina; no sperm are evident. Hematoxylin and eosin staining. Scale bars = 100 µm.

 


   Discussion
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 Abstract
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Chronic exposure of male rabbits to DBA beginning at differentiation of the fetal reproductive system impaired testicular descent, spermiogenesis, and sexual function as adults in some animals. There were no changes in anogenital distances or weights of the testes, epididymides, or accessory sex glands. This is consistent with a general lack of observed effects on serum FSH, LH, or testosterone either before or after GnRH stimulation. These findings also are consistent with the results of previous studies on haloacid exposure in rodents. Daily administration of 270 mg DBA/kg failed to alter serum testosterone (Linder et al, 1994b). More recent studies with DBA (Klinefelter et al, 2004) or BCA (Sloan et al, unpublished data) demonstrating significant body weight–independent delays in attainment of puberty (as indicated by preputial separation) did not detect any hormonal deficiencies in either serum or testicular interstitial fluid. While it is commonly accepted that a delay in puberty (ie, delay in preputial separation) is prompted by androgen insufficiency during early reproductive development, this may not always be the case. Notably, the definitive indicator of the onset of puberty in the male (ie, the first presence of sperm in the epididymis or ejaculate) has rarely been determined in studies involving laboratory animals.

DBA exposure compromised mating behavior significantly. Rabbits exposed to 50 mg DBA/kg required twice the time for ejaculation than control males. While all rabbits in the control group ejaculated at each opportunity, 10% of the males exposed to 1 or 5 mg DBA/kg and 22% of the males exposed to 50 mg DBA/kg failed at least 1 attempt. The observed deficit in mating behavior is consistent with an earlier rat study on DBA (Linder et al, 1995) in which 40%, 60%, 80%, and 90% of males exposed to 2, 10, 50, and 250 mg DBA/kg daily, respectively, for 70 days failed to successfully impregnate 2 females during 1 week of cohabitation. One of the rabbits exposed to 1 mg DBA/kg in the present study never ejaculated and in fact displayed no sexual interest and was unable to achieve an erection. Thus, it would seem that some underlying neurologic deficit following developmental exposure to DBA may account for the observed sexual dysfunction. Further support that early development is when the effect on sexual behavior is manifested comes from our observations that male rabbits exposed developmentally to chemical pollutants common in drinking water (Veeramachaneni et al, 2001) or pesticides such as vinclozolin (Veeramachaneni et al, 2006) manifest aberrant sexual behavior and function. That some of these chemicals indeed affect normal differentiation of sexually dimorphic areas of the brain that are important for regulating male sexual behavior is evident from our recent studies with vinclozolin (Bisenius et al, 2006).

Haloacid exposure consistently has been found associated with deficits in sperm quality. Toth et al (1992) reported a decline in motility and morphology of epididymal sperm following exposure of rats to dichloroacetic acid (DCA). Subsequently Linder et al (1994a,b, 1995) reported similar findings in rats exposed to DBA. More recently BCA has been shown to produce compromise in epididymal sperm motility and morphology (Klinefelter et al, 2002). In the present study, the percentage of morphologically normal sperm decreased significantly at each exposure level. Moreover, the magnitude of the changes in morphology were consistent across exposures, with 18%, 13%, and 16 % of the sperm displaying acrosomal defects in rabbits exposed to 1, 5, and 50 mg DBA/kg, respectively, compared with 2% in control males. Similarly, 13%, 9%, and 16 % of the sperm had nuclear defects compared with 2.5% for sperm in control males.

An interesting new effect described in the present rabbit study is the DBA-induced increase in volume of the seminal ejaculate resulting in an apparent decrease in sperm concentration (23%–29%) as total sperm per ejaculate in these animals was not different than that of controls. The accessory sex glands were heavier but not significantly in DBA-treated rabbits; perhaps this reflects increased secretion and/or biochemically altered constitution of seminal plasma components. Although it is not possible to delineate under the experimental conditions, if it is the impaired secretion of seminal plasma or morphologically defective sperm that contributed to impaired fertility (see below), the value of an animal model such as rabbit, which enables evaluation of seminal ejaculates, is obvious.

Using a very limited number of test females (n = 2) per male, insemination of a constant number of ejaculated sperm from DBA-treated rabbits resulted in reduced conception rates (55%–65% across treatment groups vs 85% in controls). Assuming that these conception rates hold with larger number of test females, the reduced conception rate for ejaculated sperm from male rabbits exposed to 1 mg DBA/kg is consistent with the observed reduction in fertility of cauda epididymal rat sperm following a 2-week exposure to 2 mg DBA/kg (Kaydos et al, 2004).

One aspect of sperm quality that now has been repeatedly associated with fertilizing ability is the SP22 protein and its level of expression on the sperm membrane (Klinefelter et al, 1997). It is well established that a unique testis-specific mRNA transcript for SP22 is expressed in pachytene spermatocytes and subsequent germ cells (Klinefelter, 2003). SP22 is quantified in sperm extracts either by quantitative 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis or by ELISA. In the present rabbit study, SP22 was quantified for the first time in extracts of ejaculated sperm by ELISA. Levels of SP22 were diminished significantly in each treatment group. It is likely that the reduced expression of SP22 together with the decreased number of morphologically normal sperm (discussed below) account, in part, for the reduced conception rates of male rabbits exposed to DBA. Haloacids have been shown to diminish SP22 in rodent epididymal sperm; diminished levels of SP22 were highly correlated with observed declines in fertility of rats exposed to BCA (Klinefelter et al, 2002) or a binary mixture of DBA and BCA (Kaydos et al, 2004).

Haloacid-induced deficits in sperm quality (ie, fertility, SP22, motility and morphology) likely result from an initial toxic insult on spermatogenesis. Over the years consistent histologic findings in testes have been noted in rodents exposed to haloacids. Delayed spermiation and/or retention of atypical residual bodies have been reported following exposure to DCA (Toth et al, 1992) and DBA (Linder et al, 1994a,b). Linder et al (1997, 1997) described a sequential appearance of lesions in germinal epithelium following 2- to 79-day exposure to 250 mg DBA/kg; delayed spermiation, atypical residual bodies, and spermatid fusion during the first 2 weeks were followed by misshapen spermatid nuclei, vesiculated acrosomes, and Sertoli cell vacuolation, culminating in marked atrophy of seminiferous tubules at 6 months (after 42 doses). The low-effect level in this study was 10 mg DBA/kg based on an increased incidence of spermatid retention. In a 2-generation drinking water exposure study of DBA (Christian et al, 2002), comparable alterations in spermatogenesis were seen again: atypical residual bodies and delayed spermiation were reported at 12 mg DBA/kg.

The present rabbit study is the first to characterize haloacid-induced alterations in spermatogenesis in a species other than the rat. Strikingly, at an exposure level as low as 1 mg DBA/kg, impaired spermiogenesis (acrosomal-nuclear dysgenesis) and an increased incidence of germinal epithelial lesions/loss were observed. In the adult rat study by Linder et al (1997, 1997), no abnormalities were detected at 2 mg DBA/kg.

Unique acrosomal-nuclear defects, characterized by sharing of an acrosome by 2 or more spermatids, acrosomal dysplasia, and nuclear malformations, observed in this study have also been found following shorter developmental exposures to common chemical contaminants in drinking water (Veeramachaneni et al, 2001) and fungicide vinclozolin (Veeramachaneni et al, 2006). In these studies, which were also performed in rabbits, acrosomal-nuclear defects continued to manifest long after cessation of exposure, suggesting that a permanent epigenetic change had been induced in stem-spermatogonia and/or Sertoli cells. It is not discernible if spermatogenic arrest and karyorrhectic or pyknotic changes in germ cell nuclei and consequent cell death are a direct effect of DBA on differentiating germ cells or an indirect effect via actions on Sertoli cells.

In summary, at the 3 dose levels studied in the present study, a no-effect level was not observed. In fact, the low-effect level of 1 mg DBA/kg was established on effects observed at multiple end points. Male rabbits exposed throughout reproductive development to 1 mg DBA/kg were found to have deficits in mating behavior, reduced conception rates, decreased concentrations of sperm in ejaculates, increased numbers of morphologically abnormal sperm in ejaculates, diminished SP22 levels in ejaculated sperm, and increased incidence of germ cell dysplasia and desquamation. It is reasonable to attempt to put these data in some context to human risk relevance by estimating a margin of exposure (MOE). An MOE simply attempts to relate an experimental exposure in which no observed adverse effect occurs to an estimated human exposure (ie, noeffect level/consumption). When a no-effect level is not established experimentally, it is computed by dividing the observed low-effect level by 10, yielding 0.1 mg/kg in this case. For adults, consumption is estimated based on the assumption that a 60-kg man consumes 2 L of water per day containing 20 µg DBA/L (10 times the average DBA level): (0.1 mg/kg/[0.02 mg/L x 2 L/60 kg] = 0.1/0.0007 = 142). Likewise, for a 3-kg infant drinking 1 L per day: (0.1 mg/kg/[0.02 mg/L x 1 L/3 kg] = 0.1/0.0067 = 14.9). Thus, the estimated no-observed–effect exposure in this study is 142 times the comparable exposure in men (driven by deficits in mating, sperm quality, etc) and 15 times for children (driven by compromised testicular descent). While this in itself may not be alarming, it also is not very comforting in view of the knowledge that all haloacids in drinking water appear to act similarly in the testis. Indeed, if the 9 prevalent haloacids in drinking water are considered collectively, one could essentially divide the above MOEs by a factor of 10. Moreover, it needs to be recognized that one's "cumulative" exposure to testicular toxicants occurs via myriad other sources (eg, pharmaceuticals, pesticide residues, alcohol).


   Acknowledgments
 
The authors would like to thank Colleen Kane, Jennifer Mayan, and Amanda McCulloch for help with dosing, Ty Higuchi for help with tissue collection, and Carol Moeller for formatting photomicrographs. Radioimmunoassays were performed by Dr K.-Y. F. Pau at the Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, Oregon.


   Footnotes
 
This research was supported by Environmental Protection Agency CO-OP CR826465-01-0.

Disclaimer: The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


   References
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 Materials and Methods
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 Discussion
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