Résumé français

Les composés organochlorés s’accumulent dans l’environnement, plus particulièrement dans la région Arctique, et constituent un danger potentiel à la santé et au développement des animaux sauvages et des humains. Bien que leurs effets néfastes soient reconnus, leurs mécanismes d’action dans les cellules cibles ne sont pas tout à fait compris. Cette étude a été conçue pour tester l’hypothèse qu’une mixture d’organochlorés pertinente à l’environnement endommage l’ultrastructure des ovocytes chez le modèle porcin. Des complexes ovocyte-cumulus (COCs) immatures ou partiellement matures (18h), avec ou sans traitement organochloré ou leur véhicule (0,1 % diméthylsulfoxide ; DMSO) pendant leur culture, furent préparés pour la microscopie optique ou électronique à transmission. Les organochlorés ont induit des changements majeurs à l’ultrastructure des COCs : diminution de la densité des gouttelettes lipidiques, augmentation du volume du réticulum endoplasmique lisse (SER) et augmentation de l’interaction entre le SER, les mitochondries, les gouttelettes lipidiques et les vésicules. Nous suggérons que ces changements à l’ultrastructure démontrent une formation facilitée d’énergie nécessaire pour produire des enzymes du métabolisme. D’autres changements à l’ultrastructure peuvent refléter une certaine toxicité des organochlorés : réduction des jonctions ouvertes et diminution de la densité électronique des granules corticaux. Étonnamment, le traitement contrôle DMSO a aussi induit des changements similaires à l’ultrastructure, mais à un degré moindre que la mixture d’organochlorés. Cette étude est la première à démontrer l’effet de contaminants environnementaux sur l’ultrastructure des ovocytes de mammifères.

136

4.3 Article

An environmentally-relevant mixture of organochlorines and its

vehicle control, dimethylsulfoxide, induce ultrastructural

alterations in porcine oocytes

Céline Campagna1, Janice L. Bailey1, Marc-André Sirard1,Pierre Ayotte2 and Poul Maddox-Hyttel3

1_{Centre de recherche en biologie de la reproduction, Département des sciences animales,}

Université Laval, Québec, Canada. 2Unité de recherche en santé publique, Centre hospitalier universitaire de Québec-CHUL, Département de médecine sociale et préventive, Université Laval, Québec, Canada. 3Department of Basic Animal and Veterinary Sciences, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.

This research was supported by the International Academic Mobility program of the Human Resources Development of Canada and the Toxic Substances Research Initiative Program of Health Canada.

Correspondence: Janice L. Bailey, Centre de recherche en biologie de la reproduction,

Département des Sciences animales, Pavillon Paul-Comtois, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4. FAX: 1 418 656 3766; TEL: 1 418 656 2131 ext.3354; e-mail: janice.bailey@crbr.ulaval.ca

Short title: Organochlorines and oocyte ultrastructure.

Keyword : Polychlorinated biphenyls, in vitro maturation, toxicology, subcellular

4.4 Abstract

Organochlorine chemicals accumulate in the environment, particularly in the Arctic, and constitute potential developmental hazards to wildlife and human health. Although some of their harmful effects are recognized, their mechanisms of action within the target cells need to be better understood. This study was designed to test the hypothesis that an environmentally-relevant organochlorine mixture alters oocyte ultrastructure in the porcine model. Immature cumulus-oocyte complexes (COCs), partially matured (18h) COCs without treatment or exposed to the organochlorine mixture or its vehicle (0.1% dimethysulfoxide; DMSO) during culture were processed for light and transmission electronic microscopy. The organochlorines induced major ultrastructural changes in the COCs: decreased density of the lipid droplets, increased smooth endoplasmic reticulum (SER) volume and increased interactions among SER, mitochondria, lipid droplets and vesicles. We suggest that these ultrastructural changes facilitate energy formation necessary to produce metabolizing enzymes. Other ultrastructural changes may reflect some degree of organochlorine toxicity: fewer gap junctions and decreased electron density of the cortical granules. Unexpectedly, the DMSO control treatment also induced similar ultrastructural changes, but to a lesser degree than the organochlorine mixture. This study is the first to demonstrate the effect of environmental contaminants on mammalian oocyte ultrastructure.

138

4.5 Introduction

Although the use of numerous organochlorine chemicals is now prohibited or restricted in many countries, they continue to accumulate in the environment (AMAP 2004). The known harmfulness of organochlorines to the health of populations and to the environment calls for a better understanding of their mechanisms of action within the target cells, and especially those forming the future generation, the gametes. Not only will these cells be as exposed as any other, but they might transmit organochlorine-induced damage affecting the development or health of future individuals (Swain 1991; Trosko et coll. 1987).

Since oocyte maturation is a critical pre-requisite for subsequent fertilization and embryo development, its disruption has considerable potential to impair female reproduction (Beard and Rawlings 1999; Kaya et coll. 2002). Detectable levels of persistent organochlorines have been identified in the follicular fluid of women and other mammals, at concentrations that are strongly correlated (r up to 0.9) to serum concentrations (one to threefold the follicular fluid levels depending on the compound) (Jarrell et coll. 1993; Kamarianos et coll. 2003a; Pauwels et coll. 1999; Trapp et coll. 1984; Younglai et coll. 2002). Previously, we showed that an environmentally-relevant mixture of organochlorines alters the maturation, polyspermic penetration and developmental competence of porcine cumulus- oocyte complexes (COCs; (Campagna et coll. 2001) at a concentration 1000-fold higher than found in the blood of Inuit mothers from Nunavik in Arctic Canada (4.2 µg/ml total PCBs; (Muckle et coll. 2001). It also dramatically affected cumulus expansion and increased apoptosis in cumulus cells at environmentally-relevant levels. Negative effects on maturation, fertilization or embryonic development were also observed in bovine COCs exposed to organochlorines (Alm et coll. 1998; Pocar et coll. 2001).

Negative effects of organochlorines on cells are known to be mediated by various intracellular systems. Gap junctional communication is inhibited by lindane in rat myometrial myocytes (Criswell et coll. 1995), in rat hepatocytes by PCBs (Bager et coll. 1994), and in bovine oviductal cells by DDT and lindane (Tiemann and Pohland 1999). However, no study has evaluated the possible effects of environmental contaminants on mammalian oocyte ultrastructure. The main goal of this study, therefore, was to determine the effect of our environmentally-relevant organochlorine mixture on oocyte ultrastructure

before germinal vesicle breakdown, using the porcine model. During this time, COCs undergo important phases of cytoplasmic maturation, an essential prerequisite for proper embryonic development (Funahashi et coll. 1997). Taking into account our previous results of IVM-exposed COCs (Campagna et coll. 2001), we hypothesized that this mixture would cause noticeable changes in the ultrastructure of the oocytes during cytoplasmic maturation that might help explain their impaired function.