253 LACK OF ABILITY TO REGULATE KINASE ACTIVITIES MAY BE RESPONSIBLE FOR MEIOTIC INCOMPETENCE IN THE DOG OOCYTE

Abstract

The size of donor follicles influences meiotic maturation of oocytes, including those of the domestic dog (Songsasen and Wildt 2005 Mol. Reprod. Dev. 72, 113–119). Maturation promoting factor (MPF, CDK1) and mitogen activating protein kinase (MAPK) play a pivotal role in regulating meiosis in several species (Abrieu et al. 2001 J. Cell Sci. 114, 257–267). Accordingly, we determined (1) MPF and MAPK activities, and (2) mRNA expression of cell cycle genes, including CDK1, CCNB1, and CDC25 in oocytes obtained from small (<1 mm diameter), medium (1 to 2 mm) and large (>2 mm) follicles. In Study 1, ovarian oocytes were classified into three groups (based on these sizes) and then cultured (38.5°C in 5% CO₂) for 0, 24, or 48 h in TCM-199 (+25 µm β-mercaptoethanol, 10 ng mL^–1 epidermal growth factor, 0.25 mm pyruvate, 2.0 mm glutamine, and 0.1% polyvinyl alcohol). Oocytes were denuded, assessed for nuclear status, and stored individually at –80°C until MPF and MAPK activities were assayed using a double kinase assay. Kinase activities of in vitro-matured (IVM) oocytes were expressed as a ratio of MPF and MAPK to that in metaphase I (MI) oocytes flushed from the oviducts of estrous bitches. In Study 2, oocytes (n = 20/follicular size class) were immediately frozen at –80°C; RNA was extracted, reverse transcribed, and subjected to quantitative real-time PCR analysis. Expression levels of each transcript were normalized to levels of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Statistical analyses were performed using ANOVA followed by the Holm-Sidak test for multiple comparisons. Both MPF and MAPK activities varied among follicular size classes. Kinase activities increased in oocytes from large follicles upon meiotic resumption, with germinal vesicle (GV) oocytes expressing the lowest levels compared to their metaphase II (MII) counterpart (GV: 25.9 ± 5.2% v. MII: 211.5 ± 19.5% and 36.4 ± 9.1% v. 150.7 ± 22.9% for MPF and MAPK, respectively; P < 0.01). For medium follicles, MPF increased (P < 0.01) as oocytes progressed from GV (23.3 ± 6.3%) to MII (243.4 ± 55.5%). However, MAPK levels remained constant until the MI stage, and then increased (P < 0.01) in MII oocytes. For small follicles, MPF increased (P < 0.05) at the MI stage and then remained constant until meiosis was completed, whereas MAPK activities remained constant after GVBD. Kinase activity levels were not different (P < 0.05) between MI and MII oocytes harvested from the three follicular classes. Interestingly, follicular size had no effect (P > 0.05) on expression levels of cell cycle transcripts. These findings suggest that the compromised developmental competence of dog oocytes from small follicles likely is related to the oocytes' inability to regulate MAPK activity during meiotic resumption.