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a Centre de Recherche en Biologie de la Reproduction,
b Département des Sciences Animales, and Unité de Recherche en santé Publique (CHUL-CHUQ), Département de Médecine Sociale et Préventive, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
ABSTRACT
The reproductive health risks related to exposure to persistent organic pollutants in the environment remain controversial. This debate is partly because most studies have investigated only one or two chemicals at a time, whereas populations are exposed to a large spectrum of persistent chemicals in their environment. Using the pig as a toxicological model, we hypothesized that exposing immature cumulus-oocyte complexes to an organochlorine mixture during in vitro maturation (IVM) would adversely affect oocyte maturation, fertilization, and subsequent embryo development. This organochlorine mixture mimics that which contaminates the Arctic marine food chain. Cumulus-oocyte complexes were cultured in IVM medium containing increasing concentrations of the organochlorine mixture, similar to that found in women of highly exposed populations. Organochlorines reduced the quality of cumulus expansion and the viability of cumulus cells in a dose-response manner. The proportion of apoptotic cumulus cells also increased due to organochlorine exposure. Half of the oocytes were fixed after insemination, and the remainders were cultured for 8 days. Concentrations of organochlorines did not affect the rates of oocyte degeneration, sperm penetration, and development to morula. However, incidence of incompletely matured oocytes increased and polyspermy rate decreased, both in a dose-response manner with increasing organochlorine concentrations. Blastocyst formation and number of cells per blastocyst declined with organochlorine concentration. Exposing porcine cumulus-oocyte complexes to an environmentally pertinent organochlorine mixture during IVM disturbs oocyte development, supporting recent concerns that such pollutants harm reproductive health in humans and other mammalian species.
cumulus cells, embryo, in vitro fertilization, oocyte development, toxicology
INTRODUCTION
Organochlorine chemicals such as polychlorinated biphenyls (PCBs), chlordane, dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), and lindane were greatly used worldwide during several decades for agricultural and industrial purposes. Although their use is now prohibited or restricted in many countries, they persist in the environment and constitute potential hazards to wildlife and human health, especially for populations that rely on sea products for subsistence. Their direct toxic effects on the reproductive system aside [1–5], many organochlorine compounds have been classified as hormonally active agents or endocrine-disrupting chemicals [2, 6, 7]. Some of these organochlorines are able to alter the endocrine and reproductive systems by either mimicking or antagonizing endogenous hormone action, modulating the synthesis and metabolism of endogenous hormones, or altering hormone receptor expression [8]. At present, the best-described endocrine-disrupting process is that where an exogenous chemical binds to the estrogen receptor thus preventing endogenous hormone function [9–14].
Oocyte maturation is a critical prerequisite for subsequent fertilization and development; thus, disruption of this process has considerable potential to impair female reproduction. Because folliculogenesis depends on several reproductive hormones, oocyte maturation may be sensitive to the effects of organochlorines that could disrupt their actions, either by interrupting hormone production, metabolism or receptor binding.
Populations in the Arctic are exposed to unusually high levels of organochlorines in their traditional foods [15, 16]; consequently, elevated levels of contaminants have been identified in blood and breastmilk [17, 18]. Trapp et al. [19] reported the presence of various organochlorine compounds in human follicular fluid such as PCBs, DDT, dieldrin, and hexachlorocyclohexane (HCH). To date, most studies have evaluated the toxic effects of only one or two of these organochlorine chemicals (or technical mixtures) at a time on animal models. An environmentally relevant mixture containing components that can interact with each other in an additive and/or nonadditive fashion (synergistic or antagonistic), and also interact with different receptors, was therefore needed to provide a better representation of what occurs in nature.
This study was designed to test the hypothesis that oocytes exposed to a complex mixture of organochlorine compounds in vitro would exhibit changes during maturation, or subsequent in vitro fertilization (IVF) or embryonic development. The mixture used in this study was designed to approximate that found in the Arctic marine food web. The pig was chosen as the animal model because porcine physiology is very similar to that of humans regarding the reproductive and endocrine systems [20].
MATERIALS AND METHODS
Organochlorine Mixture
The organochlorine mixture was designed to approximate levels found in ringed seal blubber [21]. Pure organochlorine compounds or technical mixtures were dissolved in dimethyl sulfoxide (DMSO) to obtain the proportions listed in Table 1. Dilutions of the stock solution were made in DMSO so that the in vitro maturation (IVM) medium contains a total concentration of 0.1% DMSO with the following concentrations of the organochlorine mixture: 0, 1x, 10x, 100x, 1000x, and 10 000x. The concentration referred to as the 1x concentration contains 4.2 ng/ml total PCBs. Concentrations of other organochlorines can be calculated from proportions listed in Table 1. The 1x and 10x doses correspond to the concentrations of total PCBs in plasma samples from Inuit women of reproductive age from Nunavik (1.0–47.9 ng/ml plasma; unpublished) and Greenland (3.0–95.3 ng/ml plasma, [22]). A negative control (no DMSO) was also included.
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Media
Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The oocyte maturation medium used was a BSA-free NCSU 37 (North Carolina State University) medium [23] supplemented with 25 µM ß-mercaptoethanol (Bio Rad, Hercules, CA), 0.1 mg/ml cysteine, 10% (v/v) porcine follicular fluid (pFF; 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]). The pFF was collected from follicles (4–6 mm diameter) of prepubertal gilt ovaries using an 18-gauge needle and a 10-ml syringe. After centrifugation at 1500 x g for 30 min at 4°C, the supernatant was filtered though 0.8-µm and 0.45-µm syringe filters and stored at -20°C until use. The pFF was not tested for organochlorine content; however, a previous study from our laboratory tested the organochlorine concentration of gilt plasma and found it to be under detection limits (<0.02 ng/ml by gas chromatography; unpublished data). The medium used for IVF was a modified Tris-buffered medium (mTBM) containing 0.1% (w/v) BSA (catalogue no. A-4503) and 1 mM caffeine [24]. Sperm washing medium was Dulbecco phosphate-buffered saline (dPBS) 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.
Oocyte Preparation
Oocytes were matured as described by Bureau et al. [25]. Ovaries were collected from prepubertal gilts at a slaughterhouse near the laboratory and transported within 30 min in a thermos flask containing sterile saline solution (0.9% NaCl) supplemented with antibiotics and antimycotics (catalogue no. A-7292), containing 100 000 IU/L penicillin G, 100 mg/L streptomycin, and 250 µg/L amphotericin B, and maintained at about 30°C. On arrival, ovaries were rinsed twice in fresh saline at 37°C. Follicles 4–6 mm in diameter were punctured and aspirated using an 18-gauge needle and a 10-ml syringe. Harvested cumulus-oocyte complexes (COCs) were washed three times with Hepes-buffered Tyrode medium containing 0.1% (w/v) polyvinyl alcohol (PVA-TL-HEPES) [26]. Those with an unexpanded, compact cumulus exhibiting uniform cytoplasm were selected for IVM.
In Vitro Maturation Assay
Selected COCs were washed three times in maturation medium. About 50 COCs were then transferred into each culture well of four-well multidishes (Nunc, Roskilde, Denmark) containing 1 ml of maturation medium with the desired concentrations of the organochlorine mixture (0, 1x, 10x, 100x, 1000x, and 10 000x). To favor organochlorine contact with the COCs, no mineral oil was layered over the medium. Cumulus-oocyte complexes were cultured for 20 h at 38.5°C in an atmosphere of 5% CO2 in air and 100% humidity. Complexes were then washed three times and transferred into 1 ml of fresh maturation medium without dibutyryl-cAMP or hormonal supplements, but containing their respective treatments, and cultured for an additional 24 h [26].
After culture, the quality of cumulus expansion was visually evaluated and classified as described by Alm et al. [27]: 4—fully expanded cumulus (widespread cumulus and corona radiata cells, zona pellucida clearly visible); 3—moderately expanded cumulus (loose cumulus and corona radiata cells, zona pellucida not clearly visible); 2—poorly expanded cumulus (partly dense pycnotic cumulus cells); 1—no expansion.
In Vitro Fertilization
After completing oocyte maturation, cumulus cells were removed with maturation medium containing 0.1% (w/v) hyaluronidase (catalogue no. H-3506). Oocytes were washed three times with fertilization medium and ca. 25 oocytes were placed in 50-µl droplets of fertilization medium covered with mineral oil (Aldrich Chemical, Milwaukee, WI) in 35- x 10-mm culture dishes (Nunc). Dishes were incubated (38.5°C, 5% CO2 in air; 100% humidity) until insemination. Two 100-µl frozen boar semen pellets, cryopreserved as described by Abeydeera and Day [28], were thawed in 2 ml of sperm-washing medium. The sperm solution was layered over a 65–70% Percoll gradient, centrifuged, and washed according to Bureau et al. [25]. The sperm pellet then was resuspended in 50–100 µl fertilization medium. Motility and concentration were each evaluated using hemacytometers, and the sperm were diluted in fertilization medium to a concentration of 300 000 motile sperm/ml. Aliquots of the sperm suspension (50 µl) were added to the 50 µl of fertilization medium containing the oocytes, giving a final sperm concentration of 75 000 motile sperm/ml. Oocytes were coincubated with the sperm for 6 h at 38.5°C in an atmosphere of 5% CO2 in air and 100% humidity.
In Vitro Development and Evaluation of Sperm Penetration and Oocyte Nuclear Maturation
At the end of the coincubation period, oocytes were washed three times in development medium and transferred into Nunc four-well multidishes containing 500 µl of the same medium covered with 500 µl mineral oil and returned to the incubator for further development. After 12 h of culture, half of the oocytes were fixed in 100 µl formalin (catalogue no. HT50-1-1) for 15 min, then rinsed in 200 µl 0.5% Triton X-100 before being mounted over a drop of Mowiol gelatin containing Hoescht 33354. The Mowiol gelatin was made of 2.4 g Mowiol (catalogue no. 324590; Aldrich) added to 6 g glycerol and 6 ml water and left to dissolve overnight at room temperature. The gelatin was then dissolved in 12 ml of 0.2 M Tris base (pH 8.5) at 50°C overnight. A concentration of 5 µg/ml Hoescht 33354 was added to the gelatin and it was kept at 4°C until use. Mounted oocytes were kept at 4°C until examined under a fluorescent microscope at x200 and x400 magnifications using ultraviolet illumination. Oocytes were considered penetrated when they had one or more features of sperm nuclei or pronuclei. The nuclear maturation of the unpenetrated oocytes was also assessed. Stages were classified as germinal vesicle (GV), GV breakdown, prometaphase I, metaphase I, anaphase I, telophase I, and metaphase II. Degenerated oocytes were also counted. Cleavage rate was evaluated 48 h postinsemination. The rates of morula and blastocyst formation at 8 days postinsemination were tabulated. Blastocysts were fixed and mounted as previously described and their total number of cells was tabulated.
Cumulus Viability Assay
Cumulus-oocyte complexes were cultured for maturation in the presence of organochlorines, as described. At the end of the maturation period, COCs were washed twice in cold PBS (4°C) and then transferred into 1 ml of cold PBS containing 5 µg/ml Hoechst 33342 and 1 µg/ml propidium iodide (PI) according to the Vybrant Apoptosis Assay Kit #5 (Molecular Probes, Eugene, OR). Cumulus-oocyte complexes were incubated on ice for 20–30 min, and the nuclei of cumulus cells were evaluated under fluorescent microscopy at x200 and x400 magnification using ultraviolet and blue-violet illumination. The incidences of live (Hoechst-positive), apoptotic (dual-stained), and dead or necrotic (PI-positive) cells were tabulated.
Statistical Analyses
Experiments were repeated five times and data were analyzed by ANOVA (for overall treatment effect), Duncan protected least significant difference test (for multiple comparisons among treatments) and by ANOVA with polynomial contrasts evaluated by regression (for dose-response effect). Probabilities greater than 5% were considered not statistically significant.
RESULTS
Effects of Organochlorines on Cumulus Cells In Vitro
The presence of 0.1% DMSO in the maturation medium reduced the quality of the cumulus expansion compared to the DMSO-free control (3.2 versus 3.8, respectively; P = 0.05). The organochlorine mixture further reduced the quality of cumulus expansion in a dose-response fashion (P < 0.001; r2 = 0.93) (Fig. 1), such that significant differences occurred between control and 100x, 1000x and 10 000x concentrations (3.2 versus 2.0, 2.0, and 1.0, respectively; P = 0.05). As determined using the Vybrant Apoptosis Assay #5, the presence of DMSO did not affect any of the viability parameters of the cumulus cells compared to the DMSO-free control. Increasing concentrations of the organochlorine mixture raised the level of apoptotic cells linearly (Fig. 2; P < 0.001, r2 = 0.98). Compared to the DMSO control, treatments with 10x, 1000x and 10 000x concentrations increased the rate of apoptosis in cumulus cells (1.44, 3.67, and 12.6% versus 1.11%; P = 0.05). No detrimental effects of the mixture were observed on the proportion of necrotic and dead cells compared to the control (P > 0.05). The total percentage of viable cells decreased in a linear manner with increasing concentrations of the organochlorine mixture (P < 0.01, r2 = 0.92). However, only the highest concentration (10 000x) produced a statistically significant increase compared to control (35.1% versus 17.1%; P = 0.05).
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Effects of Organochlorines on Nuclear Maturation In Vitro
Compared to the control without DMSO, the presence of 0.1% DMSO in the maturation medium did not affect the rate of degenerated oocytes (5.6% versus 4.1%) or the rate of incompletely matured oocytes (any stage prior to metaphase II; 2.6% versus 3.5%; P > 0.05) (Fig. 3). There was no effect of the organochlorine mixture on the degeneration rate compared to controls (P > 0.05). However, the rate of incompletely maturated oocytes increased in a linear manner with organochlorine concentrations (P < 0.01; r2 = 0.73). Furthermore, 1000x and 10 000x concentrations increased the incomplete maturation rate compared to the DMSO control (5.4% and 8.3% versus 3.5%, respectively; P = 0.05).
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Effects on IVF
Again, the presence of 0.1% DMSO in the maturation medium did not affect the rates of penetration (34.1% versus 47.9%) and polyspermy (21.7% versus 30.7%) compared to the control without DMSO (P > 0.05; Fig. 4). Sperm penetration was not affected by the organochlorine mixture added to the maturation medium (P > 0.05). The polyspermy rate decreased in a linear manner as organochlorine mixture level increased (P = 0.05; r2 = 0.77); however, on an individual basis, no treatments were different from the DMSO control (P > 0.05).
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Effects on Embryo Development
The rates of morula and blastocyst formation were not affected by the presence of DMSO in the maturation medium of oocytes (15.6% and 15.4% versus 16.2% and 17.1% without, respectively; P > 0.05). Subsequent development to morula was not affected by treatment with the organochlorine mixture (P > 0.05; Fig. 5). A linear regression analysis demonstrates that increasing levels of the mixture reduced oocyte developmental competence as reflected by decreased numbers of blastocysts (P < 0.01; r2 = 0.84). The 10 000x concentration significantly reduced the number of blastocysts formed compared to control (0% versus 15.4%; P = 0.05). The quality of the blastocysts was also affected by the treatments as shown in Figure 6. The number of cells in each blastocyst decreased as the concentrations increased in a linear manner (P = 0.05, r2 = 0.77). Yet, no treatments were different from the control when analyzed by ANOVA (P > 0.05).
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DISCUSSION
Populations are exposed to numerous congeners of persistent organic chemicals in their food sources, particularly in the Polar Regions [15]. Therefore, we have used an in vitro system to estimate certain reproductive consequences of in vivo exposure to an organochlorine mixture that is similar in composition to those found in the Arctic [21]. This investigation specifically assessed the effects of such an organochlorine mixture on the maturation of porcine oocytes and their subsequent fertility and developmental competence. The results clearly demonstrate that the organochlorine mixture is detrimental to the developmental competence of porcine oocytes and cumulus cell viability in a dose-dependent fashion.
Concentrations of several organochlorines, such as DDT, dieldrin, HCH, and PCBs, have been found in human follicular fluid [19]. The total PCB concentrations are reported to range from 4.7 to 27 ng/ml PCB [29], and similar concentrations can be found in human plasma samples [1, 17] that approximate the 1x and 10x doses in the present report. Ovaries and follicles are therefore as exposed to environmental organochlorines as most other organs in the body. Moreover, the lowest concentrations of PCBs used in the present study (4.2 ng/ml and 42 ng/ml total PCBs, referred to as 1x and 10x concentrations) are relevant to circulating levels of PCB and organochlorines currently found in Inuit women of reproductive age (unpublished). Compared to controls, only the cumulus cells were directly affected by these concentrations. However, dose-responses were noted at several endpoints (cumulus cell viability, oocyte maturation, polyspermy, and blastocyst formation), which are clearly indicative of a toxicological effect. In fact, our treatments likely underestimate the effects of in vivo exposure because the COCs were transitorily in contact with the toxins rather than being chronically exposed to increasing levels of bioaccumulated chemicals. Furthermore, the effects of chronic exposure to even low levels of organochlorines could also be exacerbated by environmental factors, including occupational, medical, or dietary contact with other toxic agents. Transgenerational effects of organochlorine exposure are well documented [30–34]. Finally, the levels of bioaccumulated organochlorines in the ecosystem will never be reduced, so these data are environmentally relevant, even at the higher doses.
Damage to Cumulus Cells Following Organochlorine Exposure
Because cumulus cells affect the processes of maturation, fertilization, and development, their presence or absence also modulates the reproductive effects of environmental toxicants. It is well known that oocytes and cumulus cells communicate and exchange numerous factors through gap-junctional channels. In porcine COCs, channel disruption decreased oocyte glutathione content, reducing male pronucleus formation and sperm nuclear envelope degradation [35]. Therefore, these channels might play an important role in regulating the cytoplasmic factors in the oocyte responsible for glutathione inflow from the cumulus cells and subsequent elimination of the sperm nuclear envelope. Although a decrease in glutathione inflow from the cumulus was considered a major pathway of oocyte disruption, the authors did not exclude the involvement of other oocyte factors such as intracellular pH and intracellular Ca2+ that may be regulated by the cumulus cells. Furthermore, in the rat, lindane (10 µM) altered folliculogenesis by inhibiting gap junction formation in the granulosa cells, thereby abolishing the oocyte-directed follicle-organizing activity [36]. Gap junction proteins of rat liver were also altered by PCB 126 [37]. In the mouse, fertilization of cumulus-free oocytes was not affected by the commercial PCB mixture Aroclor-1254 [38], but fertilization and early embryogenesis of cumulus-enclosed oocytes were significantly reduced [29].
The cumulus cells in this study were affected by in vitro exposure to the mixture of organochlorines. The quality of the cumulus expansion greatly decreased and the rate of apoptotic cells increased in a dose-dependant manner with the increasing levels of organochlorines. Surprisingly, the rate of total apoptotic, necrotic, and dead cells was not correlated with decreased cumulus expansion. At the higher concentrations, still more than 60% of the cumulus cells were alive even though cumulus expansion did not occur. The mechanisms by which the organochlorine mixture affects the cumulus cells are not known; however, Mori et al. [35] found that cumulus expansion of porcine oocytes were not affected by a gap junction inhibitor, heptanol. Progesterone production by granulosa cells was inhibited by estrogenic agents o,p'-DDT and 17ß-estradiol [39]. This suggests that bioaccumulated levels of organochlorine chemicals could adversely affect folliculogenesis, follicle degeneration, and maturation of primordial oocytes in women and females of other mammalian species.
Exposure to Organochlorines Affects Oocyte Maturation
The organochlorine mixture had no effect on oocyte degeneration during IVM; however, the number of oocytes unable to mature fully to metaphase II increased in a dose-dependant manner. These results are consistent with the experiment of Krogenaes et al. [40] on bovine oocytes. They found that PCB 153 or PCB 126 did not affect the rate of degenerated oocytes, but that 100.6 pg/ml PCB 126 increased the rate of incompletely matured oocytes whereas 84 ng/ml PCB 153 had no effect. Previously, Alm et al. [27] showed that treatment with 29.0 µg/ml DDT or lindane (
-HCH) reduced the maturation rate by 50% and increased the degeneration rate by 50% of bovine oocytes. Organochlorines can affect the gap junctions between cumulus cells [36]. The regulation of cAMP that contributes to meiotic arrest of porcine oocytes [26] is mediated by the cumulus cell gap junctions [41, 42]. Therefore, a defect in cumulus cell function, and subsequent oocyte cAMP levels, might be an explanation among others for the maturation problem in oocytes exposed to the mixture.
Polyspermy, but Not Penetration, Is Decreased after Organochlorine Exposure
This organochlorine mixture also decreased polyspermy but not the overall rate of penetration (mono- and polyspermy combined) in a linear manner. Polyspermy in porcine oocytes is not due to delayed or incomplete cytoplasmic maturation (expressed in terms of cortical granules exocytosis) but mostly to a delayed zona pellucida reaction [43], suggesting that organochlorines affect polyspermy via the zona reaction rather than cytoplasmic maturation. Because penetration was not affected, it seems that our mixture may affect the mechanisms responsible for the oocyte's block against polyspermy.
Oocyte Developmental Competence Is Reduced Following Organochlorine Exposure
Oocyte competence to embryonic development also declined following IVM exposure to the organochlorine mixture. Previous studies showed that PCB 126, DDT, and -HCH can also affect the developmental competence of bovine oocytes [27, 40]. The glutathione content of porcine oocytes, which is regulated by gap junctions [35], is closely related to cytoplasmic maturation, male pronuclear formation, and developmental competence [24, 44–48]. It is possible that organochlorines perturb the gap junction communication of the cumulus cells, reducing the glutathione content of the treated oocytes and subsequently adversely affecting embryo quality.
Several studies have investigated the influence of organochlorines on the reproductive system in vivo. In the mouse, maternal exposure to diethylstilbestrol, bisphenol A, and Aroclor 1016 induced testosterone-independent prostate growth in male fetuses by directly interfering with the reproductive organs and not via maternal or fetal endocrine systems [49]. In rabbit does, oral administration of Aroclor-1260 resulted in its bioaccumulation in blastocysts and increased mortality of preimplanted embryos [50]. Several studies assessed the in vitro effects of organochlorines on the detailed events leading to fertilization and embryo formation in mice [51–54], cattle [40], and rabbits [55]. Not only did organochlorine exposure during fertilization or development (in vivo or in vitro) affect further embryonic development and possibly cause future health problems, our results confirm that exposure during maturation is also detrimental to embryonic development. This suggests that the same effects would be observed in vivo.
In conclusion, the present study demonstrates that exposing porcine COCs to an environmentally relevant mixture of more than 15 organochlorines (similar to that found in the Arctic food chain) during IVM negatively affects the maturation of the oocytes and their subsequent developmental competence by decreasing the rate and quality of blastocysts. The quality and viability of the cumulus cells also decreased in a dose-dependant manner, which may account for the reduced maturation and developmental competence of the oocytes. Maturation of oocytes is an important step leading to fertilization, and perturbation in this process can greatly affect the development of the surviving oocyte and embryo. Some of these effects were seen at the 10x mixture concentration, which can be encountered in a substantial fraction of women of reproductive age in the Inuit population. The results of this study therefore support concerns that environmental contaminants harm reproductive health in humans and other mammalian species.
ACKNOWLEDGMENTS
Thanks to Mariève Bureau for excellent training with the protocol, to René Paradis for technical assistance, to Dr. Bruno Bérubé for help with the organochlorine mixtures and to Drs. Tiaan de Jager and Robert Sullivan for critically reviewing this manuscript.
FOOTNOTES
First decision: 26 February 2001.
1 This work was supported by the Toxic Substance Research Initiatives Programme of Health Canada. 
2 Correspondence: J.L. Bailey, Centre de Recherche en Biologie de la Reproduction,
Départment des Sciences Animales, Pavillon Comtois, Université
Laval, Sainte-Foy, PQ, Canada G1K 7P4. FAX: 418 656 3766; janice.bailey{at}crbr.ulaval.ca
Accepted: April 3, 2001.
Received: January 26, 2001.
REFERENCES
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