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BIOLOGY OF REPRODUCTION 67, 80–87 (2002)

An Environmentally Relevant Organochlorine Mixture Impairs Sperm Function and Embryo Development in the Porcine Model1 Ce´line Campagna,3 Christine Guillemette,3 Rene´ Paradis,3 Marc-Andre´ Sirard,3 Pierre Ayotte,4 and Janice L. Bailey2,3 Centre de Recherche en Biologie de la Reproduction,3 De´partement de Sciences Animales, and Unite´ de Recherche en Sante´ Publique (CHUL-CHUQ),4 De´partement de Me´decine Sociale et Pre´ventive, Universite´ Laval, Que´bec, Canada G1K 7P4 ABSTRACT

cause of reproductive dysfunction in humans and other animals [1–5]. In an earlier study, our group showed that exposure to an environmentally relevant mixture of organochlorines during in vitro maturation (IVM) negatively affects oocyte maturation and subsequent embryonic competence in the porcine model [6]. Male rat pups with lactational exposure to lindane (g-hexachlorocyclohexane) showed reproductive abnormalities that were detectable after puberty, including reduced testicular weight, sperm count, and testosterone level [7]. Commercial mixtures and individual polychlorinated biphenyls (PCBs) also negatively affected several aspects of prenatal and postnatal development, and fertility in various animal species [5]. Several investigations have been conducted on the effects of organochlorines on the development of preimplantation embryos [8–11], whereas their effect on fertilization has received less attention. However, exposure of mouse oocytes to PCB mixtures or individual PCB congeners during in vitro fertilization (IVF) reduced penetration and eventual development to two-cell embryos [12–14]. People who live in the Arctic, such as the Inuit, are exposed to a wide variety of organochlorines through their traditional diet [15, 16], and elevated levels of contaminants have been found in blood and breast milk of mothers [17, 18]. Organochlorines have also been detected in human and animal follicular fluid, genital tract secretions and tissues, and semen [19–24]. Furthermore, a very strong correlation exists between PCB levels in serum and follicular fluid in women with background exposure through a Western-style diet [25]. Therefore, because organochlorines can be present at the site of fertilization in vivo, it is important to assess the effects of these contaminants on gamete interactions. This study was designed to test the hypothesis that exposure of porcine oocytes and sperm to a complex mixture of organochlorine compounds in vitro would adversely affect IVF or subsequent embryonic development. The mixture used is an environmentally pertinent combination of organochlorines, which resembles that found in the blood of pregnant women in Arctic areas [26]. The pig was chosen as the animal model because its reproductive and endocrine physiologies are similar to those of humans [27].

We evaluated the effects of an environmentally relevant mixture of more than 15 organochlorines on the development of pig oocytes and sperm during in vitro fertilization (IVF). Oocytes were cocultured with sperm in IVF medium containing increasing concentrations of an organochlorine mixture, similar to that found in women of highly exposed populations. Exposure to the organochlorine mixture diminished oocyte penetration rates and polyspermy in a linear manner. The mixture did not affect rates of cleavage nor development to multicell embryos. However, rates of development to the blastocyst stage were lower at the highest concentration at which oocyte penetration was observed. The same experiment was performed using oocytes that were preexposed during in vitro maturation. This greater exposure to the mixture also reduced penetration in a dose-response manner and affected polyspermy. Frozen-thawed pig sperm were also cultured in IVF medium containing the same organochlorine concentrations. Sperm motility parameters were immediately reduced in a dose-dependant manner by the organochlorines, followed by diminished viability 2 h later. From these results, it appears that reduced sperm quality would account for decreases in fertilization, polyspermy, and blastocyst formation. These results suggest that exposing porcine oocytes and sperm to an environmentally pertinent organochlorine mixture in vitro disrupts the oocyte block to polyspermy, sperm fertility, and further embryonic development, and supports recent concerns that such pollutants harm reproductive health in humans and other species.

embryo, fertilization, gamete biology, in vitro fertilization, sperm, toxicology

INTRODUCTION

Because organochlorine chemicals persist in the environment, concerns have been raised about their implication in reproductive toxicity and endocrine disruption. Organochlorine chemicals and other persistent organic pollutants travel through air and water and gradually enter the food chain. Sea and coastal animals bioaccumulate high levels of these chemicals, which are then ingested by humans. Environmental accumulation of persistent organic pollutants over the last 50 years has been suggested as a major This work was supported by the Toxic Substances Research Initiative Programme of Health Canada. 2 Correspondence: J.L. Bailey, Centre de Recherche en Biologie de la Reproduction, De´partement des Sciences Animales, Pavillon Paul-Comtois, Universite´ Laval, Sainte-Foy, QC, Canada G1K 7P4. FAX: 418 656 3766; e-mail: [email protected] 1

MATERIALS AND METHODS

Organochlorine Mixture The organochlorine mixture was designed to approximate the levels found in ringed seal blubber, which is frequently consumed by the Inuit population [28]. As described previously [6], pure organochlorine compounds and technical mixtures were dissolved in dimethyl sulfoxide (DMSO) to obtain the proportions listed in Table 1. Dilutions of the stock solution were made fresh daily in DMSO so that the IVF medium con-

Received: 23 July 2001. First decision: 28 August 2001. Accepted: 18 January 2002. Q 2002 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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ORGANOCHLORINES DAMAGE SPERM AND IVF TABLE 1. Composition of the organochlorine mixture used in this study. Product Aroclor and congener neat mix† Technical Chlordane p,p9-dichlorodiphenyldichloroethylene (DDE) p,p9-dichlorodiphenyltrichloroethane (DDT) Technical Toxaphene a-hexachlorocyclohexane (HCH) Aldrin Dieldrin 1,2,4,5-tetrachlorobenzene p p9-dichlorodiphenyldichloroethane (DDD) b-hexachlorocyclohexane (HCH) Hexachlorobenzene Mirex Lindane (g-HCH) Pentachlorobenzene

Chemical Abstract Service no. 57-74-9 72-55-9 50-29-3 8001-35-2 319-84-6 309-00-2 60-57-1 95-94-3 72-54-8 319-85-7 118-74-1 2385-85-5 58-89-9 608-93-5

Origin*

Weight (%)

AccuStandard Radian Radian Aldrich Radian Radian Radian Sigma Aldrich Aldrich Radian Aldrich Radian Aldrich Aldrich

32.4 21.4 19.3 6.8 6.5 6.2 2.5 2.1 0.9 0.5 0.4 0.4 0.2 0.2 0.2

∗ AccuStandard Inc. (New Haven, CT); Aldrich Chemical (Milwaukee, WI); Radian Corp. (Austin, TX); Sigma Chemical Co. (St. Louis, MO). † Mix containing Aroclor 1260 (58.9%), Aroclor 1254 (39.3%), 2,4,49-trichlorobiphenyl (1.0%), 2,29,4,49 tetrachlorobiphenyl (0.8%), 3,39,4,49,5-pentachlorobiphenyl (PCB 126) (0.02%) and 3,39,49-tetrachlorobiphenyl (0.004%).

tained a final concentration of 0.1% DMSO with the organochlorine mixture in 0, 13, 103, 1003, 10003, and 10 0003 concentrations. The 13 concentration contains 4.2 ng/ml total PCBs. Concentrations of other organochlorines (aldrin, dieldrin, chlordane, lindane, etc.) can be calculated from proportions listed in Table 1. The 13 and 103 concentrations correspond to the concentrations of total PCBs in plasma samples from Inuit women of reproductive age in Nunavik (Canada) (1.0–47.9 ng/ml plasma [26]) and Greenland (3.0–95.3 ng/ml plasma ([29]). An IVF control (without DMSO) was also included.

Media Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Company (St. Louis, MO). The oocyte maturation medium was BSA-free North Carolina State University (NCSU) 37 medium [30] supplemented with 25 mM b-mercaptoethanol (Bio-Rad, Hercules, CA), 0.1 mg/ml cysteine, 10% (v:v) porcine follicular fluid (the same batch was used for all repetitions), 1 mM dibutyryl-cAMP, and hormonal supplements (10 IU/ml hCG; [APL; Ayerst Laboratories Inc., Philadelphia, PA] and 10 IU/ml eCG [Folligon; Intervet, Whitby, ON, Canada]). Porcine follicular fluid was collected from follicles (4–6 mm in diameter) of unexposed prepubertal gilt ovaries as described by Campagna et al. [6]. The medium used for IVF was a modified Tris-buffered medium containing 0.1% (w:v) BSA (Sigma, A-4503) and 1 mM caffeine [31]. Sperm washing medium was Dulbecco phosphate-buffered saline supplemented with 0.1% (w:v) BSA pH 7.2. The culture medium for embryonic development was NCSU 37 medium supplemented with 0.4% (w:v) BSA.

Cumulus-Oocyte Complex Preparation Cumulus-oocyte complexes (COCs) were obtained and prepared as described by Campagna et al. [6] and based on a procedure initially developed by Abeydeera and Day [32]. Briefly, ovaries were collected from prepubertal gilts at a slaughterhouse and transported to the laboratory. Follicles 4- to 6-mm in diameter were punctured and aspirated, then harvested COCs were washed 3 times with Hepes-buffered Tyrode medium containing 0.1% (w:v) polyvinyl alcohol [33]. Those with an unexpanded, compact cumulus exhibiting uniform cytoplasm were selected for IVM (about 350–400 oocytes per repetition).

In Vitro Maturation Selected COCs were washed 3 times in maturation medium. About 50 COCs were then transferred into each culture well of 4-well multidishes (Nunc, Roskilde, Denmark) containing 500 ml of maturation medium covered with mineral oil (Aldrich Chemical, Milwaukee, WI). Cumulus-oocyte complexes were cultured for 20 h at 38.58C in an atmosphere of 5% CO2 in air and 100% humidity. Complexes were then washed 3 times and transferred into 500 ml of fresh maturation medium without dibutyrylcAMP or hormonal supplements, and cultured for an additional 24 h [33].

Experiment 1: Inclusion of the Organochlorine Mixture During IVF After completing oocyte maturation, cumulus cells were removed with maturation medium containing 0.1% (w:v) hyaluronidase (H-3506; Sigma). Completely denuded oocytes were washed 3 times with fertilization medium and ;25 of these oocytes were placed into culture wells of a 96well tissue culture plate (Falcon Microtest, U-bottom with a low evaporation lid; Becton Dickinson, Oakville, ON, Canada) containing 50 ml of fertilization medium with the desired concentrations of the organochlorine mixture (i.e., 0, 13, 103, 1003, 10003, 10 0003, and IVF controls). All oocytes were distributed equally and randomly among the 7 treatments so that there were 2 groups of ;25 oocytes per treatment. To favor organochlorine contact with the oocytes, no mineral oil was layered over the medium. Pure water filled the surrounding wells in order to minimize evaporation. The culture plate was incubated (38.58C, 5% CO2 in air, 100% humidity) during semen preparation (approximately 30 min). Two 100-ml frozen boar semen pellets, cryopreserved as described by Abeydeera and Day [32], were thawed in 2 ml of sperm washing medium. The sperm solution was layered over a Percoll gradient (65%–70%), centrifuged, and washed according to the method of Bureau et al. [34]. The sperm pellets then were resuspended in 50–100 ml of fertilization medium. Sperm motility and concentration were each evaluated with hemacytometers, and the sperm were diluted in fertilization medium containing the organochlorine treatments to a concentration of 70 000 motile cells per milliliter. Aliquots of the sperm suspension (50 ml) were added to the 50ml fertilization medium containing the oocytes, giving a final sperm concentration of 35 000 motile cells per milliliter. Oocytes were coincubated with the sperm for 6 h at 38.58C in an atmosphere of 5% CO2 in air and 100% humidity.

In Vitro Development and Evaluation of Sperm Penetration At the end of coincubation, oocytes were washed 3 times in development medium, transferred into Nunc 4-well multidishes containing 500 ml of the same medium covered with 500 ml of mineral oil, and returned to the incubator for further development. From the 2 groups of ;25 oocytes per treatment, 1 was used to evaluate fertilization parameters (penetration and polyspermy) and the other was used to evaluate developmental parameters (embryo development). Fixation of the penetrated oocytes after 12 h of culture, and evaluation of penetration (expressed as a percentage of the total number of oocytes) and polyspermy (as a percentage of penetrated oocytes) were performed as in Campagna et al. [6]. Cleavage rate was evaluated at 48 h. The rates of multicell embryos and blastocyst formation 8 days after insemination were tabulated. Blastocysts were fixed and mounted as described, and their total number of cells was tabulated.

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FIG. 1. Effects of the organochlorine mixture added to the IVF medium of sperm and oocytes on their subsequent fertilization parameters. Presence of a significant linear regression for both the percentages of penetrated (open bars, P # 0.0001; r2 5 0.81) and polyspermic oocytes (closed bars, P 5 0.0016; r2 5 0.51). Each bar represents the mean of five experiments 6 SEM. The total number of oocytes used for each concentration ranged from 124 to 142. Letters indicate significant differences due to organochlorine concentration within fertilization parameters (P 5 0.05).

Experiment 2: Exposure to Organochlorines During Oocyte Maturation and Fertilization The effects of the organochlorine mixture on both oocyte maturation and fertilization were evaluated. Cumulus-oocyte complexes were harvested and prepared as described. Selected COCs were matured as described, except that they were cultured in 1 ml of maturation medium also containing the organochlorine treatments (as assayed in our earlier study; [6]). To favor organochlorine contact with the COCs, no mineral oil was layered over the medium. Matured oocytes were then completely denuded from their cumulus cells and cocultured with the sperm for 6 h in fertilization medium containing the corresponding treatments as before. They were then washed and cultured in development medium without organochlorines for 12 h, fixed, and mounted. Penetration and polyspermy rates were tabulated.

Experiment 3: Effect of the Organochlorine Mixture on Sperm Motility, Viability, and the Acrosome Reaction Because fertility of fresh ejaculated boar semen is highly variable in IVF, we use frozen-thawed ejaculated semen in our laboratory to avoid this problem [35–37]. In this study, we used frozen-thawed semen from the same ejaculate for all the repetitions to achieve a better control of gamete function in the presence of the organochlorines. To evaluate whether the effects of the organochlorine mixture on the fertilization and development parameters were mainly due to the oocytes, the sperm, or a combination of both, we also assayed sperm viability and motility during exposure to the mixture in the IVF medium without the oocytes. Five 100-ml frozen boar semen pellets (from the same batch that served for IVF) were thawed, centrifuged, and washed through Percoll as before. The pellets were resuspended in fertilization medium and sperm concentration was assessed using a hemacytometer. Semen samples were diluted to a final concentration of 25 3 106 spermatozoa per milliliter in fertilization medium containing the organochlorine treatments. Although this sperm concentration is greatly different from the one used in IVF, it is the lowest concentration possible to ensure accuracy in the sperm function assays. All incubations were carried out for 0, 2, and 4 h at 37.58C in an atmosphere of 5% CO2 in air and 100% humidity. Sperm motion parameters were assessed using a Hamilton-Thorne IVOS analyzer (version 7.4G; Beverly, MA). The analyzer settings were as follows: frames acquired, 20; frame rate, 30 Hz; minimum contrast, 7; minimum size, 5; low/high size gate, 0.6 to 2.0; low/high intensity gate, 0.3 to 1.5; nonmotile head size, 8; nonmotile brightness, 15; medium path velocity (VAP) value, 8; low VAP value, 5; slow cells motile, yes; and threshold, 60. Sperm viability was evaluated using eosin-nigrosin exclusion staining [38]. The membranes of dead sperm are permeable to eosin, which results in a pink coloration. Sperm samples were diluted 1:8 with staining solution. Ten microliters of the mixture were smeared on a microscope slide, dried, and a coverslip was fixed with Permont (Fisher, Montreal, QC, Canada) before examination. Slides were prepared in duplicate and 100

sperm per slide were scored under 4003 magnification using phase contrast microscopy. Sperm acrosome reactions were also assessed using the fluoresceinconjugated Pisum sativum agglutinin (FITC-PSA) staining [38]. Sperm samples in fertilization medium in the presence or absence of the different concentrations of the organochlorine mixture were prepared as described earlier. Each sample was divided in two; 1 received 10 mM calcium ionophore A23187 in DMSO (ionophore-induced acrosome reaction), the other received only DMSO (spontaneous acrosome reaction; negative control). All samples were incubated for 15 min at 37.58C with 5% CO2 in air. Ten microliters of sample were then mixed with 10 ml of fertilization medium, dried onto microscope slides, and fixed with 100% ethanol. Sixty microliters of FITC-PSA (100 mg/ml) were dropped on each slide, which was then incubated in a dark and humid chamber for 20 min. After repeated washes in PBS, a coverslip was fixed onto each slide with Permount. One hundred sperm per slide were analyzed with a fluorescent microscope under ultraviolet illumination.

Statistical Analyses Experiments were repeated 5 times, except for the sperm analyses, which were repeated 3–4 times. Data were analyzed by ANOVA (general linear models for overall treatment effect), the Duncan protected least significant difference tests (for multiple comparisons among treatments with P 5 0.05 considered as significant), and by general linear models with polynomial contrasts evaluated by regression (on the log values of treatments for dose-response effects). The SAS software package (SAS Institute Inc., Cary, NC) was used for all analyses. Probability values greater than 0.05 were considered to be not statistically significant.

RESULTS

Overall DMSO Effect

For all the experiments, the presence of 0.1% DMSO (solvent control) in the fertilization medium did not affect any of the parameters measured (sperm assays, penetration, polyspermy, cleavage, or morula and blastocyst formation) compared with controls without DMSO (P . 0.05). Experiment 1: Inclusion of the Organochlorine Mixture During IVF

As shown in Figure 1, sperm penetration of oocytes decreased in a linear manner as the dose of organochlorine mixture increased (P # 0.0001; r2 5 0.81). The 10003 and 10 0003 concentrations negatively affected penetration compared with controls (P 5 0.05). The proportion of polyspermic oocytes also correlated with organochlorine dose in a linear manner (P 5 0.0016; r2 5 0.51). Again, only the highest concentrations (10003 and 10 0003) decreased the frequency of polyspermy compared with controls (P 5 0.05). To separate the organochlorine effects on penetration from their effects on further development, the rate of cleavage and morula and blastocyst formation are expressed as a percentage of penetrated oocytes. Because there was no sperm penetration at the 10 0003 concentration of the mixture (Fig. 1), this treatment was not evaluated for cleavage or morula and blastocyst formation. Cleavage of embryos at 48 h after fertilization was not affected by the organochlorine mixture (data not shown, 104.9% for control vs. 110.6% for 10003, SEM 6 36.1%; P . 0.05). The high percentage (.100% of penetrated oocytes) might be due to some parthenogenic activation of the unfertilized oocytes. The rate of multicell embryo formation was also not affected by the organochlorine mixture (Fig. 2, 18.2% for controls vs. 38.4% for 10003, SEM 6 13.04%; P . 0.05). Because multicell embryos were present at the 10 0003 concentration even though there was no penetration at this concentration, we fixed some of these embryos and observed that the majority contained no more than 2 cells per

ORGANOCHLORINES DAMAGE SPERM AND IVF

FIG. 2. Effects of the organochlorine mixture added to the IVF medium of sperm and oocytes on the proportion of multicell embryos (open bars) and blastocysts (closed bars) subsequently formed, expressed as a percentage of penetrated oocytes. Presence of a significant linear regression for the percentage of blastocysts formed (P # 0.0137; r2 5 0.52). Each bar represents the mean of five experiments 6 SEM. The total number of oocytes used for each concentration ranged from 119 to 130. Letters indicate a significant difference due to organochlorine concentration (P 5 0.05).

embryo (data not shown). The nature of these cytoplasmic vesicles was not evaluated; however, simple fragmentation could describe this observation. Increasing concentrations of the mixture reduced oocyte developmental competence in a dose-response manner as reflected by proportionally fewer blastocysts formed (Fig. 2, P # 0.0137; r2 5 0.52). The 10003 concentration yielded significantly fewer blastocysts compared with controls (P 5 0.05). As shown in Figure 3, the quality of the blastocysts formed (number of cells per blastocyst) was affected by the mixture following a weak but significant cubic relationship, with a peak at the 13 concentration (P 5 0.0342; r2 5 0.06). However, no treatment was significantly different from the other (P 5 0.05). Experiment 2: Exposure to Organochlorines During Both Oocyte Maturation and Fertilization

As shown in Figure 4, the percentage of penetrated oocytes linearly decreased as the organochlorine concentrations increased (P 5 0.0034; r2 5 0.73). Penetration of oocytes was significantly lower than controls at the 10 0003 concentration (P 5 0.05). The proportion of polyspermic oocytes followed a quadratic relation as organochlorine treatment increased (P 5 0.0111; r2 5 0.72). Compared with controls, the percentage of polyspermic oocytes was higher at the 1003 concentration and lower at the 10 0003 concentration (P 5 0.05).

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FIG. 3. Effects of the organochlorine mixture added to the IVF medium of sperm and oocytes on the mean cell numbers of the derived expanded blastocysts. Presence of a significant cubic regression (P 5 0.0342; r2 5 0.06). Each bar represents the mean of 23 blastocysts 6 SEM.

the organochlorine mixture reduced the percentage of progressively motile sperm in a linear dose-dependant manner at 0 and 2 h (P 5 0.0051, r2 5 0.79; and P 5 0.0102, r2 5 0.57, respectively), but they had no effect at 4 h. At 2 h, only the 10 0003 concentration lowered the percentage of progressively motile sperm compared with controls (P 5 0.05). No organochlorine treatments were different from controls at 0 and 4 h (P . 0.05). Unlike the percentage of motile and progressively motile sperm, the organochlorine mixture diminished sperm viability at 2 h, but not at 0 and 4 h, in a linear dose-response manner, as shown in Figure 5C (P 5 0.0140, r2 5 0.85). Only the 10 0003 organochlorine concentration reduced viability after 2 h of incubation compared with controls (P 5 0.05). The ionophore-induced acrosome reaction shown in Figure 5D was calculated from the data from the ionophoreinduced acrosome reaction minus the spontaneous acrosome reaction. Neither the ionophore-induced nor spontaneous acrosome reactions were affected by treatments with the organochlorine mixture at any time (Fig. 5D; P . 0.05). DISCUSSION The results obtained from this study indicate that our environmentally relevant mixture of organochlorines added

Experiment 3: Effect of the Organochlorine Mixture on Sperm Function

As seen in Figure 5A, the percentage of motile sperm decreased due to organochlorine treatments in a linear manner at each time point, but the detrimental effects were more pronounced at 0 h (P , 0.0001, r2 5 0.93 at 0 h; P 5 0.0006, r2 5 0.67 at 2 h; and P 5 0.0019, r2 5 0.77 at 4 h). The 10003 and 10 0003 concentrations reduced the percentage of motile sperm at 0 and 4 h, and only the 10 0003 concentration reduced it at 2 h, compared with controls (P 5 0.05). The detrimental effects of the organochlorine treatments on the proportion of progressively motile sperm was more pronounced at 0 h (Fig. 5B). Increasing concentrations of

FIG. 4. Effects of the organochlorine mixture added to the IVF medium of both sperm and oocytes pre-exposed during in vitro maturation on their subsequent fertilization parameters. Presence of a significant linear regression for the percentages of penetrated oocytes (open bars, P 5 0.0034; r2 5 0.73) and quadratic for the percentage of polyspermic oocytes (closed bars, P 5 0.0111; r2 5 0.72). Each bar represents the mean of five experiments 6 SEM. The total number of oocytes used for each concentration ranged from 68 to 74. Letters indicate significant differences due to organochlorine concentration within fertilization parameters (P 5 0.05).

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can contain substantial concentrations of organochlorines [19–23, 25, 39]. Disruption of Fertilization by Organochlorine Mixture

In this study, the organochlorine mixture (at 10003 and 10 0003 concentrations) diminished overall oocyte penetration and polyspermy of oocytes treated during IVF. Fertilization can be altered at many steps, such as sperm adhesion to the zona pellucida, the acrosome reaction, spermoocyte fusion, oocyte activation, the block to polyspermy, and sperm nucleus decondensation. In all cases, organochlorines can impair fertilization by acting on sperm or oocyte functions and morphology. Here, the sperm functions were altered at these concentrations of organochlorines, supporting the hypothesis that sperm damage accounts for at least some of the disrupted fertilization. Similar results were observed in the mouse [14, 40], in which fertilization of cumulus-enclosed oocytes was reduced in the presence of 0.1, 1.0, and 10.0 mg/ml of individual PCB mixtures (Aroclor 1221, 1254, 1268, or 3,39,4,49-tetrachlorobiphenyl). The authors suggested that mechanisms involving the zona pellucida could account for this reduced fertilization, such as reduced sperm-oocyte adhesion or sperm penetration of the oocyte. The exposure to 1.0 and 10.0 mg/ml of different mixtures of commercial PCB mixtures (A: Aroclor 1016, 1232, 1248, and 1260; B: Aroclor 1221, 1242, and 1254) also reduced fertilization in mouse oocytes without any additive or synergistic effects compared with each PCB mixture alone [12]. The mechanisms by which organochlorines affect fertilization and polyspermy are presently not known. However, it is clear that PCBs disrupt Ca21 homeostasis in somatic cells [41– 43], which is essential for several fertilization parameters, including capacitation, gamete interactions, the acrosome reaction, and fertilization [44–46]. Does Organochlorine Exposure During IVF Perturb Subsequent Development?

FIG. 5. Effect of various concentrations of an organochlorine mixture on motility (A), progressive motility (B), viability (C), and induced (plain bars) and spontaneous (dotted bars) acrosome reaction (D) at 0, 2, or 4 h of frozen-thawed sperm incubated in the fertilization medium. Presence of a linear regression for motility at 0 (P , 0.0001; r2 5 0.93), 2 (P 5 0.0006; r2 5 0.67), and 4 h (P 5 0.0019; r2 5 0.77), progressive motility at 0 (P 5 0.0051; r2 5 0.79) and 2 h (P 5 0.0102; r2 5 0.57), and viability at 2 h (P 5 0.0140; r2 5 0.85). Each bar represents the mean of three (D) or four (A, B, and C) experiments 6 SEM. Letters indicate significant differences due to organochlorine concentration within sperm parameters (P 5 0.05).

to the fertilization medium impairs the ability of sperm and oocytes to fertilize. In mammals, gamete interactions, fertilization, and early embryonic development occur in the oviduct. Oocytes and sperm steep in the oviductal fluids and are in contact with the oviductal cells, both of which

Developmental competence depends on the oocyte and sperm integrity and quality. In an earlier study, we found that pretreating porcine oocytes during the period of IVM with the organochlorine mixture harms their developmental competence by reducing the proportion of blastocysts formed and the number of cells per blastocyst [6]. In the present study the organochlorine mixture added to the fertilization medium did not affect the cleavage rate or development to the multicell embryo stage, but the 10003 concentration restricted the percentage of blastocysts formed and the number of cells per blastocyst. Although we did not evaluate the nature of the cytoplasmic vesicles present in the so-called multicell embryos at the 10 0003 concentration, they had all the characteristics of fragmented (activated) oocytes (unequal cleavage and small blastomeres through the cytoplasm, with many of them enucleated) [47]. This effect can also be enhanced by chemicals such as vanadate, an inhibitor of protein tyrosine phosphatases [48]. This fragmentation hypothesis can explain the absence of blastocysts formed at the 10003 concentration, even though some oocytes were penetrated. If this is the case, it would confirm that during the short period of IVF (6 h), sperm and oocytes, which are resting at metaphase II until penetration, can be negatively affected by organochlorines at high concentrations in a manner that would not just reduce or inhibit oocyte penetration. Moreover, organ-


ochlorine treatments also limit the proportion of successfully fertilized oocytes developing to blastocysts. Although some investigators have examined the effects of organochlorines on in vitro development [8, 11, 48] or IVM [49–51], few have assayed their effects on subsequent blastocyst formation when added earlier to the fertilization medium. In their study with mixtures of commercial PCB mixtures (A: Aroclor 1016, 1232, 1248 and 1260; B: Aroclor 1221, 1242, and 1254), Kholkute et al. [12] found that exposure to 1.0 and 10.0 mg/ml of each mixture during IVF increased the percentage of abnormal mice embryos. However, they did not mention the age of the embryos or any effects on the rates of cleavage and blastocyst formation. Fertilization in the Presence of Organochlorines Following Pretreatment During IVM

In vivo, sperm, oocytes, and embryos are stage-specifically exposed to organochlorines. They are subjected to various concentrations of pollutants throughout their passage to the site of fertilization and the uterus. Stage-specific assays of organochlorine effects are necessary to identify the specific damage and to characterize the mechanisms by which gametes or embryos are affected. However, the combined effect of organochlorine exposure during the various steps of fertilization and early embryonic development in vitro can be more closely related to in vivo conditions. We have recently shown that IVM of porcine oocytes in the presence of a mixture of organochlorines reduces the frequency of matured and polyspermic oocytes, but does not affect overall penetration [6]. Because the present study shows that IVF in the presence of the same mixture generally reduces oocyte penetration and polyspermy via sperm damage, we tested the effect of a continuous exposure to organochlorines throughout IVM and IVF on the fertility parameters. The percentage of penetrated oocytes fell as organochlorine concentrations increased in the same range as was observed when the mixture was administrated only during IVF, confirming that penetration failure reflects sperm dysfunction rather than faulty oocyte maturation. However, the frequency of polyspermic oocytes increased at the 1003 concentration, but decreased drastically at higher concentrations when the mixture was present continuously during IVM and IVF, as supported by the quadratic dose-response observed. Further analyses on the oocyte block to polyspermy, such as cortical granule migration or degranulation, are required in order to elucidate the mechanisms resulting in the increase and then the decrease in polyspermy, although this decrease is probably related to the decrease in penetration rates created by sperm dysfunction. Organochlorines Affect Sperm Motility and Viability

The motility of cryopreserved ejaculated spermatozoa, which was already very low immediately after thawing, rapidly decreases during incubation [37, 52]. Isolating an enriched fraction of live and motile sperm through a Percoll gradient helps to increase the rate of fertilization in the pig [53]. Despite the relatively good fertility of thawed sperm in vitro, they are very fragile cells, as observed by the poor initial quality and the decline over time (Fig. 5). Furthermore, cryopreservation of semen clearly damages sperm, especially by affecting plasma membrane integrity (ultrastructural modifications, changes in lipid composition, elevated intracellular calcium levels, and increased produc-

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tion of reactive oxygen species [ROS]), which restricts the motility and life span of the sperm, as well as zona pellucida binding and oocyte penetration ability [44]. The plasma membrane can, therefore, be a major target for lipophilic organochlorines by which interaction with its lipid and protein components may induce structural and functional damage. In the present study, the organochlorine mixture reduced the percentage of motile, progressively motile, and viable sperm in a general pattern and at most times. These parameters were especially affected at organochlorine concentrations of 10003 and 10 0003, equivalent to 4.2 and 42 mg/ ml PCBs, respectively. The detrimental effects of the organochlorine mixture on sperm motion parameters (motility and progressive motility) were nearly immediate (0 h), whereas for sperm viability, detrimental effects were greater at 2 h. Although the sperm concentration (25 3 106 spermatozoa per ml) used in these assays was greatly superior to that used in IVF (35 000 motile cells per milliliter; i.e., about 88 000 spermatozoa per milliliter), it was the lowest concentration possible for accurate assessment of the sperm parameters measured. Even if the results cannot be clearly related to IVF, at minimum, they provide some understanding of the sperm damage involved. Also, these results can be extrapolated to the IVF system, because for the same concentration of organochlorine molecules, a low sperm concentration will cause each spermatozoon to be exposed to more organochlorine molecules than with a high sperm concentration. Therefore, the sperm assays conducted here may, in fact, underestimate the possible effect to the sperm during the IVF experiments. The proportion of motile sperm, which is an important parameter for fertility [35], is affected by the numerous physiological factors affecting the membrane integrity [44]. Therefore, we speculate that the mixture of organochlorines might reduce sperm motility and viability via these different pathways, but without any effect on the acrosome. Effectively, motility can be affected by a deficiency in the energetic (i.e., ATP) contribution from the mitochondria [35]. It has been shown that methoxychlor and dichlorodiphenyltrichloroethane (DDT), but not lindane, induce sublethal changes in transmembrane potential, ROS production, and ATP-induced intracellular Ca21 homeostasis in bovine oviductal cells [43]. However, lindane and dieldrin stimulate ROS production in neutrophils by activating phospholipase A2 [54], and lindane alters rat testicular Ca21- and Mg21-ATPase activities [55]. Perturbations in intracellular Ca21 homeostasis and in Ca21 uptake by mitochondria was also observed in rat cerebellar granule cells following PCB exposure [42, 56]. A previous study failed to demonstrate that exposure to Aroclor 1254 (1.0 and 10 mg/ml) for 1.5 h had any effect on motility, capacitation, acrosome reaction, and in vitro fertilizing ability of epididymal mouse spermatozoa [14]. In ejaculated capacitated human sperm, however, exposure to 0.1 mM lindane inhibited spontaneous acrosome reactions, and 1 mM inhibited the progesterone-induced acrosome reaction and stimulated a transient increase in intracellular Ca21 [57]. The authors pointed out that lindane inhibited sperm fertilizing potential at doses similar to those found in the cervical mucus of women from low-risk exposure areas suffering from ‘‘unexplained infertility’’ [39]. In the present study, the organochlorine mixture had no effect on either spontaneous or ionophore-induced acrosome reactions. In sea urchin sperm, exposure to dieldrin (1–30 mM) and mirex (0.03–1.0 mM) stimulated progressive motility over time, whereas exposure to lindane (0.42–

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13 mM) was inhibitory [58]. In our study, inhibitory effects of the organochlorine mixture on motility were observable at 10003, corresponding to only 0.089 mM dieldrin, 0.047 mM mirex, and 0.71 mM lindane, plus many other chemicals such as PCBs (42 mg/mL). Therefore, the variety of chemicals present in our mixture may act differently on sperm functions, resulting in an overall decrease in motility, progressive motility, and viability. The mechanisms of sperm damage are not yet elucidated, but experiments are presently underway in our laboratory. In conclusion, the present study demonstrates that exposing both porcine oocytes and sperm to an environmentally relevant mixture of more than 15 organochlorines (similar to that found in the Arctic food chain) during IVF negatively affects sperm function and competence, and subsequent embryonic development. Oocytes seem to be less damaged during this process, although there could be some enhancement of cytoplasmic fragmentation, as shown by their reduced ability to be fertilized and to develop. Even though no effect was observed at environmentally relevant concentrations of the mixture, significant dose-response effects occurred at several end points, indicating a toxicological effect. The in vitro nature of this study also underrepresents the effect of in vivo exposure, because the gametes were only transitorily exposed to the organochlorines rather than being chronically exposed to increasing levels of bioaccumulated products over a lifetime. Furthermore, environmental factors (excluded from our study), including occupational, medical, or dietary contact with other toxic agents might amplify or complicate the effects of chronic exposure to even low levels of the organochlorine mixture. The results of this study, therefore, support concerns that environmental contaminants harm reproductive health in humans and other mammalian species, and can initiate further research on the mechanisms of adverse effects on the gametes.

10. 11. 12. 13. 14.

15. 16.

17.

18. 19. 20. 21. 22.

ACKNOWLEDGMENTS

23.

The authors thank Dr. Bruno Be´rube´ for helping with the organochlorine mixtures, and a local slaughterhouse for graciously providing the porcine ovaries.

24.

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