Reproductive Toxicology 22 (2006) 51–55

Mancozeb adversely affects meiotic spindle organization andfertilization in mouse oocytes

Gianna Rossi a, Maria Grazia Palmerini b, Guido Macchiarelli b,Roberto Buccione c, Sandra Cecconi a,∗

a Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di L’Aquila, 67100 L’Aquila, Italyb Dipartimento di Medicina Sperimentale, Universita degli Studi di L’Aquila, 67100 L’Aquila, Italy

c Dipartimento di Biologia Cellulare ed Oncologia, Consorzio Mario Negri Sud, Santa Maria Imbaro, 66030 Chieti, Italy

Received 10 August 2005; received in revised form 7 November 2005; accepted 14 November 2005Available online 10 January 2006

Abstract

In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilizationwere analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to1Ascpo©

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�g/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium.lthough, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in

pindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter cultureondition, oocytes matured in the presence of 0.1 and 1 �g/ml mancozeb showed a significant reduction also in the formation of male and femaleronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubularrganization during meiotic maturation.

2005 Elsevier Inc. All rights reserved.

eywords: Meiotic maturation; Mouse; Oocyte; Fertilization; Spindle; Chromosome organization

. Introduction

Many environmental toxicants, e.g. pesticides, dioxins,lkylphenolic chemicals, phtalates and synthetic estrogens, areeferred to as endocrine disruptors, due to their ability toodify processes governed by hormones such as estrogens,

ndrogens and thyroid hormones [1]. Both male and femaleeproductive systems are particularly vulnerable to these chem-cals, likely by disruption of feedback mechanisms along theypothalamus/pituitary–gonadal axis [2–4].

More than 80% of pesticides available for various homend agricultural use are either organophosphate or carbamateompounds. Dithiocarbamates (DTCs) and ethylenebisdithio-arbamates (EBDCs) are generally used to protect many fruits,egetables and field crops against a large spectrum of fungaliseases. Despite their short environmental persistence, one of

∗ Corresponding author. Tel.: +39 0862 433459; fax: +39 0862 433433.E-mail address: cecconi@univaq.it (S. Cecconi).

their main degradation products, ethylenethiourea (ETU), hasbeen found in soil even after 5–10 weeks from its distribution[5]. Beside occupational risks [6], the potential effects of thesechemicals on the general population derives from chronic dietaryexposure to residues left on foods [7] and on tobacco, so thatsmoke can represent a chronic source of absorption for smokersand non-smokers [8].

Among EBDCs, mancozeb is one of the most widely usedbecause of its low toxicity (LDB50B = 8 g/kg/day in rats) [9].Although its mutagenic effect is still controversial [10,11], itis considered a potent carcinogenetic agent [12]. In exposedrodents, two-generation fertility studies [13] evidenced thatmancozeb dose-dependently impairs male and female reproduc-tive potential by damaging testes and increasing the number ofatretic follicles [14–17].

An in vitro assay to assess mancozeb toxicity has beendeveloped by Greenlee et al. [18], who demonstrated that thiscompound increases apoptosis of mouse blastomeres. How-ever, nothing is known about the potential effects exerted bythe fungicide on the ability of mammalian oocytes to complete

890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2005.11.005

52 G. Rossi et al. / Reproductive Toxicology 22 (2006) 51–55

meiotic maturation and to be fertilized. In this paper we haveaddressed these issues by using an in vitro approach, in whichmouse preovulatory oocytes have been matured in the presenceof increasing concentrations of mancozeb and then assessed formeiotic maturation and fertilization rate.

2. Materials and methods

2.1. Experimental animals

Swiss CD1 mice (Harlan Italy, Udine, Italy) were housed in individual cageswith 12 h:12 h light:dark regimen, and at a room temperature of 21 ± 1 ◦C. Allanimal experimentation described in this article was conducted in accordancewith accepted standards of humane animal care. All experimental protocolswere approved by the local committees on animal care and use and accordingto accepted veterinary medical practice.

2.2. Chemicals

All the reagents were obtained from Sigma Chemical Co. (St. Louis, MO,USA), unless stated otherwise. Mancozeb (>99% pure) was purchased fromAccuStandard, Inc. (New Heaven CT). Fluorescein (FITC)-conjugated affin-ity pure donkey anti-mouse IgG was obtained from Jackson ImmunoResearchLaboratories, Inc. (West Grove, PA, USA).

2.3. In vitro maturation and fertilization of oocyte-cumulus cellcomplexes in the presence of mancozeb

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2.5. Immunofluorescence microscopy

Oocytes were fixed as described by Cekleniak et al. [26]. Briefly, cellswere incubated at 37 ◦C for 30 min in a microtubule stabilizing buffer (2%formaldehyde, 0.1% Triton-X-100, 1 �M of taxol, 10 IU/ml of aprotinin, and50% deuterium oxide), were washed and incubated with monoclonal anti �,�-tubulin (1:150), and then with donkey anti-mouse IgG-FITC (1:50), for 1 h at37 ◦C. Finally the oocytes were labelled with propidium iodide (50 �g/ml) andmounted on slides. Labelled oocytes were analyzed using an Olympus Flu-oview FV500 confocal laser scanning biological microscope, equipped with60× objective, and images were recorded using Fluoview Software.

2.6. Statistic analysis

All the experiments were repeated at least three times. Data were expressedas the mean ± S.E.M. and compared by ANOVA followed by Tukey–Kramer test(GraphPad InStat 2.03, GraphPad Software) for comparison of multiple groups;data expressed as percentages were compared by chi-square test. P < 0.05 wasconsidered statistically significant.

3. Results

3.1. Effects of mancozeb on oocyte meiotic maturation andspindle formation

In order to test whether mancozeb could adversely affectoocyte meiotic maturation, OCCs were subjected to IVM in thepresence of increasing concentrations of the fungicide (0.001,0ccDo

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Oocyte-cumulus cell complexes (OCCs) were retrieved from 23- to 25-day-ld Swiss CD1 mice (Harlan Italy, Udine, Italy). Follicular development wastimulated by 5 IU of Pregnant Mare Serum Gonadotropin (PMSG; Folligon,ntervet Italia, Milan, Italy). OCCs were matured in vitro (IVM) at 37 ◦C, and% CO2 in air in alpha MEM supplemented with 0.23 mM pyruvate, 2 mM l-lutamine, in the absence (control) or in the presence of low concentrations ofhe pesticide, ranging from 0.001 to 1 �g/ml. These concentrations were selectedn the basis of results on mouse embryo toxicity at 0.003 �g/ml mancozeb [18]nd on the capacity of inducing lymphocyte proliferation at 0.1 and 1 �g/mlancozeb [19]. Moreover, these concentrations are based on the 1× reference

ose (RfD) value reported in the U.S. EPA [20], and converted from RfD units toicrograms per milliliter as reported by Greenlee et al. [18]. A stock solution ofancozeb (50 �g/ml, 50×) was prepared by resuspending the fungicide either in

ulture medium or vehicle (DMSO: dimethyl sulfoxide); in the latter case, finalorking concentrations were obtained by diluting with culture medium. Thenal volume of vehicle added to the samples never exceeded 0.1% (v/v), ando adverse effects on maturation were observed [21]. After 16 h, control andxposed oocytes were deprived of cumulus cells and scored for their meiotictatus. Oocytes arrested at metaphase II (MII), as evidenced by extrusion ofhe first polar body (PB1), were sorted into two groups: one was analyzed forssessment of meiotic spindle and chromosome organization, the other one wastilized for in vitro fertilization (IVF) experiments.

.4. Preparation of spermatozoa and in vitro fertilization

Spermatozoa, obtained from the cauda epididymis of adult male mice, wereapacited in T6 medium [22] for 2 h at 37 ◦C, 5% CO2 in air. Only suspensionshowing excellent sperm motility, evaluated by using phase-contrast microscopy,ere used for insemination at a final concentration of 5 × 104 spermatozoa ml−1.

For in vitro fertilization, oocytes were deprived of the zona pellucida by arief treatment with Tyrode’s solution (pH 2.5) in order to synchronize fertiliza-ion process [23], transferred for 1 h in M16 medium and then inseminated forh. After careful washing, oocytes were further cultured at 37 ◦C, 5% CO2 inir in T6 medium for 6 h. At this time, the eggs were examined for the presencef the male and female pronuclei (2PN) [24,25].

.01, 0.1 and 1 �g/ml). At the end of maturation period, the per-entage of oocytes extruding PB1 was similar under all cultureonditions tested (about 95%; P > 0.05). The presence of 0.1%MSO, either in control or in mancozeb samples, had no effectn meiotic maturation.

In order to determine whether the fungicide could affectpindle microtubule integrity and/or chromosome alignmentn metaphase plate, oocytes were analyzed by immunofluores-ent staining. About 98% of all control oocytes showed nor-ally assembled spindles, located at the periphery of the cell,ith well-aligned chromosomes on metaphase plate (Fig. 1A).y contrast, mancozeb induced a dose-dependent increase in

pindle morphology alterations, that was significantly higherhen 1 �g/ml of the fungicide was diluted in culture medium

40 ± 2%; Table 1) than in DMSO (31 ± 1%; Table 2). Elon-ation of microtubules (Fig. 1B) was the most common formf spindle alterations; however, a reduction spindle length was

able 1pindle morphology of mouse oocytes matured in vitro in the presence of

ncreasing concentrations of mancozeb resuspended with culture medium

ancozeb�g/ml)

No. ofoocytes

Spindle morphology

Normal (%) Altered (%) Abnormal (%)

(control) 83 98 ± 2 a 0 2 ± 1 a.001 78 97 ± 2 a 1 ± 1 a 2 ± 1 a.01 75 95 ± 2 a 4 ± 2 a 1 ± 1 a.1 80 74 ± 3 b 23 ± 2 b 3 ± 1 a

80 56 ± 2 c 40 ± 1 c 4 ± 1 a

ifferent letters within the same column indicate significant differenceP < 0.05).

G. Rossi et al. / Reproductive Toxicology 22 (2006) 51–55 53

Fig. 1. Representative images of spindles and chromosomes in IVM oocytes. (A) Normally assembled spindles, located at the periphery of the cell, with well-alignedchromosomes on metaphase plate. (B) Elongated and (C) short spindles with aligned chromosomes. (D) Abnormal spindle structure with dispersed chromosomes.Bar = 10 �m.

noted in 15% at 1 �g/ml treated oocytes (Sp Fig. 1C). As aconsequence, the interpolar distance of 26 ± 2 �m recorded forcontrol MII (Fig. 1A) ranged from to 34 ± 2 to 17 ± 2 �m(P < 0.05). The percentage of oocytes showing a completelyabnormal spindle configuration (Fig. 1D) was very low and notsignificantly different under both experimental conditions (seeTables 1 and 2).

Table 2Spindle morphology of mouse oocytes matured in vitro in the presence ofincreasing concentrations of mancozeb resuspended with DMSO as vehicle

Mancozeb (�g/ml) No. ofoocytes

Spindle morphology

Normal (%) Altered (%) Abnormal (%)

0 (solvent-control) 76 98 ± 2 a 0 2 ± 1 a0.001 85 97 ± 2 a 1 ± 1 a 2 ± 1 a0.01 72 94 ± 3 a 4 ± 3 a 2 ± 2 a0.1 75 72 ± 2 b 25 ± 2 b 3 ± 1 a1 70 65 ± 2 c 31 ± 2 c 4 ± 2 a

Different letters within the same column indicate significant difference(P < 0.05).

3.2. Effects of mancozeb on oocytes fertilizability

Given previous results, only OCCs exposed to 0.1 and1 �g/ml mancozeb diluted in culture medium during IVM, werein vitro fertilized. As reported in Fig. 2, while about 80% ofcontrol oocytes formed both male and female pronuclei, thefungicide negatively affected IVF rates, as the proportion of

Fig. 2. Effects of increasing mancozeb concentrations on the % of PB1 emissionand of 2PN formation in IVM exposed oocytes (n = 100–120 oocytes/treatment).Different superscripts indicate significant difference (P < 0.05).

54 G. Rossi et al. / Reproductive Toxicology 22 (2006) 51–55

oocytes forming 2PN was 58 ± 2 and 43 ± 1%, respectively(P < 0.05).

4. Discussion

Here we demonstrate that exposure of mouse oocytes to man-cozeb during IVM can interfere, in a dose-dependent manner,with the formation of normal meiotic spindle and with fertiliza-tion rate. Furthermore, the negative effect of the fungicide onspindle organization was increased when the fungicide powderwas resuspended in culture medium instead of vehicle.

Results from in vivo studies support the idea that man-cozeb and related dithiocarbamates exert detrimental effects onthe reproductive process, by inducing a generalized hormonalimbalance that affects gonadotropin secretion [27] and, conse-quently, reproductive performance of both sexes [28–30]. Onthe other hand, in vitro experiments designed to evaluate theeffects of various pesticides on the process of meiotic matura-tion in cultured mammalian oocytes [3,21,31,32], can representa rapid model system to assess cell vulnerability to low doses ofcontaminants.

A recent study by Greenlee et al. [18] has demonstratedthat mouse embryos cultured for several days in a very lowconcentration of mancozeb (0.003 �g/ml), considered harm-less to humans, undergo significant apoptosis of blastomeres.Our experiments, in which OCCs were exposed to low con-ciidoaossii[tSdowrOfmzaido

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reported that mammalian oocyte and embryo viability can beaffected also through the induction of massive cumulus cellapoptosis. Indeed, Campagna et al. [35] have demonstrated thatthe significant decrease in the number and quality of porcineblastocysts obtained from oocytes treated with increasing con-centrations of organochlorines during IVM is due to the induc-tion of massive cumulus cell apoptosis. Although under ourexperimental conditions we did not observe mancozeb-inducedcumulus cell apoptosis (data not shown), we cannot exclude theoccurrence of this process at higher concentrations of the fungi-cide.

Concerning possible exposure for humans, our experimentshave been performed using mancozeb concentrations rang-ing from that used by Greenlee et al. (0.003 �g/ml; [18]) formouse embryo toxicity test to those reported by Colosio et al.(0.1–1 �g/ml; [19]) and capable of inducing in vitro prolifer-ation of human lymphocyte. In a recent paper, using a wholeblood assay, Colosio et al. [36] found similar immunomodula-tory effects also in agricultural workers. Thus, it is possible forhumans to be exposed, although for a short-time, to mancozebconcentrations capable of inducing health impairment. Sincein vitro maturation process is very short, lasting 17 h in miceand 24 h in humans, it could be possible for female workers,especially in rural area, to be exposed to such concentrations incoincidence with oocyte maturation process. To our knowledge,no reports are present in literature concerning mancozeb con-ct[

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entrations of mancozeb (from 0.001 to 1 �g/ml) during then vitro maturation period, show that the fungicide does notnterfere with the process of polar body emission per se, butose-dependently interferes with the formation of normal mei-tic spindles and, after IVF, with the formation of 2PN. Spindlelterations here recorded are represented mainly by elongationf microtubules, but at the highest concentration also by shorterpindles. Both alterations are more frequent when the stockolution of mancozeb is prepared directly into culture mediumnstead of DMSO. This observation confirms that the fungicides more soluble and stable in aqueous solution than in DMSO18,33], and strongly suggests that studies assessing mancozeboxicity should be performed with culture medium as a vehicle.imilar results in terms of IVM rate and percentage of spin-le deformations have been obtained when commercial mixturef mancozeb (78% pure) was used (unpublished results), orhen IVM was performed in the presence of BSA (unpublished

esults). Spindle alterations have been described also for mouseCCs exposed during IVM to diethylstilbestrol [32] and to the

ungicide methyl 2-benzimidazolecarbamate [21]. In this case,ouse OCCs not only exhibit aberrant meiotic spindle organi-

ation, evidenced by small or completely disrupted spindles, butlso impairment of cell cycle progression. These observationsndicate that different pesticides can affect the same target inifferent ways, and that the simultaneous exposure to mixturesf these chemicals can dramatically increase cell injury.

Although in our study we did not analyze complete preim-lantation development, the observed reduction in the percent-ge of 2PN formation, that results from a series of orderedetabolic and regulative processes [34], can be considered as

rognostic of subsequent developmental failure [24]. It has been

entration in follicular fluid. However, since for other pesticideshe transfer from blood to follicular fluid has been demonstrated18], we could suppose that this is possible also for mancozeb.

In conclusion, in vitro maturation of mouse oocytes and spin-le analysis can be used as a sensitive test for assessing cellnjury due to pesticides, even when used at low concentrations. Itas been demonstrated that during the peri-ovulatory period thencreased follicular vascularization [37] may facilitate the trans-er of these chemicals to the maturing oocytes, and in fact manyesticides have been detected in human follicular fluid [38].his finding indicates that, besides the different mechanisms ofction, EBDCs can exert important toxic effects on mammalianocytes, and their role as inducers of infertility should not bender-rated.

cknowledgments

This study was granted by Ministero della Salute (Progettoi ricerca finalizzato anno 1998) and by MIUR 60% to S.C.nd G.M. Thanks are due to Prof. Mauro Maccarrone, Fulvioandolfi and Dott. Alberto Mantovani for critical reading of theanuscript.

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