Long-term effects on male reproduction of early exposure to common chemical contaminants in drinking water

D.N.R. Veeramachaneni1,, J.S. Palmer and R.P. Amann

Animal Reproduction and Biotechnology Laboratory, Department of Physiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1683, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We evaluated sequelae to early exposure of male rabbits to drinking water containing chemicals typical of ground water near hazardous waste sites. The mixture (p.p.m. at 1x) was 7.75 arsenic, 1.75 chromium, 9.25 lead, 12.5 benzene, 3.75 chloroform, 8.5 phenol and 9.5 trichloroethylene. Dutch-Belted does received mixture at 0x (deionized water; control), 1x or 3x as drinking water from day 20 pregnancy through weaning. Exposure of individual males (7–9/treatment) continued until 15 weeks (adolescence); then, all males received deionized water. At 57–61 weeks of age, ejaculatory capability and seminal, testicular, epididymal and endocrine characteristics were evaluated. At 10 opportunities with a female teaser, all seven control males ejaculated every time, but 12 of the 17 treated males failed to express interest, achieve erection and/or ejaculate on one to five occasions; four of the 12 accomplished ejaculation with a second male teaser. Total spermatozoa/ejaculate and daily sperm production were unaffected. However, treatment caused (P < 0.03) acrosomal dysgenesis and nuclear malformations. Baseline serum concentrations of LH were lower, but with borderline significance (P = 0.05). Testosterone secretion after exogenous human chorionic gonadotrophin (P < 0.04) was low. Thus, even at 45 weeks after last exposure to drinking water pollutants, mating desire/ability, sperm quality, and Leydig cell function were subnormal.

Key words: acrosomal dysgenesis/drinking water pollutants/seminal quality/sexual dysfunction/testicular function


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Exposure of adults to toxic agents, either in the workplace or home environment, can cause sexual dysfunction, infertility or sterility (Christian, 1983Go; Lamb and Foster, 1988Go; Working, 1989Go; Wyrobek et al., 1997Go). Exposure to workplace hazards can be eliminated or controlled. However, causal exposure to complex mixtures of toxicants typically is undetected and even when detected may be difficult to eliminate. Low-level contamination of drinking water is one example of this problem. Because reproductive function in humans is much more vulnerable to effects of environmental pollutants than that in common animal models (Amann, 1982Go, 1986Go; Working, 1989Go) or sentinel animals, it is important to consider reproductive sequelae of chronic exposure of humans to low-level contamination of drinking water. Surprisingly, little is known about chronic exposure of infants to relatively low levels of a mixture of common chemical contaminants.

Long-term sequelae of infantile exposure to certain toxic agents could remain undetected for many years until manifestation as idiopathic subfertility or testicular pathology in adult men. Such sequelae might be one cause of a generally perceived decline in reproductive capacity of the human male over the past 4–5 decades (Carlsen et al., 1992Go; Andersen et al., 2000Go).

We postulated that exposure of boys to low concentrations of environmental toxicants during infancy, as in drinking water, can alter development of reproductive function, but remain undetected until after sexual maturity or attainment of advanced age. If this hypothesis is correct, it would (i) heighten efforts to identify aetiological agents acting long before clinical manifestation of infertility, and (ii) provide a partial explanation for sub-fertility of many human males.

We selected a rabbit model to test the hypothesis. Rabbits have a long quiescent period before puberty, mimicking human development better than rodents (Amann, 1982Go). In rabbits, at about day 20 of gestation, morphological and biochemical changes indicative of endocrine activity occur in fetal interstitial (future Leydig cells) and indifferent supporting (future Sertoli cells) cells (Bjerregaard et al., 1974Go; Catt et al., 1975Go). Final differentiation of Leydig cells starts around 7 weeks postnatally (Gondos, 1975), the first spermatids are formed at ~12–13 weeks, and the first mature spermatids are released near 15 weeks of age (Amann and Lambiase, 1967Go; Gondos et al., 1973Go; Gondos, 1974Go). Thus, although the infantile period in rabbits is much shorter than that in humans, it is of a relative length comparable with that in humans and is of sufficient length to allow exposure to a potential reproductive toxicant. Furthermore, use of rabbits (in contrast to rodents) facilitates multiple evaluations of sexual capacity and seminal quality. Herein we report long-term reproductive sequelae of prenatal plus infantile exposure of male rabbits to a mixture of chemicals in drinking water.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The experiment compared reproductive parameters for three groups of male rabbits, exposed starting on day 20 of gestation through parturition, infancy and adolescence (15 weeks of age) to a 0x (control), 1x or 3x concentration of a chemical mixture in deionized drinking water (prepared from city drinking water). Treatment was not begun until gestation day 20 to ensure that the major organogenesis was not perturbed. This exposure window also corresponds with the major events associated with differentiation and development of the male reproductive tract. From 16 weeks postnatal to 61 weeks of age, when they were terminated, all males received deionized water.

The chemical mixture
The chemical mixture, as p.p.m. for the 1x concentration, was: 7.75 arsenic, 1.75 chromium, 9.25 lead, 12.5 benzene, 3.75 chloroform, 8.5 phenol, and 9.5 trichloroethylene. Concentrations of metals were for the ionic forms, which were provided as arsenic trioxide, chromium chloride hexahydrate, and lead acetate trihydrate. This mixture represented seven of the most frequently detected ground water pollutants (Yang et al., 1989Go). At the 1x concentration, arsenic, lead, and phenol were present at ~0.25x and the other four chemicals at ~2.5x the average Environmental Protection Agency (EPA) survey value for ground water samples taken at or near hazardous waste disposal sites in the USA (Yang et al., 1989Go). Because of substantially deviant outlier values in the EPA survey data, the 1x concentrations were thought to best estimate a typical baseline mixture.

The general procedures for mixing inorganic and organic stock solutions and the dosing solutions have been reported (Yang et al., 1989Go). To minimize loss of volatile organics, stock solutions were prepared every 2 days and individual water bottles were refilled daily. Amber, borosilicate glass water bottles were fitted with a fluorocarbon septum containing a stainless steel sipper tube, equipped with balls to minimize water dripping. Bottle systems were steam-cleaned twice weekly. Water consumption by each male was measured every second week during the last 10 weeks of infantile exposure to contaminated water.

Animals
Forty nulliparous, 6 month old, female Dutch-Belted rabbits were obtained from a specific-pathogen-free breeding colony free of Pasteurella multocida, Bordetella bronchiseptica, Encephalitozoon cuniculi, and Eimeria stiedae. Animals were housed in standard stainless-steel cages and exposed to a 14:10 h light:dark photoperiod at ~20°C and ~40% humidity, in a facility with animal care and use programmes approved by the Association for Assessment and Accreditation of Laboratory Animal Care, International. Animals were fed certified rabbit ration (#7009; Harlan Teklad, Madison, WI, USA), provided water ad libitum from hanging water bottles (see above), and bedded with kiln-dried aspen shavings ~3 cm below the stainless-steel mesh floor.

After acclimatization for a month, each of the 40 female rabbits was artificially inseminated with 20x106 spermatozoa, pooled from semen collected from six contemporaneous males. Ovulation was induced by an i.m. injection of 10 µg gonadotropin-releasing hormone (GnRH). Females were palpated for pregnancy 19 days after insemination, and at least 10 pregnant does were randomly allocated to one of the three treatment groups. Starting on day 20 after insemination, the water supply of each pregnant doe was changed from city water to water representing the assigned treatment.

On day 28 of gestation, females were provided with nest boxes. After parturition, typically on day 30 after insemination, rabbit pups were allowed to nurse their dam and self-wean at 4–5 weeks of age. At 6 weeks of age, one male pup was randomly selected to represent each litter. This provided seven to nine male pups per treatment group. Pups were individually caged, received the appropriate treated water through 15 weeks postnatal, and received deionized water from 16 to 61 weeks of age. Body weights were recorded biweekly.

Evaluation of sexual capacity and semen characteristics
Rabbits were trained for semen collection using one of several female teasers and an artificial vagina (Bredderman et al., 1964Go), and one ejaculate was collected every second day between 57 and 60 weeks (total 10 ejaculates/animal). Every time an ejaculate was collected, sexual behaviour and capacity were evaluated by monitoring the outcome and recording: (i) subjective evaluation of interest in mounting and copulation; (ii) number of mounts to accomplish ejaculation; and (iii) time between introduction of a female teaser and ejaculation or cessation of the session. After a randomly selected female (one of six) was placed into the male's cage, 3 min were allowed for the male to accomplish ejaculation. If a male failed to express interest or accomplish ejaculation within 3 min with the female teaser, she was removed and a second teaser (a male) was introduced and sexual behaviour monitored for an additional 3 min.

Volume (after discarding any gel) of each ejaculate was recorded to the nearest 0.1 ml, from graduations on the collection tube (inaccurately graduated tubes had been culled). A 50 µl aliquant of semen was diluted in 1 ml of buffer (10 mmol/l Tris, 0.15 mol/l NaCl, 1 mmol/l EDTA, 0.05% Triton-X) and refrigerated for determination of sperm concentration. Concentration of spermatozoa was determined by haemocytometer and total spermatozoa per ejaculate was calculated. An aliquant of semen was fixed in phosphate-buffered formal saline for evaluation of morphological features of spermatozoa, and the remaining semen was fixed in 4% glutaraldehyde in 0.1 mol/l sodium cacodylate. The first set of samples was evaluated as wet smears, in a treatment-blinded manner, using an Olympus phase-contrast microscope. Two hundred spermatozoa/ejaculate were evaluated for normalcy of the acrosome, head, mid-piece and principal piece, and retention of a proximal or distal cytoplasmic droplet (criteria similar to those for bulls) (Harasymowycez et al., 1976Go; Veeramachaneni et al., 1986Go). The second set of samples was embedded in Poly/Bed 812 (Polysciences Inc., Warrington, PA, USA) and thin (60–80 nm) sections, stained with uranyl acetate and lead citrate, were examined using a Jeol JEM-1200EX transmission electron microscope (Veeramachaneni et al., 1993Go). Evaluation was limited to characterization of acrosomal and nuclear lesions, because examination of sufficient spermatozoa for quantitative analysis is impractical using transmission electron microscopy.

Responsiveness of the pituitary-gonadal endocrine axis
During week 61, a blood sample was taken from each male (by jugular puncture) for later measurement of basal serum concentration of LH. Immediately thereafter, GnRH was injected (10 µg i.m.) to test normalcy of the anterior pituitary gland to secrete LH, as evidenced by LH concentration in a blood sample taken 20 min later. It was anticipated that injection of this pharmacological dose of GnRH would result in at least a 3-fold increase in circulating LH. Two days after GnRH challenge, a baseline blood sample was taken to quantify testosterone and immediately thereafter HCG was injected (50 IU i.m.) to test normalcy of Leydig cell function as evidenced by testosterone concentration in a blood sample taken 60 min later. It was expected that exogenous HCG would result in at least a 3-fold increase in circulating testosterone.

Tissue sampling
One or 2 days after the HCG injection, late in week 61, each rabbit was weighed and euthanized. The hypothalamus was removed by incisions caudal to the mamillary bodies, through the preoptic area rostrally, and in the hypothalamic sulci laterally. A cut was made just dorsal to the anterior commissure. The hypothalamus was weighed, placed into methanol:formic acid (9:1, v/v) (Nett and Adams, 1977Go), and stored at –80°C. The pituitary gland also was removed, weighed and frozen at –80°C until extracted and assayed. Reproductive organs were removed and examined for gross abnormalities. The testes and epididymides were individually weighed. The right testis was decapsulated, and parenchyma was weighed and frozen at –80°C for determination of daily sperm production. The right epididymis was frozen at –80°C for determination of epididymal sperm reserves. The left testis was sliced in two, and one piece was further cut into 2x2x2 mm pieces, fixed in 4% glutaraldehyde and processed for transmission electron microscopy (Veeramachaneni et al., 1993Go).

Hormone assays
Each hypothalamus was homogenized in its storage medium and the concentration of GnRH was measured by an established radioimmunoassay (Nett and Adams, 1977Go). The anterior pituitary gland was extracted and assayed for LH using an established radioimmunoassay (Matteri et al., 1987Go). Serum LH and testosterone were measured using standard radioimmunoassays (Berndtson et al., 1974Go; Matteri et al., 1987Go). All assays were validated for rabbit tissues. All samples for each hormone were assayed as a single batch and intra-assay coefficients of variation were <6.75%.

Daily sperm production and epididymal sperm reserves
To determine daily sperm production and cauda epididymal sperm reserves (Amann and Lambiase, 1969Go), frozen tissues were thawed and homogenized in a semi-micro Waring blender using 50 ml buffer [0.145 mol/l NaCl containing 4 mmol/l NaN3 and 0.05% (v/v) Triton X-100]. The number of homogenization-resistant (elongated) spermatid nuclei in each suspension was determined by haemocytometer counts. Data for the testis were converted to daily sperm production using a time divisor of 5.35 days (Amann et al., 1974Go). The efficiency of sperm production was calculated by expressing daily sperm production on a per gram of testicular parenchyma basis. Data for the cauda epididymidis were expressed as total sperm nuclei per organ.

Statistical analyses
Data for each attribute were subjected to one-way analysis of variance (Minitab release 12.23; State College, PA, USA) with water treatment as the factor and rabbits within-treatment group providing replication. Effects of 1x and 3x treatments were compared against the control (0x) using Dunnett's two-tailed test with {alpha} = 0.05. Also, concentrations of serum LH before and after injection of GnRH and of serum testosterone before and after injection of HCG were compared using a 3x2 model, isolating effects of water treatment and before or after administration of secretogogue.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Masses of water contaminants transferred to fetuses or nursing pups are unknown. Consumption of water by rabbit does on day 25 of gestation was similar for 0x and 1x females (137 and 154 ml), but ~15% lower for 3x does (119 ml). When measured during weeks 8, 10, 12 and 14, average water consumption by rabbit pups in the 1x group was reduced by 9-17% relative to consumption by 0x rabbits. For the 3x group the reduction ranged from 24 to 45%. Thus, the difference in exposure of males in the 3x and 1x groups to the toxic agents was ~2.2-fold, not 3-fold. Consumption of chemicals is summarized in Table IGo. The declines in water consumption did not affect growth as indicated by similar body weights (P > 0.2) for the 0x, 1x and 3x groups at 16 weeks (end of the exposure) and 61 weeks (termination of the experiment); the weights were 1.74, 1.62 and 1.64 kg at 16 weeks, and 2.21, 2.14 and 2.10 kg at 61 weeks (pooled SD were 0.15 and 0.23 kg respectively).


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Table I. Average amounts of chemicals (mg/kg body wt/day) consumed at various periods of exposure
 
Sexual behaviour and capacity
There was an adverse effect of early exposure to treated water on sexual behaviour evaluated between 42 and 45 weeks after cessation of treatment (57–60 weeks of age). This was evident in both subjective and objective observations. Excluding episodes culminating in failure (see below), the interval between placement of a teaser into the male's cage and ejaculation averaged <30 s for each of seven control males (Table IIGo). However, response time averaged >30 s for eight of nine 1x and seven of eight 3x males.


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Table II. Effects of prepubertal exposure to a chemical mixture on sexual behaviour and capacity at 57–60 weeks of age
 
Failure to ejaculate during 6 min exposure to teaser animals occurred only with 1x and 3x rabbits. Although each of the seven control males ejaculated promptly during each of 10 opportunities with a female teaser, 12 of the 17 treated males failed to express interest, achieve erection and/or ejaculate on one to five occasions during 3 min exposure to a female teaser (Table IIGo). Eight of these 12 males failed to accomplish an ejaculation during subsequent exposure for an additional 3 min to a male teaser. Collectively, this constituted 19% (32/170) failures to achieve ejaculation with a female teaser and 12% (20/170) with either a female or a male teaser. Eight of these 20 failing episodes were due to lack of sexual interest in the teaser and 12 were due to lack of penile erection.

Neuroendocrine axis
Endocrine measurements at 61 weeks of age (Table IIIGo) revealed that hypothalamic content of GnRH was not affected by early exposure to chemicals in water; although not statistically significant, the average values in treated animals were 27–44% lower than in controls. Content of LH in the anterior pituitary gland also was unaffected by treatment. However, there was a lower baseline concentration of LH in blood serum for 1x or 3x rabbits but the significance level was borderline (P = 0.05) (Table IIIGo). Injection of GnRH evoked a marked increase (P < 0.01) in concentration of serum LH, but the response was ~10 ng/ml for all three groups of males. Clearly, water treatment did not abolish responsiveness of the anterior pituitary gland to hypothalamic stimulation via GnRH.


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Table III. Effects of prepubertal exposure to a chemical mixture on the neuroendocrine axis at 61 weeks of age
 
In contrast to production and secretion of LH by the anterior pituitary gland, production and secretion of testosterone by Leydig cells of the testis were affected (P = 0.04) by water treatment. Unexpectedly, the mean baseline testosterone concentration for 1x rabbits was greater than those for 0x or 3x males (Table IIIGo). Despite this high baseline value, 1 h after injection of HCG serum testosterone was increased in all groups (P < 0.01). The magnitude of the increase in 1x and 3x males was less (P < 0.05) than that in 0x males. The interactive effect of water treatment and sampling (before or after HCG) affected (P < 0.05) concentration of serum testosterone. We concluded that the increase in blood concentration of testosterone after injection of HCG was attenuated by prepubertal exposure to the chemical mixture.

Production of spermatozoa
Exposure to chemical mixture did not affect spermatogenesis on a quantitative basis as evidenced by similarity among the 0x, 1x, and 3x groups for testis parenchyma weight, daily sperm production per gram of testis, and total number of spermatozoa per ejaculate during every-other-day collection (Table IVGo). Further, the number of spermatozoa in the cauda epididymis was not affected by treatment.


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Table IV. Effects of prepubertal exposure to a chemical mixture on sperm production and semen characteristics at 57–60 weeks of age
 
Prepubertal exposure of male rabbits to contaminated water, however, did affect quality of sperm produced 45 weeks later. This was evident by light microscopic evaluations of sperm morphology (Table IVGo, lower portion). There were almost twice as many abnormal spermatozoa for the 1x or 3x group versus 0x group, 22 and 25 versus 12% (P < 0.03). The >2-fold increase (P < 0.03) in acrosomal-nuclear defects accounted for much of this change (Table IVGo); the morphogenesis of these defects is described in detail in next section.

Nature of induced acrosomal-nuclear defects
Defects in morphogenesis of spermatozoa in animals exposed to this chemical mixture frequently involved both the acrosome and nucleus. At the light microscopic level, acrosomal lesions appeared as a knob or cyst at the apex or on the lateral aspect of the rostral end of the sperm head (Figure 1a,bGo). The affected nuclei were misshapen and often two nuclei were fused.





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Figure 1. Phase contrast (a, b) and transmission electron (c) micrographs of ejaculated spermatozoa from a 61 week old rabbit exposed to 1x chemical mixture in utero and during the first 15 weeks of life. (a, b) Note presence of knobbed/cystic acrosomes (arrow heads) and fused sperm heads (arrows). Bar = 10 µm. (c) Sagittal section of two fused sperm heads (similar to the profile seen in b) sharing a common acrosome. Note dysplastic acrosome shared between two spermatozoa (arrows) displaying vesiculation and inclusions (arrow heads). Bar = 500 nm.

 
Ultrastructurally, the knobbed acrosomes were characterized by dysplastic spread of acrosomal matrix, resulting in vesiculation enclosing cytoplasmic inclusions (Figure 1cGo). While some of these inclusions appeared to have resulted from incorporation of excess Golgi-associated vesicles (Figure 2aGo), most inclusions contained cytoplasmic constituents which resembled residual cytoplasm of a differentiating germ cell (Figure 1cGo) and/or that of an adjacent Sertoli cell (Figure 2cGo). The vesicles were bounded by inner and outer acrosomal membranes and the plasma membrane of the spermatid (Figures 2b,cGo). In instances of fused sperm heads, an acrosome conjoining two (Figure 1cGo) or more (not shown) spermatozoa was always evident. This manifestation began with a common Golgi apparatus shared by two (Figure 3aGo) or more spermatids resulting in spread of acrosomal matrix all around affected spermatids (Figure 3bGo). As the spermatids condensed and elongated, depending on the orientation of involved nuclei relative to the shared acrosome, nuclear distortions resulting in malformations occurred (Figure 3cGo). Shared acrosomes frequently were dysplastic and vesiculated.





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Figure 2. Transmission electron micrographs of testicular sections from a 61 week old rabbit exposed to 3x chemical mixture in utero and during the first 15 weeks of life depicting morphogenesis of acrosomal vesiculation and nuclear inclusions. Bar = 1 µm. (a) Spherical spermatid in acrosome phase. Despite advanced spread of the acrosomal cap, the Golgi apparatus has not moved to the caudal aspect of the cell and prominent Golgi-associated vesicles are evident. Note membranous inclusions in the nucleus (arrow). (b) Condensing spermatids with acrosomal vesiculation (arrow head) and nuclear invasion (arrow). In the lower right cell, the vesicle contains membranous structures and is bounded by inner and outer acrosomal membranes juxtaposed to the nuclear membrane. The bulging causes distortion of sperm nucleus. In the upper left cell, a portion of the nuclear membrane is broken down with apparent invasion of the acrosomal matrix into the nucleus of the second spermatid (arrow). (c) Elongated spermatids shortly before spermiation. Note dysplastic acrosomes (arrow heads) with excess acrosomal material and inclusions. The cytoplasmic constituents of the inclusions resemble those of a Sertoli cell.

 




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Figure 3. Transmission electron micrographs of testicular sections from a 61 week old rabbit exposed to 3x chemical mixture in utero and during the first 15 weeks of life depicting morphogenesis of acrosomal sharing, vesiculation, and nuclear distortion. Bar = 1 µm. (a) Two spherical spermatids sharing a common acrosome (arrows). Note placement of the Golgi apparatus (large arrow) which, despite the advanced stage of acrosomal development, is still closely associated with the cranial aspect of the spermatids. (b) Spermatids with a condensing nucleus. As the spermatids sharing a common acrosome (arrow) condense, the acrosomal matrix surrounds both nuclei resulting in the development of two spermatozoa as a single unit. Note vesiculation and inclusions in the acrosome (arrow head). (c) Acrosomal-nuclear malformations. As nuclei such as those in (b) further condense, both elongated spermatids become distorted and misshapen because of the meandering, shared acrosomal matrix (arrow) and bulged acrosomal vesicles (arrow head).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Late gestational plus infantile exposure of male rabbits to the combination of water toxicants used in this study affected mating behaviour and testicular function 45 weeks after cessation of exposure. Clearly long-term changes occurred. Because we did not study perinatal or adolescent endocrine features or sexual behaviour or sperm morphology shortly after puberty (e.g. between 15 and 20 weeks of age), it is uncertain if the changes were expressed concurrent with final maturation of the reproductive system or after a longer interval of latency. Design of this study also precludes a conclusion as to what combination of the seven toxicants, or individual toxicant, caused the problems.

Certain chemicals in the mixture might have induced changes culminating in diminished sexual interest and impediment of ejaculation while others induced testicular changes evidenced as malformed spermatozoa. For example, adverse changes in the sperm count and morphology with minimal changes in endocrine parameters have been associated with increased blood levels of lead under experimental conditions in rabbits (Moorman et al., 1998Go). Similarly, elevated levels of lead in seminal plasma have been associated with significant impairment of sperm function and fecundity rates in humans (Benoff et al., 2000Go). Regardless, our study demonstrated that a combination of seven chemicals at concentrations typical of water near waste disposal sites in the 1980s adversely affects reproductive function in rabbits.

The chemicals used in this study were a subset of a more complex mixture of 25 ground water contaminants that was used in rodent studies (Chapin et al., 1989Go; Heindel et al., 1995Go). After 90 days of exposure of adult male mice to three different dose levels of this complex mixture (some concentrations were comparable with ours), no adverse effects on spermatogenesis were found (Chapin et al., 1989Go). These researchers conjectured that the lack of reproductive toxicity observed in their study may have been due to the relative brevity of the exposure compared with that necessary to reach biological equilibrium for some chemicals, actual ingested dose, or species tested. Using the same mixture and concentrations, it was found (Heindel et al., 1995Go) in continuous breeding (and exposure) studies using rats and mice that fertility was unaffected with only minimal reproductive effects. Neither of these studies examined spermatogenesis qualitatively. Their evaluations of sperm morphology were less comprehensive than ours. Changes similar to those reported herein might have escaped detection. Likewise, use of rodent models precludes objective monitoring and evaluation of sexual capacity, and therefore detection of possible sexual dysfunction.

The observation that fertility in rats is not sharply reduced by exposure to a chemical mixture in drinking water should not give false comfort. As detailed elsewhere (Amann, 1982Go, 1989Go), the efficiency of sperm production in men is at best ~15% that of a rabbit or other animal models. For a typical animal model, a <90% reduction in production of fertile spermatozoa probably would not lower fertility after natural mating; a reduction in percentage of spermatozoa with complete fertilizing capability could have the same effect as a reduction in actual number of cells produced. However, for a man producing only 50–60x106 spermatozoa per day, of which <25% might be normal, an 85% reduction to 8x106 spermatozoa, or <2x106 morphologically normal spermatozoa, per day probably would adversely affect fertility.

Extrapolating these data for mice, rats and rabbits to humans, it is unlikely that inadvertent exposure of boys or men to chemical mixtures at the low doses used in these studies was the sole cause of reported declines in seminal quality (Carlsen et al., 1992Go; Andersen et al., 2000Go) and sexual dysfunction (Laumann et al., 1999Go). Rather, if declines in male reproductive health (Skakkebaek, 1998Go) are in part associated with environmental agents, the problem might be a variety of other chemical contaminants such as pesticides, phthalates, and xeno-oestrogens (Colborn et al., 1993Go; Sharpe and Skakkebaek, 1993Go; Toppari et al., 1996Go; Gray et al., 1999Go, 2000Go). This does not mean, however, that third-trimester gestational and/or infantile exposure of human males to low concentrations of chemicals in water is without potential deleterious sequelae. Potential effects on sexual behaviour or seminal quality should not be trivialized.

It is unlikely, in the rabbits in this study, that reduced desire to mount and copulate or lack of penile erection were consequences of physical ailments involving the musculoskeletal system. Further, there has been no case of impotentia coeundi, or inability to copulate, among ~200 untreated males in our colony of Dutch-Belted rabbits during the past 10 years.

We assume that late prenatal or infantile exposure to this mixture permanently altered certain elements of the central nervous system (i.e. behavioural centres), possibly to an extent greater than it affected the hypothalamus. Further, the problem probably is not a consequence of insufficient circulating testosterone. All afflicted males were in the 1x or 3x groups, and baseline mean values for testosterone in these groups were greater than those of 0x rabbits (Table IIIGo). Perhaps non-endocrine-mediated physiological mechanisms involved in penile erection, such as nitric oxide cascades (Burnett et al., 1992Go), might have been affected. We concluded that the agent(s) in this mixture which acted on the central nervous system or neurons innervating the penile corpus cavernosum to cause depressed sexual capability 45 weeks later was not associated with insufficiency of circulating testosterone.

In other studies (Palmer et al., 2000Go; Veeramachaneni, 2000Go; Veeramachaneni et al., 2000Go) evaluating effects of perinatal exposure to dibromoacetic acid (a by-product of a standard water disinfection process) or vinclozolin (an anti-androgenic fungicide), individually- treated rabbits failed to show interest in females, or ejaculate, just as in the present study. Hence, it appears that a variety of agents might cause long-lasting impairment of sexual behaviour.

Both testosterone production by Leydig cells and function of the seminiferous epithelium apparently were altered by prepubertal exposure to this chemical mixture. Unfortunately, we did not study endocrine features in prenatal or infantile rabbits, or the numbers of Leydig cells and area of smooth endoplasmic reticulum in Leydig cells at 15 and 61 weeks. For these and other reasons, we consider our endocrine data as indicative of a terminal state, but uninformative in respect to proximate cause or temporal onset of any change. Nevertheless, Leydig cells in normal males have the capability to synthesize and secrete testosterone in amounts substantially in excess of amounts in typical episodic discharges. Therefore, a deficiency of testosterone in the milieu of the seminiferous tubules is unlikely. We did not study pituitary or serum concentrations of FSH, although it is known that a sharp reduction in FSH available to Sertoli cells adversely affects normalcy of spermatozoa produced (Sharpe, 1994Go). However, we consider it unlikely that a deficiency of FSH existed in these rabbits. Detailed endocrine evaluation of similarly treated rabbits would be appropriate.

Formation of morphologically normal spermatozoa requires complex interactions of Sertoli cells and the developing spermatid, using mRNA and proteins primarily produced while the latter cells were spermatocytes. Given that spermatozoa with acrosomal-nuclear defects were produced 45 weeks after cessation of ingestion of contaminated water, if one discounts deficiencies in trophic hormones (i.e. FSH and testosterone; see above), it is likely that a genetic change was responsible. We speculate that within a given rabbit some but not all of a specific cell type were permanently altered, and that the lesion is in Sertoli cells or stem-spermatogonia. Irrespective of whether DNA has an active role in sperm nuclear shaping, it has been postulated that it may act as a resistive force to shaping because of its viscous properties and its continuous linear structure (Meistrich, 1993Go). It is often stated (Russell et al., 1986Go) that, during spermiogenesis in normal animals, the acrosome assumes a shape dictated by nucleus. However, with reprogramming of function of Sertoli or germ cells by a reproductive toxicant, as postulated for this study, the converse is plausible and any aberration in acrosomal morphogenesis could affect nuclear morphogenesis. Although these two events can be inter-dependent, it cannot be ruled out that manifestations of reprogramming, due to toxicants, are direct and independent of each other.

Ultrastructural analyses revealed that there were clusters of abnormal spermatids reasonably close to other clusters of normal spermatids. However, we did not attempt three-dimensional reconstructions of seminiferous tubules to define the volume of affected areas of the seminiferous epithelium or distances among affected clusters. Examinations to exclude common envelopment of normal and abnormal spermatids within a given Sertoli cell(s) or presence of inter-spermatid bridges conjoining a cohort of cells might be possible, but ultimately techniques of molecular biology will be required to resolve the cause of the observed defects. Nonetheless, it is possible that inherent damage to either Sertoli cells or to differentiating spermatids (produced from affected stem-spermatogonia) could result in the acrosomal-nuclear defects observed in our study, since connections between the plasma membrane of the Sertoli cell and that of the spermatid adjacent to the acrosome are known to occur during spermiogenesis (Russell et al., 1988Go).

Sperm acrosomal-nuclear defects identical to the ones observed in this study have been observed in rats exposed to dibromoacetic acid (Linder et al., 1997Go). We have seen (Veeramachaneni and Sawyer, 1996Go) identical defects in subfertile/infertile stallions, that perhaps were unknowingly exposed to drinking water contaminants similar to the ones we tested in rabbits. These observations indicate that acrosomal-nuclear defects of spermatozoa are inducible and lasting. A `knobbed acrosome' defect, which is transmitted to male progeny, has been described for domestic animals (Saacke et al., 1968Go; Barth and Oko, 1989Go). This defect has many manifestations during spermiogenesis similar to those depicted herein. Is it possible that this defect arose from an agent-induced change rather than a random mutation? For example, N-ethyl-N-nitrosurea, a known mutagen, induces similar aberrations during spermiogenesis (Rinchik et al., 1995Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to C.Moeller and A.DeVincent for technical assistance. This research was supported by NIEHS Superfund program project P42-ES-05949 and the College Research Council of the College of Veterinary Medicine and Biomedical Sciences, Colorado State University.


    Notes
 
1 To whom correspondence should be addressed at: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO 80523-1683, USA. E-mail: rao{at}cvmbs.colostate.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on August 15, 2000; accepted on February 14, 2001.