285 REGULATION OF OOCYTE MEIOTIC RESUMPTION USING cAMP MODULATORS IN BOVINE IN VITRO MATURATION

Abstract

In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP). Currently, IVP has low efficiency due to an inadequate IVM system where premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3′,5′-cyclic adenosine monophosphate (cAMP). Some successful methods of improving IVM have utilised cAMP modulators to maintain high intraoocyte cAMP, delaying the onset of nuclear maturation and allowing cytoplasmic maturation to occur. The current experiment is a follow-up to previous work in which an extended 2-step maturation system was examined. In the previous experiments, we found that meiotic resumption was significantly delayed, but the overall maturation rates of extended IVM were about half those of standard IVM, suggesting that the effects of the modulators on the oocytes were not completely reversible. The current experiment compares cAMP concentrations of oocytes in this extended IVM to standard IVM to determine whether high cAMP is the cause of the low maturation rate. Bovine oocytes (n = 686) were obtained from mixed-breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into 2 groups: standard IVM and extended IVM. Standard IVM consists of a 23-h maturation composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured at 39°C in 5% CO₂. Extended IVM consists of 2 steps: a pre-IVM phase composed of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 h at 39°C, followed by an extended IVM phase composed of standard IVM media supplemented with 20 µM cilostamide for 31 h (39°C, 5% CO₂). Additionally, oocytes in the extended IVM treatment group where held in HEPES-TALP media with FSK and IBMX during the 2-h oocyte collection period in order to prevent any decrease in cAMP before the oocytes could be placed in the extended IVM media. Oocytes in standard IVM were sampled at 0, 8, and 23 h of maturation, while oocytes in extended IVM were sampled at 0, 8, 18, and 33 h of maturation. Cumulus cells were removed from all oocytes by vortexing in hyaluronidase solution. Oocytes were stored in groups of 10 at –80°C. A cAMP enzyme immunoassay (GE Healthcare) was performed to determine cAMP concentrations throughout standard and extended IVM. Assay results were analysed using an ANOVA followed by a Tukey's pairwise test (Sigma Stat 3.5) to detect significant differences (P < 0.05). Results indicated significantly higher cAMP levels in extended IVM oocytes during the first 3 h after collection using FSK and IBMX in the holding media (0.505 v. 1.006 fmol/oocyte, P = 0.035) but cAMP levels were not maintained in the cilostamide-only extended IVM medium. This suggests that high cAMP levels were not the cause of low maturation rates in extended IVM, since cAMP concentrations did decrease after 3 h. Possible negative effect of cilostamide on these oocytes may be suggested and need to be analysed.