Regulation of the aryl hydrocarbon receptor activity in bovine cumulus-oocyte complexes during in vitro maturation: The role of EGFR and post-EGFR ERK1/2 signaling cascade

Highlights

• Inhibition of EGFR-ERK1/2 signaling downregulates AhR-mediated expression of CYP1A1.

• Modulation of AhR activity during oocyte maturation affects meiotic resumption.

• The AhR/EGFR crosstalk might play a major role in sustaining oocyte’s maturation.

Abstract

The aryl hydrocarbon receptor (AhR) has been extensively characterized as an environmental sensor with major roles in xenobiotic-induced toxicity. Evidence is accumulating that these functions serve as adaptive mechanisms overlapping its physiological roles. We previously described a critical role of constitutive AhR activation for the correct progress of mammalian oocyte maturation but the signaling pathway through which AhR controls maturation remains unclear.

The aim of this study was to investigate whether the AhR interacts with the epidermal growth factor receptor (EGFR) and p42/44 extracellular regulated kinases (ERK1/2), both key factors in the signaling network that finely regulates the oocyte maturation. As experimental model we used bovine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM).

Blocking ERK1/2 signaling in COCs during IVM with the specific EGFR inhibitor AG1478 or the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 downregulated the expression of the AhR-target gene Cyp1a1. Inhibition of AhR activity was associated with a reduction in the oocytes’ ability to progress in meiosis resumption. In contrast, exposure to the AhR antagonist resveratrol reduced both CYP1A1 expression and the oocytes’ maturation competence, without affecting ERK1/2 signaling.

These findings strongly indicate the EGFR/ERKs signaling network as an upstream regulator of the AhR activation in COCs, offering a new understanding of the finely tuned physiological mechanism leading to oocyte maturation. This information may provide fresh opportunities for improving oocyte in vitro maturation, and therefore boosting the efficiency of assisted reproduction techniques in mammals.

Introduction

Female fertility relies on proper maturation of the oocyte. Oocyte maturation is a highly complex cellular process involving the meiotic cell cycle progression and cytoplasmic changes that determines the subsequent successful fertilization, zygote formation, attainment of blastocyst stage, normal embryo growth and development, as well as appropriate implantation [[1], [2], [3]]. In mammals, the molecular machinery governing oocyte maturation is controlled by multiple interactions among different signaling pathways [4,5]. Despite significant advances the molecular mechanisms underpinning the process of oocyte maturation, yet many key questions remain to be resolved.

In this context, we previously reported that the aryl hydrocarbon receptor (AhR) is constitutively activated in mammalian cumulus-oocyte complexes (COCs) during in vitro maturation (IVM), as shown by the upregulation of the two main target genes CYP1A1 and CYP1B1 in the absence of exogenous ligands [6,7]. In addition, AhR activation appears to be necessary for correct progressing of meiosis resumption, since treatment with specific AhR antagonists during IVM negatively affects the oocytes’ ability to reach the metaphase II [7]. The molecular mechanisms by which AhR exerts its effect in the mammalian cumulus-oocyte complex are still not understood.

The AhR is a ligand-activated transcription factor of the basic helix-loop-helix/per-ARNT-Sim (bHLH/PAS) superfamily. Since the early 1990’s, the AhR has been defined as an environmental sensor with major roles in xenobiotic-induced toxicity and carcinogenicity [8]. The AhR can in fact be activated by planar aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (B[a]P), dibenzofurans and planar polychlorinated biphenyls (PCBs), all of which are widely detectable in the environment [9,10].

In its resting state AhR is sequestered in the cytosol in a multiprotein complex with heat shock protein 90 (hsp90), p23 and the AhR interacting protein (AIP) [11]. Upon ligand binding, AhR translocates into the nucleus where it forms a heterodimer with the AhR nuclear translocator (ARNT). The AhR/ARNT complex then binds to a specific DNA sequence (xenobiotic responsive element - XRE) in the promoter of target genes and triggers their expression. The AhR gene battery consists of genes encoding for phase I and II drug metabolizing enzymes, with cytochrome P450 (CYP) 1A1 as the main one, as well as for proteins involved in regulating cell growth and differentiation [9,11,12].

The view that the AhR is exclusively a promiscuous cytosolic sensor of xenobiotic chemicals is changing. Evidence is accumulating that xenobiotic-dependent AhR functions are an adaptive mechanism, overlapping its physiological roles. In fact, a significant number of studies support its contribution to the proper functioning of the immune, hepatic, cardiovascular and reproductive systems [[13], [14], [15], [16]]. However, little is known about its physiological function and its endogenous ligands.

To date, a number of putative endogenous AhR-activating factors have been identified [[17], [18], [19]]. However, the physiological consequences of AhR activation by these ligands still need to be elucidated. AhR-dependent transcriptional activity is both ligand- and cell-specific. For a given cell type, it may even depend on the tissue milieu, such as in an ongoing immune response [20,21]. Furthermore, gene microarray studies have reported that different AhR ligands can induce a distinctly different, ligand-specific set of gene products [22,23].

Activated AhR signaling impinges on numerous molecular pathways in eukaryotic cells [24]. There is strong evidence of two-way cross-talk between AhR and the RAS-RAF-MEK-ERK1/2 pathway which is the most important pathway mediating the biological response of the epidermal growth factor receptor (EGFR-ErbB1) [25].

In bovine COCs, EGFR and ERKs are both key factors in coordinating oocyte maturation. Their activation is necessary for gonadotropin-induced oocyte meiotic resumption, regulation of microtubule organization and meiotic spindle assembly [26,27] and for the maintenance of metaphase II arrest [28,29]. Given the proven interactions between AhR and the RAS-RAF-MEK-ERK1/2 pathway and the central role of EGFR/ERKs in COCs, it is logical to hypothesize that AhR might interact with ERKs and EGFR in the complex signaling network regulating the progression of mammalian oocyte maturation. We set out to find proof of this cross-talk in bovine COCs during IVM. The COC is a morpho-functional unit and the relationship between the two compartments allow for important physiological processes such as oocyte growth, meiotic resumption, maintenance of meiotic arrest and acquisition of developmental competence. In previous studies, we showed that CYP1A1 expression pattern was similar in oocytes and cumulus cells during IVM (Pocar et al., 2004), thus in the present study we elicited to consider the entire COC as physiological entity.

Clarifying the biological role of the AhR during IVM can contribute to our understanding of the complexity of mammalian oocyte maturation and, in turn, provide fresh information on the xenobiotic-independent activity of the AhR.