246 INDUCTION OF MEIOTIC ARREST IN IMMATURE FELINE OOCYTES WITH ROSCOVITINE AND DIBUTYRYL CYCLIC-AMP

Abstract

Successful application of some assisted reproduction technologies in feline species requires the use of competent, meiotically mature ova, which can be difficult to obtain in sufficient quantities. Meiotic arrest of immature feline oocytes would allow the accumulation of oocytes over time to maximize laboratory resources. Two meiosis inhibitors commonly used in other species, roscovitine (ROS) and dbcAMP, were evaluated for the ability to maintain a germinal vesicle (GV) in immature feline oocytes after 24 h of in vitro culture. Feline ovaries were obtained from routine ovariohysterectomies and oocytes with homogenous cytoplasm and at least 2 layers of cumulus cells were selected. All oocytes were cultured in a basic medium modified from Gomez et al. (2000 Reprod. Fertil. Dev.) consisting of TCM-199 with Earle’s salts, 0.3% fraction V bovine serum albumin, 2.0 mm L-glutamine, 1.12 mm L-cysteine, 2.2 mm calcium lactate, 0.36 mm sodium pyruvate, 100 μm cysteamine, 10 ng μL^–1 epidermal growth factor, 1 μg mL^–1 estradiol, and 50 μg mL^–1 gentamicin, with or without meiotic inhibitor. After 24 h of culture, cumulus cells were removed; oocytes were fixed, permeabilized, and stained with 5 μg mL^–1 of Hoechst 33342; and chromatin configuration was assessed under ultraviolet fluorescence. In 4 replicates of Experiment 1, oocytes were cultured with either 25 nm ROS, 1 mm dbcAMP, both ROS and dbcAMP (Both), or without inhibitor (Control). A greater proportion of oocytes retained the GV when cultured with Both compared with ROS, dbcAMP, or Control (90.6, 67.8, 25.0, and 14.8%, respectively; chi-square P < 0.05), and ROS alone was superior to dbcAMP or Control. Culture of oocytes in the base medium plus 0.5 IU mL^–1 eCG and 1.0 IU mL^–1 hCG after arrest showed that pretreatment with Both did not decrease their ability to resume meiosis (87.9 v. 87.0%, respectively). Given the low percentage of oocytes with a retained GV in the presence of 1.0 mm dbcAMP, Experiment 2 evaluated the concentration of dbcAMP required to inhibit meiosis. Oocytes were cultured over 4 replicates in the base media containing 0, 0.1, 0.5, 1.0, 2.0, and 10.0 mm dbcAMP. After 24 h of culture, a greater number of oocytes were arrested at the GV stage when cultured in 10 mM dbcAMP (39.8%) than in 0, 0.1, 1.0, and 2.0 mM dbcAMP (22.0, 20.4, 25.8, and 26.3%, respectively; Fisher’s exact test P < 0.05), but there was no difference from those cultured in 0.5 mm dbcAMP (28.1%). For many species, 1.0 mm dbcAMP is commonly used to inhibit meiosis successfully for 24 h. However, dbcAMP alone did not effectively arrest meiosis, but when combined with ROS, it tended to improve meiotic arrest. Experiment 2 indicates that at least 10.0 mm dbcAMP is required to show a significant effect of meiotic inhibition in feline oocytes. Under these culture conditions, dbcAMP at levels up to 10 mm were not effective at inducing meiotic arrest. Interestingly, ROS and dbcAMP may act synergistically to successfully induce a reversible inhibition of meiosis in a high percentage of feline oocytes.