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Biol Reprod 2003, 10.1095/biolreprod.103.020586
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BIOLOGY OF REPRODUCTION 70, 980–985 (2004)
DOI: 10.1095/biolreprod.103.020586
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

In Vivo Effects of Arsenite on Meiosis, Preimplantation Development, and Apoptosis in the Mouse1

Paula A.A.S. Navarro3,4,5, Lin Liu3,4, and David L. Keefe2,3,4,6

Department of Obstetrics & Gynecology,3 Women & Infants Hospital, Brown University, Providence, Rhode Island 02905 Marine Biological Laboratory,4 Woods Hole, Massachusetts 02543 Department of Obstetrics & Gynecology,5 Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14049-000, Brazil Department of Obstetrics & Gynecology,6 Tufts-New England Medical Center, Boston, Massachusetts 02111


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

apoptosis, early development, embryo, environment, meiosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Inorganic arsenic is ubiquitous in the environment, where it occurs mainly as compounds of arsenite (As3+) and arsenate (As5+). Small quantities of arsenic appear naturally in rocks, soil, water, air and foods [1] and higher levels can result from environmental contamination. Humans are exposed to arsenic from natural sources, with daily intake typically in the range of 12–40 µg/day [2, 3]. However, certain areas, such as parts of Taiwan, Mexico, Chile, Argentina, India, and the United States [4, 5], contain natural mineral deposits from which enough inorganic arsenic may leach into the water supplies to present a hazard to human health. In certain parts of Bangladesh and West Bengal, India, as many as 5% of sampled drinking wells have arsenic levels exceeding 1 mg/L, and 27% of wells have levels exceeding 300 µg/L [6], six times higher than the current U.S. maximum contaminant level of 50 µg/L. Although water supplies in the United States are generally low in arsenic, there have been reports of arsenic contamination of groundwater in the Southwest, with levels in the hundreds and, in few cases, more than 1,000 µg/L [7, 8]. Moreover, arsenic is a contaminant at a number of sites on the U.S. National Priorities List of hazardous waste sites [9] and the U.S. Environmental Protection Agency has placed arsenic at the top of its Superfund Contamination List [10].

In addition to chronic exposure from food and drinking water, inhalation exposure also may occur in individuals working in or living near sources of arsenic air pollution [11, 12]. Recently many countries and international agencies have lowered (from 50 µg/L to 10 µg/L) the acceptable levels for arsenic in drinking water set by the World Health Organization in 1994 (see Note Added in Proof) due to their increased preoccupation with the environmental contamination by this heavy metal and its consequences to human health.

Epidemiological studies have shown that chronic exposure to arsenic can result in liver injury, peripheral neuropathy, and increased incidence of cancers of the lung, skin, bladder, and liver [13]. Inorganic arsenic also has been extensively studied as a teratogenic agent in several mammalian species, and its potential for developmental toxicity has been experimentally investigated in several species of laboratory animals [14–20]. Data derived from well-conducted laboratory animal investigations can serve as a surrogate to predict human risk due to the nonexistence of good human data. Although humans most commonly are exposed to arsenite via the oral route, published developmental toxicity studies of inorganic arsenicals largely employ treatment by i.v. or i.p. routes, to ensure reliable levels of intake. However, most published developmental toxicity studies used doses of arsenite that were so high they were often maternally toxic.

Despite the large number of studies of the developmental toxicology of inorganic arsenic, data about its effect on meiosis, preimplantation development and embryonic apoptosis are lacking. Because arsenite uncouples mitochondria [21–26], and we previously demonstrated toxic effects of mitochondria uncoupling on oocyte and embryo development [27–29], we hypothesized that arsenite may disrupt oocyte and embryo development. We studied arsenite rather than arsenate because of its higher toxicicity [30]. The dose of 8 mg/kg, the maternal no-observed-adverse-effect level (NOAEL), previously established in mice [31] was used in addition to vehicle controls.

To test whether arsenite affects oocyte and embryo development in mammals, we investigated the effects of in vivo treatment with arsenite on meiotic progression of oocytes and on early embryo development and apoptosis in mice. Environmental arsenite exposure can occur by various routes, but we chose to deliver arsenite by i.p. injection to ensure reliable delivery and because most published reports employed this route of administration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Reagents and Animals

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Equine chorionic gonadotropin (eCG) used for superovulation was purchased from Calbiochem (La Jolla, CA). Mice were subjected to a 14L:10D cycle for at least 1 wk before use. Animals were cared for according to procedures approved by Marine Biological Laboratory and Women and Infants Hospital Animal Care Committees. Six-week-old CD-1 mice (Charles River Laboratory, Boston, MA) were treated with vehicle or 8 mg/kg or 16 mg/kg sodium arsenite every 2 days for a total of seven i.p. injections over a period of 14 days. Mice were superovulated by i.p. injection of 5 IU of eCG, followed 46–48 h later by i.p. injection of 5 IU hCG.

We avoided doses of arsenite that cause significant maternal toxicity, because such maternal toxicity might camouflage more specific effects of arsenite on meiosis and preimplantation embryo development. All doses of arsenite used in this study did not induce obvious systemic toxicity to animals.

Collection of Oocytes and Embryos and In Vitro Culture

To obtain ovulated oocytes, females were killed by cervical dislocation 14 h after hCG injection. Oocytes enclosed in cumulus masses were released from the oviductal ampullae into the modified Hepes-buffered potassium simplex optimized medium (HKSOM) containing 14 mM Hepes and 4 mM NaHCO3. Cumulus cells were removed by gentle pipetting in HKSOM containing 0.03% hyaluronidase [32]. Cumulus-free oocytes were washed in HKSOM three times, washed again in pre-equilibrated modified KSOM three times, and then cultured in vitro in KSOM at 37°C in 7% CO2 and humidified air for 30 min. Oocytes were imaged live using the Pol-Scope (Cambridge Research & Instrumentation, Woburn, MA) or after fixation and immunostaining using fluorescent microscopy, as described elsewhere [33, 34].

For in vivo fertilized oocytes, after injection of hCG, female mice were mated individually with 2-to 3-mo-old CD-1 males of proven fertility. To collect zygotes, successfully mated females were harvested 20–21 h after hCG injection. Zygotes enclosed in cumulus masses were released from the ampullae into HKSOM, with 0.03% hyaluronidase; then cumulus cells were removed by gentle pipetting. In vitro manipulation of embryos was carried out in HKSOM at 34–37°C on a heating stage. Modified KSOM, supplemented with nonessential amino acids and 2.5 mM Hepes, was used for in vitro culture [35–37]. Embryos were washed and cultured in 50 µl droplets of KSOM under mineral oil at 37°C in a humidified atmosphere of 7% CO2 in air. Embryos were assessed for cleavage at 24 h, morula formation at 72 h, and development to blastocyst at 96 h. The number of apoptotic cells was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in some embryos at96 h.

Immunofluorescence Microscopy of Tubulin and Chromatin

Denuded oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer [33, 34]. Oocytes were washed extensively and blocked overnight at 4°C in the wash medium (PBS supplemented with 0.02% NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% goat serum, 2% BSA, and 0.1 M glycine). Afterwards, oocytes were incubated with ß-tubulin mouse monoclonal antibodies (1:150), washed, then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:200; Molecular Probes, Eugene, OR) at 37°C for 2 h. After being washed, the samples were stained for DNA with Hoechst 33342 (10 µg/ml) in Vectashield mounting medium (H-1000; Vector Laboratories, Burlingame, CA) on a glass slide and sealed. The samples were observed under a Zeiss Axiovert 100TV (Carl Zeiss, Inc., Oberkochen, Germany) inverted fluorescence microscope.

Pol-Scope Imaging

To confirm effects of arsenite on spindles using a noninvasive method that has been shown to correlate highly with the more invasive immunofluorescence method, mouse oocytes were imaged using a Zeiss Axiovert 100TV inverted microscope equipped with a Cohu analogue video camera and Pol-Scope hardware consisting of liquid crystals and electro-optical controller (Cambridge Research & Instrumentation) [38, 39]. Settings of the liquid crystals were computer-controlled through MetaMorph/Pol-Scope imaging software (Universal Imaging Corp., Boston, MA). Oocytes were imaged at 37°C in HKSOM in a plastic Petri dish with a cover glass bottom (MatTek Corp., Ashland, MA). The chambers and the microscope were enclosed in a custom-made, insulated, heated box for optimal thermal control.

Detection of Apoptosis by TUNEL Assay

Embryos were fixed in 3.7% paraformaldehyde in Dulbeccos PBS containing 0.1% polyvinylpyrrolidone. Nuclear DNA fragmentation in embryos was detected by TUNEL method using the In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN), and nuclei were counterstained with propidium iodine (PI; 50 µg/ml; Molecular Probes), as described previously [32, 40]. Fluorescence was detected using a Zeiss Axiovert 100TV inverted fluorescence microscope. In addition, cytoplasmic fragmentation, cell shrinkage, and nuclear condensation were measured as indicative of apoptosis [41].

Statistical Analysis

Each experiment was repeated twice, and six mice were treated in each group. One-way ANOVA and Fisher protected least-significant difference using StatView software (1998; SAS Institute Inc., Cary, NC) were used for comparisons of treatment means, such as cell number. The chi-square test was used for analysis of differences in the case of percentage comparison, such as rate of development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 
Arsenite Disrupts Meiosis in Mouse Oocytes

The incidence of oocyte meiotic abnormalities, characterized by spindle disruption and/or chromosomal misalignment or fragmentation visualized under fluorescent microscopy after immunostaining, was significantly (P < 0.0001) increased in arsenite-treated groups. Even the previously established NOAEL dose was found to disrupt spindles: 25% of oocytes exhibited abnormalities after exposure to the 8 mg/kg dose and 62.5% of oocytes exhibited abnormalities after exposure to 16 mg/kg, compared to the vehicle control group, in which all oocytes exhibited normal spindles. Arsenite also disturbed the meiotic cell cycle: after 16 mg/kg arsenite, 5% of oocytes remained at germinal vesicle stage, 37.5% displayed abnormalities of metaphase (15% of oocytes with abnormalities of metaphase of meiosis I and 27.5%, of metaphase of meiosis II) and 20% exhibited fragmentation, whereas only 32.5% were normal metaphase II oocytes (Table 1 and Fig. 1A). In all control oocytes, chromosomes were well aligned over the metaphase plate of the meiotic spindle. The 8 mg/kg arsenite treatment caused spindle disruption and/or chromosome misalignment, which may lead to chromosome missegregation (Fig. 1B). The 16 mg/kg arsenite treatment produced chromosome misalignment and/or condensed chromosomes, as well as spindle disruption and extensive formation of cytoplasmic microtubule asters (Fig. 1, C and D). Moreover, cytoskeletal misarrangements, characteristic of early oocyte fragmentation, were observed in this arsenite-treated group.


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  		TABLE 1. Immunofluorescent microscopy of spindle and chromatin of in vivo mature oocytes from mice treated with arsenite.*



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  		FIG. 1. Representative immunofluorescence images of spindles and chromosomes of oocytes from control and arsenite (As)-treated CD-1 mice. Meiotic II spindles were stained by anti-ß-tubulin and FITC-conjugated 2nd antibody and chromosomes stained by Hoechst 33342. A) Control mouse oocyte with chromosome alignment on the metaphase spindle. B) Mouse oocyte treated with 8 mg/kg As, showing missegregated chromosomes at telophase (arrow). C and D) Mouse oocyte treated with 16 mg/kg As, showing misaligned chromosome (arrow) and spindle disruption (C), and microtubule asters (D) of the same oocyte but different focus plane. Bar = 10 µm

As described previously, the Pol-Scope allowed visualization of abnormal as well as normal spindles in living oocytes, without need for fixation and staining. As depicted in Figure 2, the Pol-Scope retardance image shows details of the 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. The lower percentage of meiotic abnormalities detected by this methodology (8 mg/kg, 15.4%; 16 mg/kg, 46.2% vs. no meiotic abnormalities following injection of vehicle) when compared to fluorescent microscopy after immunostaining (low dose, 25%; medium dose, 62.5%) presumably was due primarily to the limited ability of the Pol-Scope to image details of chromosomal alignment.



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  		FIG. 2. Pol-Scope imaging of oocytes from control and arsenite (As)-treated CD-1 mice. Control: normal meiotic II spindle. Treatment of 8 and 16 mg/kg As causes spindle shortening, elongation, or disruption. Bar = 10 µm

It is important to emphasize that in the two replicates of these experiments data were very similar, indicating that effects of in vivo treatment with arsenite on meiosis were repeatable in the different mice treated.

Arsenite-Induced Zygotic Cell Death and Compromised Preimplantation Development

Zygotes derived from arsenite-treated animals exhibited significantly lower (P < 0.05) rates of cleavage and development to blastocyst than those from the control group (Table 2 and Fig. 3). The effects of arsenite on cleavage and preimplantation development also exhibited dose-dependence. The percentage of cytofragmentation at 24 h was significantly higher (P < 0.05) after arsenite treatment, but did not significantly differ between the two arsenite doses.


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  		TABLE 2. Development of control and arsenite-treated mouse zygotes.*



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  		FIG. 3. Effects of arsenite (As) on cleavage and early development of mouse zygotes. In untreated control culture, an average of 87.3% of zygotes (n = 79) cleaved to 2-cell stage at 24 h and developed to blastocysts at 96 h. As-treated mouse zygotes showed compromised cleavage and development, and exhibited morphological hallmarks of apoptotic cell death, characterized by cytofragmentation. Arrows indicate shrunken or fragmented (24 h), and arrested or degenerated embryos (96 h). Bar = 50 µm

Blastocysts developed from the vehicle-treated control group had an average of 58.9 nuclei, with an average of 2.5% apoptotic nuclei observed by TUNEL stain (Table 3), a result similar to that reported previously [40]. The average number of nuclei was significantly decreased (P < 0.01) in the 16 mg/kg arsenite treated group compared to the vehicle and 8 mg/kg groups (Table 3). The percentage of apoptotic nuclei did not differ significantly between the vehicle- and 8 mg/kg arsenite-treated groups. The 16 mg/kg arsenite-treated group, on the other hand, exhibited apoptosis in 6.3% of nuclei, which was significantly higher than that exhibited in vehicle or 8 mg/kg treatment groups (P < 0.01).


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  		TABLE 3. Apoptosis in blastocysts developed from zygotes of control and arsenite-treated mice.*

It is important to emphasize that in the two replicates of these experiments data were highly similar, indicating that effects of arsenite on preimplantation development and apoptosis were repeatable in the different mice treated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
NOTE ADDED IN PROOF
 REFERENCES
 
Our data demonstrate that in vivo treatment with arsenite produces meiotic aberrations on in vivo-matured mouse oocytes, in some cases even with doses at the maternal no-observed-adverse-effect-level (8 mg/kg) [31]. Moreover, the effects of arsenite on meiosis clearly were dose-dependent and robust: even with arsenite at only the 16 mg/kg dose, 62.5% of oocytes evaluated showed evidence of meiotic abnormalities. Meiotic abnormalities can contribute to developmental failure by various pathways: 1) meiotic incompetence or inability to resume or complete meiotic maturation resulting in oocytes incapable of fertilization and 2) errors in meiotic maturation that compromise embryo viability [42–44].

Decreased cleavage rates, compromised preimplantation development, cell shrinkage, cytoplasmic fragmentation, and pronuclear condensation, consistent with the classical morphological definition of apoptosis [41, 45], also were induced by arsenite. Previously we demonstrated the ability of mitochondrial uncouplers and reactive oxygen species (ROS) to induce apoptosis in embryos [28]. The present results extend these findings to demonstrate that an environmental toxicant, also capable of increasing ROS, can promote apoptosis in preimplantation embryos. Indeed, considerable evidence suggests that ROS are involved in the genotoxicity of arsenite [21–23]. Using Chinese hamster ovary cells and x-ray-hypersensitive, DNA repair-deficient mutant XRS-5, Wang and Huang [24] showed that arsenite induces a dose-dependent increase in micronuclei that is blocked by exogenous catalase. In addition, heme oxygenase, an oxidative stress protein, and peroxidase are induced by sodium arsenite in various human cell lines [25]. Furthermore, antioxidant enzymes, such as superoxide dismutase, reduce the incidence of sister chromatid exchanges induced by arsenite in cultured human lymphocytes [26]. Mitochondria play a crucial role in the early stages of apoptosis. In many systems, the release of proapoptotic factors, such as cytochrome c or apoptosis-inducing factor, from the mitochondrial intermembrane space into the cytosol have been demonstrated to be a primary event in caspase activation and nuclear apoptosis [46–50]. Both loss of outer mitochondrial membrane integrity, leading to cytochrome c release, and inner membrane depolarization are caspase-activating agents that trigger the apoptotic cascade downstream of Bcl-XL [51]. Also in support of a major rule for mitochondria in apoptosis is the finding that oxidatively-stressed cytoplasm can transmit apoptotic potential to untreated, healthy nuclei after reconstitution by nuclear transfer [50]. Most likely, the significantly higher percentage of apoptotic nuclei detected in mouse blastocysts after in vivo treatment with arsenite was due to mouse zygote cytoplasmic dysfunction caused by arsenite. The extent to which arsenite exposure contributes to oocyte dysfunction in women is unknown at present, because we do not know the levels of arsenite that reach the reproductive tract of exposed women.

Our data demonstrate that in vivo treatment with arsenite induces meiotic abnormalities, compromised preimplantation development, and apoptosis in mouse zygotes in a dose-dependent manner. Presumably, these various effects of arsenite are correlated, because meiotic abnormalities can impair subsequent preimplantation development and increase apoptosis in embryos. These data open new perspectives about possible effects of arsenite on the reproductive system in mammals, including women, that require further investigation. Furthermore, this study may provide a rationale for evaluating the meiotic and preimplantation effects of other heavy metals.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
REFERENCES
 
For acceptable levels of arsenic in drinking water set by the World Health Organization, Guidelines for drinking water quality, vol. 1. Recommendations. Geneva: WHO. 1994.


   ACKNOWLEDGMENTS
 
We thank Dr. James R. Trimarchi for encouraging discussion.


   FOOTNOTES
 
1 This work was supported in part by the Women and Infants Hospital Faculty Research Fund and by a scholarship from Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Brazil to P.A.A.S.N. Back

2 Correspondence: David L. Keefe, Women and Infants Hospital, 101 Dudley Street, Providence, RI 02905. FAX: 401 453 7599;dkeefe{at}wihri.org Back

Received: 23 June 2003.

First decision: 28 July 2003.

Accepted: 15 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 NOTE ADDED IN PROOF
 REFERENCES
 

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