Arsenite induces aberrations in meiosis that can be prevented by coadministration of N-acetylcysteine in mice

Arsenite induces aberrations in meiosis that can be prevented by coadministration of N-acetylcysteine in mice Paula A. A. S. Navarro, M.D., Ph.D.,a,b,c Lin Liu, Ph.D.,a,b Rui A. Ferriani, M.D., Ph.D.,c and David L. Keefe, M.D.a,b a

Department of Obstetrics and Gynecology, Women and Infants Hospital, Brown University, Providence, Rhode Island; Marine Biological Laboratory, Woods Hole, Massachusetts; and c Department of Obstetrics and Gynecology, University of São Paulo, Ribeirão Preto School of Medicine, Ribeirão Preto, Brazil

b

Objective: To evaluate in vitro effects of arsenite and of arsenite plus N-acetylcysteine on mouse oocyte meiosis. Design: Morphological study using mouse oocytes submitted to in vitro maturation (IVM). Setting: Laboratory of reproductive biology. Animal(s): Six-week-old CD-1 mice superovulated with pregnant mare serum gonadotropin. Intervention(s): During IVM, mouse oocytes were exposed to arsenite alone or to arsenite plus N-acetylcysteine. Main Outcome Measure(s): Meiotic anomalies were assessed using immunofluorescence microscopy and PolScope (Cambridge Research and Instrumentation, Boston, MA) imaging. Result(s): In vitro arsenite administration produced dose-dependent and time-dependent meiotic anomalies, characterized by spindle disruption or chromosome misalignment. After 12–14 hours of IVM, exposure to 2 ␮g/mL of arsenite for 12–14 hours or to 8 ␮g/mL of arsenite for 2 hours arrested oocyte maturation at the germinal vesicle or germinal-vesicle breakdown stage. Exposure to 4 ␮g/mL of arsenite for 2 hours arrested oocyte maturation at metaphase I stage in 95% of exposed oocytes (80% exhibiting abnormalities) after 12–14 hours in IVM. After 12–14 hours in IVM, of the oocytes exposed to 2 ␮g/mL of arsenite for 2 hours, only 15% reached the meiosis II stage (5% exhibiting abnormalities). After 15–17 hours in IVM, however, of the oocytes exposed to 2 ␮g/mL of arsenite for 2 hours, 65.2% reached the meiosis II stage (43.5% exhibiting abnormalities). Co-administration of N-acetylcysteine prevented the arsenite-induced meiotic abnormalities and the delayed IVM. Conclusion(s): In vitro arsenite exposure caused meiotic abnormalities that were prevented by co-administration of N-acetylcysteine, suggesting that arsenite-induced meiotic aberrations are mediated by reactive oxygen species. (Fertil Steril威 2006;85(Suppl 1):1187–94. ©2006 by American Society for Reproductive Medicine.) Key Words: Arsenite, meiosis, oocytes, growth and development, acetylcysteine

Inorganic arsenic is ubiquitous in the environment, in which it occurs mainly as compounds of arsenite (As3⫹) and arsenate (As5⫹). However, in certain parts of the world, including the United States (1, 2), this heavy metal is a serious environmental contaminant and represents a human health hazard. Epidemiological studies have shown that chronic exposure to arsenic can result in liver damage, peripheral neuropathy, and increased incidence of cancer of the lung, skin, bladder, and liver (3). Inorganic arsenic has also been extensively studied as a teratogenic agent in several mammalian species, and its potential for developmental toxicity has been investigated in laboratory animals (4 –10). Data on the reproductive and developmental toxicity of inorganic arsenic in humans are limited to a few studies of Received March 3, 2005; revised and accepted August 11, 2005. P.A.A.S.N. was supported by a scholarship granted by Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasília, Brazil. Reprint requests: Paula A. A. S. Navarro, M.D., Ph.D., Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo 14049-900, Brazil (FAX: 55-16-633-0946; E-mail: [email protected]).

0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.08.060

populations exposed to arsenic in drinking water or from working at or living near smelters (11–14). In more than one of these studies, arsenic exposure was correlated with spontaneous abortion, stillbirth, and low birth weight, although it is difficult to draw definitive conclusions because study populations usually were exposed to multiple chemicals (11–14). Despite the large number of studies assessing the potential developmental toxicity of inorganic arsenic, there are few data regarding its effect on meiosis. Meiosis is a cell cycle phase that is particularly sensitive to external agents. The first meiotic division is initiated during fetal life but is completed only before ovulation in the adult. The duration of the first meiotic prophase can be quite prolonged, spanning more than 50 years in some women. During this process, oocytes can be susceptible to a number of agents, including environmental pollutants such as arsenite. Moreover, meiotic errors are a major cause of aneuploidies in humans and can contribute to developmental failure through various pathways (15–19). There is considerable evidence that reactive oxygen species (ROS) are involved in the genotoxicity of arsenite

Fertility and Sterility姞 Vol. 85, Suppl 1, April 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.

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(20 –23) and that antioxidant enzymes, such as superoxide dismutase, can reduce the incidence of arsenite-induced sister chromatid exchanges in cultured human lymphocytes (23). Co-administration of the antioxidant L-ascorbate has been shown to protect against arsenite-induced ovarian toxicity in rats (24). These data suggest that the co-administration of antioxidants might prevent some of the toxic effects of arsenite. In a previous study, we demonstrated that arsenic-induced oxidative stress promotes chromosome instability, telomere attrition, and genotoxicity in mouse embryos treated with arsenite in vitro (25). We also have previously demonstrated that oocytes from mice treated in vivo with arsenite exhibit meiotic abnormalities and that mice treated in vivo with arsenite produce embryos presenting compromised early development and a significantly higher level of apoptosis (26). To further determine whether meiotic abnormalities could result directly from the effects of arsenite on germ cells, we investigated the impact of different arsenite concentrations on spindle and chromosome alignment of mouse oocytes during in vitro maturation (IVM). In addition, we evaluated whether an antioxidant, N-acetylcysteine, could prevent arsenite-induced meiotic anomalies. MATERIALS AND METHODS Reagents and Animals All reagents were purchased from Sigma Chemical Co. (St Louis, MO), unless otherwise noted. Pregnant mare serum gonadotropin (PMSG), used for superovulation, was purchased from Calbiochem (La Jolla, CA). Mice were subjected to a 14:10 hour light-dark cycle for at least 1 week before use. Animals were cared for according to procedures approved by the Animal Care Committees of the Marine Biological Laboratory and the Women and Infants Hospital. Institutional review board approval was obtained from Marine Biological Laboratory and the Women and Infants Hospital. Six-week-old CD-1 mice were purchased from Charles River Laboratory (Boston, MA) and superovulated by intraperitoneal injection of 5 IU of PMSG. Arsenite and N-acetylcysteine Solutions Stock solutions (1 mL/mL) of sodium arsenite were prepared in double-distilled water and sterilized with a 0.22-␮msyringe filter. Working arsenite concentrations of 2, 4, and 8 ␮g/mL were obtained by dissolving arsenite stock solution in IVM media. Stock solutions were stored in the dark at 4°C and used within 1 week. Dose–response and exposure time curves to arsenite treatment (first 2 hours or 12–14 hours) were generated to establish effects of in vitro treatment with varying arsenite concentrations and exposure times in meiosis. A stock solution (100 mg/mL) of N-acetylcysteine was prepared in double-distilled water and sterilized with a 0.22-␮m syringe filter. A working solution of N-acetylcysteine (30 1188

Navarro et al.

Arsenite induces aberrations in meiosis

mM) was obtained by dissolving N-acetylcysteine stock solution in IVM media containing 2 ␮g/mL of sodium arsenite. Collection of Oocytes and IVM Isolation and culture of immature oocytes was performed according to accepted scientific procedures (27). To obtain immature oocytes, females were killed by cervical dislocation 44 – 48 hours after PMSG injection. Cumulus– oocyte complexes were isolated by puncturing ovarian follicles into a modified N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid (HEPES)– buffered potassium simplex optimized medium (KSOM) containing 14 mM of HEPES and 4 mM of sodium bicarbonate (NaHCO3). Nude oocytes with no cumulus cell layers were excluded from experiments. Cumulusintact oocytes at the germinal vesicle (GV) stage were washed three times in minimum essential medium (MEM; GibcoBRL, Grand Island, NY) containing 10% fetal bovine serum and 5 IU of PMSG per milliliter (IVM media). They then were cultured in IVM media under mineral oil at 37°C in an atmosphere of 7% CO2 in humidified air for 12–14 hours or 15–17 hours. After culture, cumulus cells were removed by gentle pipetting in HEPES-buffered KSOM containing 0.03% hyaluronidase, and denuded oocytes were imaged by using a PolScope (Cambridge Research and Instrumentation, Boston, MA) or fixed for immunostaining and fluorescent microscopy, as described elsewhere (28, 29). Immunofluorescence Microscopy of Tubulin and Chromatin Denuded oocytes were fixed and extracted for 30 minutes at 37°C in a microtubule-stabilizing buffer. Oocytes were washed extensively and blocked overnight at 4°C in the wash medium (phosphate buffered saline (PBS) supplemented with 0.02% NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% goat serum, 2% bovine serum albumin (BSA) and 0.1 M of glycine) (28, 29). They were then incubated with ␤-tubulin mouse monoclonal antibodies (1:150), washed again, and incubated with fluorescein isothiocyanate– conjugated anti-mouse IgG (1:200; Molecular Probes, OR) at 37°C for 2 hours. After being washed, the samples were stained for DNA with Hoechst 33342 (10 ␮g/mL) in Vectashield mounting medium (H-1000, Vector, Burlingame, CA) on a glass slide and sealed. The samples were observed under a Zeiss Axiovert 100T (Thornwood, NY) inverted fluorescence microscope. PolScope Imaging To determine the effects of arsenite on spindles, we used the noninvasive PolScope method that has been shown to have a high degree of concordance with the more invasive immunofluorescence method. Mouse oocytes were imaged with a Zeiss Axiovert 100T inverted microscope equipped with a Cohu analogue video camera and PolScope hardware consisting of liquid crystals and electro-optical controller (30 –32). Settings of the liquid crystals were computer controlled Vol. 85, Suppl 1, April 2006

FIGURE 1 Immunofluorescence images of spindles and chromosomes of CD-1 mouse oocytes from control (A) and in vitro arsenite-treated groups (B–D). (A) Chromosome alignment over the metaphase II plate of a normal spindle. (B) Chromosome misalignments over a disrupted meiosis I spindle 12–14 hours in culture after in vitro treatment with 2 ␮g/mL of arsenite for 2 hours. (C) Chromosome misalignments over a disrupted telophase I spindle 12–14 hours in culture after in vitro treatment with 2 ␮g/mL arsenite for 2 hours. (D) Severe chromosome spreading over a disrupted meiosis II spindle 15–17 hours in culture after in vitro treatment with 2 ␮g/mL arsenite for 2 hours. Green, meiosis spindles stained by anti-␤-tubulin and fluorescein isothiocyanate– conjugated secondary antibody; blue, chromosomes stained by Hoechst 33342; red, actin-filaments. Scale bar ⫽ 10 ␮m.

Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

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1189

5.0 43.5 10.0 21.7 15.0

Exposure to 2 ␮g/mL of arsenite for 12–14 hours or to 8 ␮g/mL of arsenite for 2 hours arrested oocyte maturation at the GV stage (64.7% and 80%, respectively) or at the GV breakdown stage (35.3% and 20%, respectively) after 12–14 hours of IVM culture, indicating marked blockade of meiotic maturation. Exposure to 4 ␮g/mL of arsenite for 2 hours arrested oocyte maturation at metaphase I stage in 95% of exposed oocytes (80% exhibiting abnormalities) after 12–14 hours in IVM. After 12–14 hours in IVM, of the oocytes exposed to 2 ␮g/mL of arsenite for 2 hours, only 15% reached the meiosis II stage (5% exhibiting abnormalities). After 12–14 hours in culture, exposure to 2 or 4 ␮g/mL of arsenite for 2 hours caused, respectively, meiotic anomalies in 75% and 80% of treated oocytes and aberrations in 60% and 80% of meiosis I (MI) oocytes. As shown in Figure 1, exposure to 2 ␮g/mL of arsenite for 2 hours caused chromosome misalignment and spindle disruption in metaphase I (Fig. 1B) and telophase I (Fig. 1C) oocytes after 12–14 hours in IVM culture. After 15–17 hours in IVM culture, 65.2% of oocytes treated with 2 ␮g/mL of arsenite for 2 hours reached the MII stage, with 43.5% exhibiting abnormalities (Fig. 1D). This indicates that arsenite delayed and compromised the IVM process (Table 1).

3 10.0

Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

15–17 h in IVM culture. All others: 12–14 h in IVM culture. a

5.0 1 80.0 20

5

20.0

2 10.0 2 35.3 5.0 6 1 11.1 64.7 3 11

27 17 20 23 20 25 12–14 12–14 2 2 2 2 0 2 2 2a 4 8

n

RESULTS In Vitro Treatment With Arsenite Induces Meiotic Aberrations During IVM Among the control group oocytes, 88.9% had normal meiosis II (MII) spindles. The chromosomes were well aligned in the mid region of normal spindles after 12–14 hours in IVM culture (Fig. 1A).

2 5 60.0 34.8 80.0 12 8 16

88.9 24

% n % n % n % %

n

Normal GV

Arsenite (␮g/mL)

Exposure time (h)

Total no. of oocytes

Navarro et al.

1 10

% n % n % n

Abnormal Normal Abnormal

Normal

Abnormal

Metaphase II Metaphase I Telophase I

GV breakdown

Immunofluorescence microscopy of spindle and chromatin of mouse oocytes exposed in vitro to varying arsenite concentrations after IVM culture.

TABLE 1 1190

through MetaMorph/PolScope imaging software (Universal Imaging Corp., Boston, MA). Oocytes were imaged at 37°C in HEPES-buffered KSOM in a plastic Petri dish with a glass bottom (MatTek Corp., Ashland, MA). The chambers and the microscope were enclosed in a custom-made, insulated, heated box for optimal thermal control.

Arsenite induces aberrations in meiosis

As previously described, the PolScope allowed visualization of abnormal as well as normal spindles in living oocytes, without the need for fixation and staining. As depicted in Figure 2, the PolScope retardance image shows details of birefringent metaphase spindle fibers oriented parallel to the cortical membrane. The chromosomes are minimally birefringent and therefore appear as dark regions across the mid-region of the spindle. Among the control oocytes, 89.5% exhibited characteristic barrel-shaped spindles (Fig. 2A). Exposure to 2 ␮g/mL of arsenite for 12–14 hours or to 8 ␮g/mL of arsenite for 2 hours arrested oocyte maturation at the GV (80%) or GV breakdown (20%) stage after 12–14 hours in IVM culture. After 12–14 hours in culture, exposure to 2 or 4 ␮g/mL of arsenite for 2 hours resulted in, respectively, meiotic anomalies in 74.9% and 90.9% of oocytes and aberrations in 58.3% and 81.8% of MI spindles. As shown in Figure 2B, exposure to 2 ␮g/mL of arsenite for 2 hours caused spindle aberration in some MI oocytes after 12–14 Vol. 85, Suppl 1, April 2006

FIGURE 2 PolScope imaging of CD-1 mouse oocytes from control (A) and in vitro arsenite-treated groups (B, C). (A) Normal meiosis II spindles. (B) Disrupted meiosis I spindles 12–14 hours in vitro maturation (IVM) culture after in vitro treatment with 2 ␮g/mL arsenite for 2 hours. (C) Disrupted meiosis II spindles at 15–17 hours in IVM culture after in vitro treatment with 2 ␮g/mL arsenite for 2 hours. Scale bar ⫽ 10 ␮m.

Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

hours in IVM culture. After 15–17 hours in IVM culture, 72.7% of oocytes treated with 2 ␮g/mL of arsenite for 2 hours reached the MII stage, with 45.4% exhibiting spindle abnormalities (Fig. 2C). These data were consistent with those from fluorescence microscopy imaging (Table 2). N-Acetylcysteine Protection Against In Vitro Arsenite-Induced Meiotic Abnormalities Although exposure to 2 ␮g/mL of arsenite for 2 hours caused meiotic anomalies (characterized by spindle disruption and/or chromosomal misalignment) in 74.9% and 75% (PolScope and immunofluorescence imaging, respectively) of treated oocytes after 12–14 hours in IVM culture, co-administration of N-acetylcysteine prevented the meiotic abnormalities and the delayed IVM induced by arsenite, normalizing 81.8% and 89.3% (PolScope and immunofluorescence imaging, respectively) of MII oocytes after 12–14 hours in IVM culture, analogous to that seen in control oocytes (Table 3; Fig. 3). DISCUSSION Our data demonstrate that in vitro treatment with arsenite caused oocyte meiotic abnormalities and that in vitro coadministration of the ROS scavenger N-acetylcysteine prevented these abnormalities. Therefore, we can presume that arsenite effects on the meiotic process are ROS mediated and could thus be prevented by administration of antioxidants. Data on the reproductive and developmental toxicity of inorganic arsenic in humans are limited to a few studies of populations exposed to this environmental pollutant (11–14), and interpretation of these data is restrictive because study Fertility and Sterility姞

populations were typically exposed to multiple chemicals (11–14). The arsenite-induced meiotic abnormalities seen in the present study could explain, at least in part, the potential negative effects of arsenite on reproduction. It is well established that meiotic abnormalities can contribute to developmental failure through several pathways (18 –20). Some errors in meiotic maturation lead to genetic abnormalities that compromise embryo viability or impair cleavage at the preor post-implantation stages of development (18 –20). Indeed, in vivo administration of arsenite to mice disrupts development and increases apoptosis in mouse embryos (26), which could, hypothetically, be responsible for increasing spontaneous abortion, as has been described in some populations exposed to this environmental pollutant (11–14). The mechanism underlying the effects of arsenite on meiosis is not completely clear at this time but presumably involves mitochondrial dysfunction and oxidative stress (21– 24). We previously have demonstrated that arsenite promotes oxidative stress and that ROS could be important mediators that link arsenite toxicity, mitochondrial dysfunction, telomere shortening/erosion, genomic instability, and compromised cell viability (25). Oxidative stress damages proteins (33, 34), and the damage may involve microtubules and small molecules important for spindle organization (35–37). Oxidative stress also directly damages DNA (38, 39) and induces chromosome misalignment (25). The fact that in vitro supplementation of N-acetylcysteine prevented arsenite-induced meiotic aberrations in mouse oocytes suggests that the effects of arsenite are likely ROS mediated and may potentially be prevented by administration of antioxidants. This finding also provides new perspectives on possible antioxidant protection 1191

1192

TABLE 2 Navarro et al.

PolScope imaging of spindles of mouse oocytes exposed in vitro to varying arsenite concentrations after IVM culture.

Arsenite induces aberrations in meiosis

Arsenite (␮g/mL)

Exposure time (h)

Total no. of oocytes

12–14 12–14 2 2 2 2

19 10 12 11 11 10

0 2 2 2a 4 8 a

GV n

%

n

%

2 8

10.5 80.0

2

20.0

8

Telophase I

GV breakdown

80.0

2

Normal n

Metaphase I

Abnormal

%

n

Normal

%

1

8.3

1

8.3

1

9.1

1

9.1

n

Abnormal

%

1

Metaphase II

n

8.3

%

7 3 9

58.3 27.3 81.8

Normal n

%

17

89.5

1 3

8.3 27.3

Abnormal n

%

1 5

8.3 45.4

20.0

15–17 h in IVM culture. All others: 12–14 h in IVM culture.

Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

TABLE 3 Immunofluorescence microscopy and PolScope imaging of spindles in mouse oocytes demonstrating the protection conferred by in vitro co-administration of N-acetylcysteine against arsenite-induced meiotic toxicity.

Imaging method Vol. 85, Suppl 1, April 2006

Fluorescence PolScope

Treatment

Exposure time (h)

Total no. of oocytes

As As ⫹ NAC As As ⫹ NAC

2 2 2 2

20 28 12 11

GV breakdown

Telophase I Normal

Metaphase I

Abnormal

n

%

n

%

n

%

1 3

5.0 10.7

2

10.0

2

10.0

1

8.3

1

8.3

2

18.2

Normal n

1

%

8.3

Abnormal

Normal

Abnormal

n

%

n

%

n

%

12

60.0

5.0

58.3

10.0 89.3 8.3 81.8

1

7

2 25 1 9

1

8.3

Note: Dose: 2 ␮g/mL of arsenite for 2 h; NAC: 30 mM for 2 h; IVM culture: 12–14 h. As ⫽ arsenite; NAC ⫽ n-acetylcysteine. Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

Metaphase II

FIGURE 3 PolScope (upper panel) and immunofluorescence images (lower panel) of CD-1 mouse oocytes 12–14 hours in vitro maturation (IVM) culture. Control oocytes show normal meiosis II (MII) spindles and chromosome alignment at the metaphase plate. Oocytes treated with 2 ␮g/mL of arsenite (As) for 2 hours exhibit disrupted spindles and/or chromosome misalignment (aberrant meiosis I is shown). Oocytes treated with 2 ␮g/mL of arsenite plus 30 mM N-acetylcysteine (NAC) for 2 hours (As ⫹ NAC) display normal MII spindles and chromosome alignment at the metaphase plate, comparable to those of controls. Green, spindles stained by anti-␤-tubulin and fluorescein isothiocyanate– conjugated secondary antibody; blue, chromosomes stained by Hoechst 33342. Scale bar ⫽ 10 ␮m.

Navarro. Arsenite induces aberrations in meiosis. Fertil Steril 2006.

against arsenite-induced meiotic toxicity in various mammalian species, including humans. This potential merits further study. Our data demonstrate that disrupted spindles, detected through both fluorescence microscopy and PolScope imaging, generally were accompanied by chromosome misalignment. Both methods revealed similar percentages of meiotic abnormalities, although the PolScope images only spindle characteristics. This suggests the possibility of using noninvasive measures to diagnose meiotic aberrations in mammalian oocytes through PolScope image observation of spindle morphology. Fertility and Sterility姞

It has been shown that human oocytes with birefringent spindles produce higher rates of fertilization and development than those with no spindles (40). Considering the facts that in the present study, the PolScope successfully differentiated between oocytes with visible spindles and those without, and that the majority of oocytes with intact spindles presented normal alignment of metaphase chromosomes, the PolScope may prove useful in screening for arsenite effects on spindle architecture in arsenite-exposed women undergoing IVF procedures. This also warrants further investigation. Our findings demonstrated that in vitro arsenite exposure caused meiotic abnormalities that were prevented by in vitro 1193

co-administration of the ROS scavenger N-acetylcysteine. Furthermore, this study may provide a model for evaluating the meiotic effects of other heavy metals. REFERENCES 1. Borum DR, Albernathy CO. Human oral exposure to inorganic arsenic. Environ Geochem Health 1994;16:21–30. 2. Cantor KP. Arsenic in drinking water: how much is too much? Epidemiology 1996;7:113–5. 3. International Agency for Research on Cancer [IARC]. IARC Monogr Eval Carcinog Risks Hum Suppl 1982;Suppl 4:50 –1. 4. Ferm VH, Carpenter SJ. Malformations induced by sodium arsenate. J Reprod Fertil 1968;17:199 –201. 5. Hood RD. Effects of sodium arsenite on fetal development. Bull Environ Contam Toxicol 1972;7:216 –22. 6. Beaudoin AR. Teratogenicity of sodium arsenate in rats. Teratology 1974;10:153– 8. 7. Hood RD, Harrison WP. Effects of prenatal arsenite exposure in the hamster. Bull Environ Contam Toxicol 1982;29:671– 8. 8. Hood RD, Vedel GC, Zaworotko MJ, Tatum FM, Meeks RG. Uptake, distribution, and metabolism of trivalent arsenic in the pregnant mouse. J Toxicol Environ Health 1988;25:423–34. 9. Domingo JL, Gomez M, Sanchez DJ, Llobet JM. Effects of monoisoamyl meso-2, 3-dimercaptosuccinate on arsenite-induced maternal and developmental toxicity in mice. Res Commun Mol Pathol Pharmacol 1995;89:389 – 400. 10. Nemec MD, Holson JF, Farr CH, Hood RD. Developmental toxicity assessment of arsenic acid in mice and rabbits. Reprod Toxicol 1998; 12:647–58. 11. Beckman L. The Ronnskar smelter. Occupational and environmental effects in and around a polluting industry in northern Sweden. Ambio 1978;7:226 –31. 12. Nordstrom S, Beckman L, Nordenson I. Occupational and environmental risks in and around a smelter in northern Sweden. V. Spontaneous abortion among female employees and decreased birth weight in their offspring. Hereditas 1979;90:291– 6. 13. Aschengrau A, Zierler S, Cohen A. Quality of community drinking water and the occurrence of spontaneous abortion. Arch Environ Health 1989;44:283–90. 14. Borzsonyi M, Bereczky A, Rudnai P, Csanady M, Horvath A. Epidemiological studies on human subjects exposed to arsenic in drinking water in Southeast Hungary. Arch Toxicol 1992;66:77– 8. 15. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996;11:2217–22. 16. Angel R. First-meiotic-division nondisjunction in human oocytes. Am J Hum Genet 1997;61:23–32. 17. Volarcik K, Sheean L, Goldfarb J, Woods L, Abdul-Karim FW, Hunt P. The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum Reprod 1998;13:154 – 60. 18. Armstrong DT. Effects of maternal age on oocyte developmental competence. Theriogenology 2001;55:1303–22. 19. Lonergan P, Monaghan P, Rizos D, Boland MP, Gordon I. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization and culture in vitro. Mol Reprod Dev 1994;37:48 –53.

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