Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocyte in vitro maturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs during in vitro maturation has been investigated. Results indicate that exposure of cumulus -oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested. Reproduction (2005) 130 857-868

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxin-like compounds. However, it has also been reported that the AhR may exert a role in ovarian physiology. In the present study, porcine cumulus-oocyte complexes (COCs) were matured in vitro in the presence of 10% follicular fluid. Expression of AhR and its partner, AhR nuclear translocator occurs in immature COCs. After in vitro maturation (IVM), an up-regulation of AhR and cytochrome P450 1A1 (CYP1A1; the main AhRtarget gene) was observed. To explore the role of the AhR during IVM, we exposed the COCs to 50 mM b-napthoflavone (bNF). The treatment induced a marked up-regulation of CYP1A1 mRNA, indicating both constitutive and inducible AhR activity. However, in contrast to what was observed in other cell types, no sign of toxicity was observed in COCs. To investigate if components of porcine follicular fluid may exert a protective role against AhR ligands, we exposed porcine COCs to bNF, in the absence of follicular fluid. In these conditions, a marked decrease in the percentage of matured oocytes, concomitant with an increase in oocyte degeneration, was observed. Furthermore, bNF increased apoptosis in cumulus cells in the absence of follicular fluid, whereas bNF has no effects when COCs were treated in the presence of porcine follicular fluid (pFF). In conclusion, these results suggest the presence of unknown endogenous AhR-ligand(s) during porcine IVM and that a dysregulation of this mechanism may result in ovotoxicity by inducing apoptosis in cumulus cells. However, this phenomenon is interrupted by the presence of follicular fluid, indicating a putative protective role for follicular fluid components against exogenous insults.

Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696-1701. The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579-585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 µM BLI (B) or 6.25 µM BLI + 12.5 µM ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 µg mL −1 FSH, 50 µg mL −1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40-50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL −1 EGF). After IVM, all groups (140-150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 × 10 6 sperm cells mL −1 ) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5 • C under 5% CO 2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 ± 1%, P < 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 ± 2 and 80 ± 1%, respectively; P > 0.05) than C (100%, P < 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 ± 1-83 ± 2%, P > 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 ± 1-89 ± 1 for 21 h and 85 ± 2-96 ± 8 for 24 hP > 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 ± 3-84 ± 3%, P > 0.05) and Day 7 blastocyst rates (26 ± 4-37 ± 4%, P > 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 ± 4-40 ± 4%, P > 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 ± 4%) only when compared with C (40 ± 4%, P < 0.05). Hatching rates were similar (10 ± 3-16 ± %3, P > 0.05) as were total cell numbers (141 ± 5-170 ± 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in...

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