Effect of an environmentally relevant metabolized organochlorine mixture on porcine cumulus–oocyte complexes

Abstract

Organochlorine compounds and their metabolites bioaccumulate and have been detected in follicular and genital tract fluids of humans and animals. This study was designed to investigate the effect of a metabolised organochlorine mixture, extracted from plasma of sows treated with an environmentally relevant organochlorine mixture in the course of a previous study, on porcine cumulus–oocyte complexes (COCs) in vitro. The major component of the metabolised mixture is 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p′-DDE) at 15.1 mg/l, which accounts for 40.7% of the total extract. Polychlorinated biphenyls (PCBs) account for 30.8% of the extract and hydroxylated PCB metabolites (OH-PCBs) for 11.8%. Exposure of COCs to the metabolised mixture induced a decrease of apoptotic cumulus cells at low concentrations and an increase at higher concentrations following a U-shaped curve (p = 0.0106), with the intermediate treatment (3.6 μg/l OH-PCBs) significantly reducing apoptosis compared to the extraction control (p = 0.05). However, the metabolised mixture did not affect cumulus expansion, oocyte maturation, penetration, development to blastocyst, or the number of cells per blastocyst. This study also indicates that organochlorine metabolites similar in concentrations to levels found in Arctic populations can affect growing cumulus–oocyte complexes without inducing an overt toxicological response.

Introduction

Organochlorine compounds accumulate and bioamplify in the aquatic food web of circumpolar regions [1]. Several organochlorines such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), lindane, dichlorodiphenyl-trichloroethane (DDT) and hexachlorocyclohexane (HCH) are now formally banned or restricted under the United Nation's Stockholm Convention because of their physical properties and adverse effects on human health [1]. However, very high levels of these chemicals are still detected in wildlife species that are part of the traditional diet of aboriginal people in the Arctic region [1], [2], [3], [4].

In the body, organochlorine compounds are biotransformed into metabolites that may also be toxic and accumulate in the individual [2], [3], [4]. Exposure to organochlorines during pregnancy or lactation can adversely affect the reproductive system of the female offspring. In humans, exposure of mothers to DDT and its main metabolite p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) increased time to pregnancy and decreased fertility in the daughters [5]. In the rat, maternal PCB exposure decreased folliculogenesis in the female pups [6]. Exposing mice to lindane before and during early pregnancy lowered the number of blastomeres per morula and reduced the mitotic index of blastomeres [7].

Organochlorine compounds or their metabolites have been detected in semen, follicular fluid and genital tract fluid of humans [8], [9], [10], [11], [12], [13] and animals such as pigs, cattle and sheep [14], [15]. Levels of PCBs and organochlorines in follicular fluid are correlated to levels in plasma: follicular fluid concentrations were about one third of those in the serum [9], [12]. Developing oocytes are therefore directly exposed to organochlorines and their metabolites in the follicle.

Hence, exposing porcine oocytes during maturation to a mixture of organochlorines metabolized in vivo by sows and extracted from their plasma would be physiologically relevant. This would allow the oocytes to be exposed in vitro to the contaminants likely present in the ovaries in vivo. Therefore, not only would the oocytes be exposed to an environmentally relevant mixture of organochlorines as in our previous study [16], they would also be exposed to the mixture of organochlorine metabolites generated in vivo through biotransformation reactions. These metabolites could be ovotoxic by themselves or interact with the parent compounds to modulate their toxicity.

In an earlier study, we observed that exposing porcine oocytes to an environmentally relevant mixture of parent organochlorines decreased cumulus expansion, maturation and blastocyst formation [16]. The mixture also increased apoptosis in exposed cumulus cells and reduced the average number of cells in the blastocysts. Although the effects of organochlorines on oocyte maturation are becoming more established, no study has yet investigated the effects of direct exposure of a relevant organochlorine mixture containing both the parent compounds and their metabolites on oocyte competence during in vitro maturation.

This study was therefore designed to investigate the effect of a mixture of in vivo metabolized organochlorines, i.e. organochlorine metabolites extracted from sows to which the mixture was orally administered [17], [18] on porcine cumulus–oocyte complexes. We hypothesized that exposing oocytes to organochlorine metabolites would decrease maturation and subsequent development, as observed in our previous work with the parent organochlorine mixture [16].