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Effects of an Environmentally Relevant Organochlorine Mixture and a Metabolized Extract of This Mixture on Porcine Sperm Parameters In Vitro

PIERRE AYOTTE

From the ^* Centre de Recherche en Biologie de la Reproduction, Département de Sciences Animales; and the Unité de Recherche en Santé Publique, Centre Hospitalier Universitaire de Québec-CHUL, Département de Médecine Sociale et Préventive, Université Laval, Québec, Canada.

Correspondence to: Dr Janice L. Bailey, Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Québec City, Québec, Canada G1V 0A6 (e-mail: janice.bailey{at}crbr.ulaval.ca).

Received for publication August 1, 2008; accepted for publication January 2, 2009.

	Abstract

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Organochlorine chemicals are present in the environment worldwide; however, populations living in the Far North are particularly at risk because their traditional diets are mainly composed of contaminated animals (fish, seals, whales, and polar bears). It has been suggested that male fertility is globally declining, possibly because of chronic, low-level exposure to environmental contaminants. This study was designed to assess the effects on fresh sperm fertility parameters using the porcine model of 1) an environmentally relevant mixture of 15 organochlorines and 2) the metabolized extract of this mixture. In the first experiment, the organochlorine mixture (at relative concentrations of 10.5, 14.7, and 21 µg/mL polychlorinated biphenyls [PCBs]) reduced sperm total motility, progressive motility, and viability, and increased capacitation, spontaneous acrosome reaction rates, and cytosolic calcium levels, suggesting that the mixture alters the sperm membrane and is detrimental to sperm function. In the second experiment, the metabolized extract of this organochlorine mixture (at relative concentrations of 0.9, 1.8, 2.7, 3.6, and 4.5 µg/L OH-PCBs) tended to decrease only sperm total motility. In an in vitro porcine model, the mixture of organochlorines, as found in the Arctic food chain, was rapidly detrimental to sperm function at concentrations above environmental levels. In contrast, short and physiologically relevant exposure to the metabolized extract of this mixture induced only limited adverse effects on sperm motility.

     Key words: Metabolites, PCB, spermatozoa, DDE

There is currently much concern that endocrine disruptors and other environmental contaminants negatively affect the male reproductive tract and subsequent fertility (National Research Council, 1999; Safe, 2000). Although most studies have tested the effects of only 1 or 2 chemicals (Pflieger-Bruss and Schill, 2000; Kuriyama and Chahoud, 2004), populations are simultaneously exposed to a variety of contaminants. In earlier studies, we demonstrated that in utero and lactational exposure to an environmentally relevant organochlorine mixture adversely affected male reproduction, including decreased sperm motility, in the rat and pig models even at low, environmentally pertinent concentrations (Bailey, 2002; Anas et al, 2005). We also showed that in vitro exposure of frozen-thawed boar sperm to the original organochlorine mixture decreased sperm motility and viability in a dose-dependent manner (Campagna et al, 2002).

Using the porcine model, this study was therefore designed to investigate the effects of an environmentally relevant mixture of 15 organochlorines on the function of fresh boar sperm in vitro. The effects of the same mixture metabolized in vivo (ie, organochlorine metabolites extracted from sows to which the mixture was orally administered) were also examined. We hypothesized that the original organochlorine mixture as well as its metabolized extract would reduce sperm functional parameters after incubation in vitro in conditions that mimic the female reproductive tract. We chose the pig as a model because of the remarkable similarity between the 2 species' physiology of many systems and organs (Petters, 1994; Prather et al, 2003).

	Materials and Methods

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Chemicals

The medium used for diluting semen was the capacitating medium Krebs-Ringer-bicarbonate (KRB; 1.19 mM KH₂PO₄, 94.6 mM NaCl, 4.78 mM KCl, 5.56 mM glucose, 25.07 mM NaHCO₃, 2 mM CaCl₂, 2 mM pyruvate, and 0.4 % bovine serum albumin [Toyoda and Chang, 1974]). The organochlorine mixture was designed to approximate the profile of organochlorines found in Arctic ringed seal blubber (Muir et al, 2000), which is frequently consumed by the Inuit population in Nunavik, Québec, Canada. Pure organochlorine compounds (15 components in total) or technical mixtures were dissolved in dimethylsulfoxide (DMSO) as described previously (Campagna et al, 2001) to obtain the proportions listed in Table 1. The metabolized extract was isolated from the plasma of untreated sows and sows highly exposed to the organochlorine mixture obtained from a previous study in which prepubertal female pigs were fed corn oil (control) or 100 µg of PCB equivalents of the organochlorine mixture per kg body weight per day for 10 months (Bilrha et al, 2004). Frozen, pooled plasma from 3 each of these control untreated and treated sows was processed in parallel for the extraction. The final extracts were dissolved in DMSO to obtain proportions summarized in Table 2. The extraction procedure is described fully in an earlier report (Campagna et al, 2007).

Sperm Preparation

The sperm-rich semen fraction from fertile boars (Duroc breed) was collected in the morning at a local insemination center, transported undiluted to the laboratory within an hour, and kept at room temperature until used. The semen was diluted to a final concentration of 40 x 10⁶ sperm/mL in KRB medium.

     Experiment 1: Exposing Sperm to the Original Organochlorine Mixture— The stock organochlorine mixture was diluted with DMSO so that the treated semen contained increasing concentrations of the mixture: 0, 10.5, 14.7, and 21 µg/mL PCBs in a final concentration of 0.1% DMSO. Concentrations of other organochlorines can be calculated from the proportions listed in Table 1. Prepared samples were placed in unsealed tubes during incubation (39°C; 5% CO₂ and 100% humidity), and sperm function was assessed on aliquots removed at 0, 2, and 4 hours.

     Experiment 2: Exposing Sperm to a Metabolized Organochlorine Mixture— Stock dilutions of the plasma extract containing the mixture of organochlorine metabolites were prepared in DMSO as previously described (Campagna et al, 2007). Stock extract from the control (unexposed) sow plasma was used as an extraction control and is designated as 0 µg/L OH-PCBs. All stock dilutions were diluted to a final concentration of 0.1% DMSO in diluted semen. The increasing concentrations of the metabolized extract in diluted semen are expressed as the sum of OH-PCBs: 0.9, 1.8, 2.7, 3.6, and 4.5 µg/L. Concentrations of other organochlorines and metabolites can be calculated from the proportions listed in Table 2. The range of the concentrations of OH-PCBs (0–4.5 µg/L) and other metabolites (0–15 µg/L p,p'-DDE; Table 2) used in this study corresponds to concentrations of organochlorines and metabolites found in plasma samples from Inuit men of reproductive age in Nunavik, Québec, Canada (0.12–11.6 ng/g whole-blood wet weight OH-PCBs and 0.3–13.3 µg/L p,p'-DDE [Sandau et al, 2000; Muckle et al, 2001]) and Greenland (0.5–29.9 µg/L p,p'-DDE [Bjerregaard and Hansen, 2000]). An untreated control (no DMSO) and a vehicle control (0.1% DMSO) were also included. Prepared samples were placed in unsealed tubes during incubation (39°C; 5% CO₂ and 100% humidity), and sperm function was assessed on aliquots removed at 0, 6, and 12 hours.

Analysis of Motility and Progressive Motility

The percentages of motile and progressively motile sperm were evaluated by computer-assisted semen analysis (CASA; Hamilton Thorne, Beverly, Massachusetts). Diluted samples were loaded in a prewarmed (39°C) Makler counting chamber (10 µm depth; Sefi-Medical Instruments, Haifa, Israel). The image was digitized and analyzed using the Hamilton Thorne software version 7.4G set as in Dubé et al (2004). Five fields were analyzed in duplicate for each sample. CASA is preferred over manual counting methods for sperm motility analysis because it can rapidly and objectively calculate the different patterns of motility and velocity of each sperm cell within an aliquot.

Measurement of Sperm Viability

Sperm viability was evaluated using eosin-nigrosin exclusion staining as described previously (Tardif et al, 1999a). The membranes of dead sperm are permeable to eosin, which results in pink coloration. Sperm samples were diluted with staining solution and then smeared on a microscope slide, dried, and mounted. Samples were prepared in duplicate, and 100 cells per slide were scored for dead/viable sperm using phase-contrast microscopy (x400).

Measurement of Sperm Functional State

Sperm functional state was evaluated using the chlortetracycline (CTC) fluorescent assay as described previously (Dubé et al, 2004). Briefly, freshly prepared CTC solution was held at 4°C and protected from light until used. Sperm samples were diluted 1:1 with CTC solution, and 15 µL of this mixture were placed on a microscope slide before the samples were fixed with 0.1 µL of 12.5% glutaraldehyde. Slides were covered with coverslips, placed in a humid container, and stored at 4°C until scoring (storage never exceeded 24 hours). Sperm were scored according to 3 different CTC fluorescence patterns (capacitated, uncapacitated, and acrosome reacted) using ultraviolet illumination at x400 as described by Wang et al. (1995). Two slides of each sample were prepared, and 100 cells per slide were tabulated.

Evaluation of Sperm Cytosolic Calcium Levels

Cytosolic calcium levels of the sperm were assessed using the cell-permeant fluorescent calcium indicator, indo-1-AM, as described previously (Dubé et al, 2003). Briefly, sperm samples were supplemented with 2.5 µM indo-1-AM and left in the dark for 30 minutes at room temperature. Excess indo-1-AM was removed by centrifugation, and indo-1–loaded sperm were resuspended in KRB and then incubated for 3 hours (39°C; 5% CO₂ and 100% humidity).

Flow cytometry was used to detect indo-1-AM fluorescence intensity in individual sperm as described previously (Dubé et al, 2003). Briefly, indo-loaded sperm preparations were diluted in KRB supplemented with 2.4 mM propidium iodide (to exclude dead or damaged sperm) and 1 mg/mL protamine sulphate (to prevent agglutination). Flow cytometry was performed using an EPICS ELITE ESP (Beckman Coulter, Miami, Florida). The relative cytosolic calcium level of each sperm was expressed as the ratio of blue (calcium-bound indo-1) to violet (calcium-free indo-1) fluorescence. Analysis was conducted at 37°C, and 10 000 cells were scored for each aliquot. Propidium iodide–labeled sperm, debris, and cell aggregates were excluded from the analysis.

Statistical Analyses

Each experiment was repeated 4 times for experiment 1 and 3 times for experiment 2 with ejaculates from different boars (1 boar per repetition). Statistical analyses were performed using the general linear model procedure of the SAS software package (SAS Institute Inc, Cary, North Carolina). The different times (0, 2, and 4 hours for experiment 1; 0, 6, and 12 hours for experiment 2) were analyzed separately using complete block design (each time representing a different block). Student-Newman-Keul's multiple comparison test was used to compare treatments. Differences with values of P .05 were considered statistically significant. Data are presented as means ± SEMs.

	Results

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Experiment 1: Exposing Sperm to an Environmentally Relevant Organochlorine Mixture

Total motility (Figure 1A; P = .0001) and progressive motility (Figure 1B; P = .0001) were reduced by exposure to the organochlorine mixture at all times. Sperm viability was reduced by the organochlorine mixture at 0 and 2 hours (Figure 1C; P = .0013 and P = .01, respectively). The mixture increased capacitation, as assessed by CTC pattern B fluorescence, at 2 and 4 hours (Figure 1D; P = .0082 and P = .028, respectively) and spontaneous acrosome reactions, as assessed by pattern AR, at 2 hours only (Figure 1E; P = .02).

View larger version (21K): [in this window] [in a new window]   Figure 1. Sperm quality parameters are reduced after 0, 2, and 4 hours of exposure to increasing concentrations of an environmentally relevant mixture of organochlorines (n = 4). (A) Percentage of motile sperm (P = .0001). (B) Percentage of progressively motile sperm (P = .0001). (C) Percentage of viable sperm (P = .001 for 0 hours, P = .01 for 2 hours, and P = .13 for 4 hours). (D) Percentage of capacitated sperm as assessed by chlortetracycline (CTC) pattern B (P = .08 for 0 hours, P = .008 for 2 hours, and P = .03 for 4 hours). (E) Spontaneous acrosome reaction rates as assessed by the CTC pattern AR (P = .02 for 2 hours and P > .07 for 0 and 4 hours). Different letters indicate significant differences (P < .05). Error bars represent SEMs.

Experiment 2: Exposing Sperm to a Metabolized Extract of the Organochlorine Mixture

In contrast to Experiment 1 with the organochlorine mixture, preliminary experiments showed that the metabolized extract was not deleterious to sperm function during 4 hours of incubation (data not shown). We therefore conducted much longer incubations (6 and 12 hours) to assess whether the metabolized extract would eventually alter sperm function.

Total motility tended to be reduced after 12 hours of exposure to the metabolized extract (Figure 2A; P = .061), whereas progressive motility (Figure 2B; P = .13) was not significantly affected by the metabolized extract. There was also a tendency toward reduced motility owing to the DMSO and 2.7 and 3.6 µg/L OH-PCBs compared with the control (Figure 2A; P = .05). Exposure to the metabolized extract did not affect the viability, capacitation, or acrosome reaction rates of the sperm (Figure 2C through E; P = .21, P = .77, and P = .64, respectively).

View larger version (36K): [in this window] [in a new window]   Figure 2. Sperm quality parameters after 0, 6, and 12 hours of exposure to increasing concentrations of a metabolized extract of the organochlorine mixture (n = 3). (A) Percentage of motile sperm (P = .061). (B) Percentage of progressively motile sperm (P = .13). (C) Percentage of viable sperm (P = .21). (D) Percentage of capacitated sperm as assessed by chlortetracycline (CTC) pattern B (P = .77). (E) Spontaneous acrosome reaction rates as assessed by the CTC pattern AR (P = .64). Error bars represent SEMs.

	Discussion

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In humans, sperm can be exposed to contaminants via seminal fluid or secretions in the female reproductive tract (Wagner et al, 1990). The concentrations of these secretions in the cervix, the site of sperm deposition, can be as much as 20 times greater than those found in the seminal fluids and have been associated with sterility in couples (Wagner et al, 1990). This study revealed that exposing freshly ejaculated sperm to an environmentally relevant organochlorine mixture impairs their fertility parameters in a dose-dependent manner in most cases. To our knowledge, this is the first study to evaluate the effects of an organochlorine mixture on fresh sperm parameters in vitro. In an earlier study, we revealed that exposing frozen-thawed sperm to this mixture also reduced motility, progressive motility, and viability, and concomitantly reduced penetration and development of porcine oocytes (Campagna et al, 2002). In contrast, the metabolized extract of the organochlorine mixture used here only had limited effects on sperm motility.

The Organochlorine Mixture Harms Sperm Function Possibly via Altered Calcium Flux

In this study, the organochlorine mixture reduced motility, progressive motility, and viability, and increased capacitation, spontaneous acrosome reaction rates, and cytosolic calcium levels in fresh pig sperm. Motility is a sperm function necessary for normal fertilization in vivo. Other investigations have also reported adverse effects in adult rats of different organochlorines on sperm parameters, such as induced spontaneous acrosome reactions and decreased sperm motility by PCB 77 (Hsu et al, 2004) and increased incidence of dead or damaged sperm by hexachlorocyclohexane (Samanta et al, 1999). Few studies have evaluated the impact of in vitro organochlorine exposure on human sperm, and most determined that, when tested individually, organochlorines (PCB 77, PCB 126, PCB 118, PCB 153, or lindane) do not affect motility, viability, or acrosome reaction rates (Pflieger-Bruss et al, 2006a,b).

In contrast, when human sperm are exposed to physiologically relevant lindane concentrations, the plasma membrane is rapidly and transiently depolarized, opening voltage-dependent calcium channels and thereby inducing an increase in intracellular calcium levels (Silvestroni et al, 1997). Lindane has also been shown to reduce the acrosomal responsiveness of human sperm to progesterone (Silvestroni and Palleschi, 1999). Increased calcium levels may also provide insight to the mechanism of the organochlorine-induced damage. We also observed a large increase in sperm cytosolic calcium levels following organochlorine exposure, which is likely related to the concomitant increase in the CTC patterns B and AR (Figure 1). Porcine sperm capacitation is calcium dependent (Tardif et al, 2003), and increased cytosolic calcium levels occur during capacitation (Dubé et al, 2003). It is well known that the acrosome reaction is calcium dependent (Tardif et al, 1999b; Yanagimachi, 1994). In this context, we speculate that the organochlorine mixture alters the sperm membrane, enabling an inappropriate and nonregulated influx of calcium that would mimic the calcium increase that normally occurs during capacitation and the acrosome reaction but ultimately compromises sperm motility and viability.

Limited Effects of the Metabolized Extract on Sperm

Exposure of the sperm to the metabolized extract of the organochlorine mixture only tended to decrease total motility (Figure 2; P = .061) and did not affect any of the other assessed parameters, possibly because of the very low yet physiologically relevant concentrations used. Effectively, the range of concentrations of OH-PCBs (0–4.5 µg/L) and other metabolites (0–15 µg/L p,p'-DDE) used here corresponds to concentrations of organochlorines and metabolites found in plasma samples from Inuit men of reproductive age in Nunavik, Québec, Canada (0.3–13.3 µg/L p,p'-DDE and 0.12–11.6 ng/g whole-blood wet weight OH-PCBs [Muckle et al, 2001; Sandau et al, 2000]) and Greenland (0.5–29.9 µg/L p,p'-DDE [Bjerregaard and Hansen, 2000]).

Some studies reported that in the human population, organochlorine (DDT, PCB 153, p,p'-DDE, or OH-PCBs) levels in the blood can be correlated with reduced sperm motility in some cases (Dallinga et al, 2002; Rignell-Hydbom et al, 2004; Toft et al, 2004; De Jager et al, 2006; Toft et al, 2006). In those studies, however, organochlorine intake by the men came from low and chronic environmental exposure, implying that the sperm and reproductive organs were exposed to the organochlorines. Similar results were observed in previous studies on the rat and pig exposed in utero and during lactation to the organochlorine mixture—sperm motility decreased dose dependently, with concomitant alteration in the male reproductive organs (Bailey, 2002; Anas et al, 2005). In all of those studies, the entire reproductive system was exposed to the organochlorines during prenatal and early postnatal development, as well as during spermatogenesis as adults. Indeed, reproductive function is particularly sensitive to exposure to environmental contaminants during such developmental stages. In the present study, mature, ejaculated sperm were only transiently exposed to the organochlorines and the metabolized extract of the organochlorine mixture, which may explain why we only observed mild alteration to the sperm function.

It is also possible that at these low concentrations, organochlorines or their metabolites will not have any direct effects on mature sperm. We would have liked to test the impact of higher levels of the metabolized extract; however, we were limited by the starting concentration of our stock. Perhaps higher levels of the metabolized organochlorine extract would have direct toxic effects on the sperm, but it is more likely that the observed effects on sperm function following in vivo treatments were due to altered spermatogenesis, epididymal maturation, or other indirect influence moreso than effects on mature sperm directly (Bailey, 2002; Anas et al, 2005). Other sensitive assays of essential sperm functions, such as zona-binding assays, in vitro fertilization of unexposed oocytes, and resistance to freezing-thawing or to osmotic stress, will be considered in future experiments to further analyze the impact and mechanisms of low concentrations of the metabolized extract on sperm function. Nonetheless, we must emphasize that the physiologically relevant metabolized extract used in the present study contains organochlorines concentrations 5000- to 10 000-fold lower than the environmentally relevant mixture that induced marked effects on male reproductive function in vivo (Bailey, 2002; Anas et al, 2005).

Summary and Conclusion

In summary, exposure to the environmentally relevant organochlorine mixture altered normal sperm fertility parameters in vitro. The concentrations used, however, were far higher than those found in human body fluids. The metabolized extract of the organochlorine mixture slightly affected only total sperm motility at the low and physiologically relevant concentrations used. Organochlorines can therefore damage sperm membranes and functions, but low and short exposures in vitro have limited effects on freshly ejaculated boar sperm. Based on our previous studies and other reports in the literature, we conclude that in vitro sperm from unexposed males are relatively resistant to environmentally relevant organochlorines and their metabolites. In vivo, however, reduced sperm function is likely due to the effect of organochlorines or their metabolites on processes related to spermatogenesis.

	Acknowledgments

 
The authors thank the Centre d'Insémination Porcine du Québec (St-Lambert, Canada) for donating boar semen and the reviewers for excellent suggestions.

	Footnotes

 
This research was supported by the Northern Contaminants Program of Indian and Northern Affairs of Canada, Toxic Substances Research Initiative Program of Health Canada and NSERC of Canada's Discovery Grant Program.

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