306 SPONTANEOUS AND PARTHENOGENETIC ACTIVATION OF RAT OOCYTES

Abstract

Oocyte activation is one of the critical steps for the success in a mammalian cloning program. In rats, information regarding parthenogenetic activation of oocytes is limited and further studies are required to elucidate the phenomenon of spontaneous activation and to develop effective protocols of induced activation. The objectives of the study were (1) to characterize the kinetics of spontaneous activation and (2) to examine different activation regimens for rat oocytes. Oocytes were collected from 3–4-wk-old PMSG-primed Sprague Dawley rats at 14 h after hCG injection. HEPES-mR1ECM was used for oocyte collection and mR1ECM for in vitro culture (Miyoshi et al., 1995 J. Reprod. Fert. 103, 27–32). Experiment 1: Cumulus-denuded oocytes were fixed and stained with bisbenzimide (Hoechst 33342) at 10, 40, 70, 100 and 130 min after recovery. Most oocytes (92.9%) were still arrested in the metaphase II stage at 10 min after recovery, but the proportion of activated oocytes increased in a time-dependent manner, i.e. at 40 min after recovery 50% were at anaphase. At 70 min, 32.4%, 40.5%, 8.1%; at 100 min, 4.8%, 57.1%, 23.8%; and at 130 min, 0%, 3.3%, 86.7% had progressed to anaphase II, telophase II and extruded a 2nd PB, respectively. Experiment 2: Oocytes were exposed to the following activation treatments: Two sets of electrical stimuli (ES) 1 h apart composed of 3 DC pulses, 1 s apart of 1.2 kV cm⁻¹ field strength and 60 μs duration in 0.25 M mannitol solution (Roh et al., 2003 Reprod. Fert. and Dev., 15, 135–140). After ES treatment, oocytes were exposed for 3 h to either cycloheximide (CHX, 10 μg mL⁻¹, Group 1), 6-dimethylaminopurine (DMAP, 2 mM, Group 2), or CHX (10 μg mL⁻¹) and DMAP (2 mM) (Group 3). Group 4 was exposed to Strontium (10 mM) for 1 h and then incubated with CHX (10 μg mL⁻¹) and DMAP (2 mM) for 3 h. Oocytes were washed thoroughly after treatment, transferred to 50-μL droplets of mR1ECM and cultured at 37°C in a humidified atmosphere of 5% O₂, 5% CO₂ and 90% N₂. Pronuclear formation and cleavage rates were significantly higher in Groups 2 and 3 than in Groups 1 and 4 (92.6%, 88.9% and 84.6%, 80.8% v. 49.1%, 45.6% and 62.8%, 55.8%, respectively). First, these results show that rat oocytes undergo spontaneous activation very rapidly after recovery from the oviducts. Second, activation protocols with two sets of triple DC electrical stimulation followed by exposure to DMAP or CHX/DMAP are effective means of activation. In conclusion, the above information will be useful in establishing effective protocols for cloning rats. (Financial support by NSERC and Canada Research Chairs.)