290 MODULATION OF ARYLHYDROCARBON RECEPTOR ACTIVITY DURING IN VITRO MATURATION OF BOVINE OOCYTES
Authors: Nestler D. , Risch M. , Fischer B. , Pocar P. 
Published: December 2004

The arylhydrocarbon receptor (AhR), a ligand activated transcription factor, has been extensively characterized from a toxicological point of view due to its ability to mediate the adverse effects of a variety of halogenated aromatic hydrocarbons. Recent reports on AhR knockout mice suggest that the AhR may play a role in ovarian physiology. We have previously demonstrated that AhR activity (as indicated by the up-regulation of the target gene cytochrome p450 1A1: CYP1A1) is stimulated during bovine oocyte IVM in the absence of exogenous ligands. Furthermore, exposure to specific AhR antagonists, besides down-regulating the expression of CYP1A1, significantly impairs the ability of the oocyte to complete maturation until the metaphase II stage (Pocar et al. 2004 Endocrinology 145, 1594–1601). The aim of the present study was to further investigate the mechanisms underlying the AhR activation during IVM. Several reports point to a critical role of phosphorylation in the regulation of the AhR-complex. Furthermore, the mitogen-activated protein kinase (MAPK, extracellular regulated kinase (ERK 1 and 2)) cascade has been shown to play a crucial role in regulating meiotic cell cycles during bovine oocyte maturation. A total of 572 bovine cumulus-oocyte complexes were used to investigate the potential role of the MAPK in modulating the activity of the AhR during IVM. The effect of the broad-spectrum serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the induction of CYP1A1 during oocyte maturation was investigated. As expected, exposure to 6-DMAP induced meiotic arrest (at the stage of germinal vesicle/germinal vesicle breakdown) and down-regulated the expression level of phosphorylated ERK 1 and 2. Interestingly, a significant down-regulation of the target genes CYP1A1 and CYP1B1 (9.5% and 26.8% of control, respectively) and an up-regulation of the AhR (199.4% of control) were observed at the mRNA level. This phenomenon was partially reversible after a period of further 24 h of culture in the absence of 6-DMAP. In this condition, besides a recovery of oocyte maturation and phosphorylation status of ERK 1 and 2 to levels comparable to control, a significant up-regulation of CYP1A1 mRNA was observed (68.3% of control). Finally, to confirm the role of serine/threonine kinases in modulating the activity of AhR during resumption of meiosis, we exposed the oocytes to cycloheximide, a protein synthesis inhibitor, also known to arrest oocyte maturation. Furthermore, although cycloheximide exposure induced meiotic arrest, no significant differences in the expression levels of AhR or its target genes compared to control were observed. Each experiment was replicated at least three times. Data were assessed using ANOVA followed by Duncans multiple rance test. The criterion for significance was set at P < 0.05. In conclusion, our results strongly suggest that 6-DMAP-sensitive kinase(s) is (are) involved in the regulation of AhR during bovine oocyte maturation. Further analyses are necessary to understand the biological significance of these observations.