234 PARTHENOGENETIC ACTIVATION OF DOMESTIC CAT OOCYTES USING STRONTIUM

Abstract

Cloning domestic cats is useful in comparative medicine programs as it may provide insight into unique disease mechanisms and facilitate investigation of new therapeutic options. It is also believed to be beneficial for the conservation of precious animal models. However, as in many species, low birth rates after nuclear transfer remain a formidable challenge. One potential reason for the low efficiency is poor embryo development following activation of the reconstructed oocytes. The number of methods available to induce a transient increase in the oocytes' cytosolic free calcium level to stimulate development is rather limited. Although strontium has been reported to successfully activate the developmental program of mature mouse and rat oocytes, it was without effect in all other species studied. Here we investigated the effect of strontium on mature cat oocytes. Oocytes collected from the cat ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mm cysteine, 0.1 mm cysteamine, 1 IU mL^–1 eCG, 2 IU mL^–1 hCG, 25 ng mL^–1 epidermal growth factor (EGF) for 24 h. For intracellular calcium measurements, mature oocytes were incubated in the presence of 2 µ m fura-2Am, a calcium indicator dye, and 0.02% pluronic F-127 for 40 min. Individual oocytes were transferred into calcium-free HEPES, and SrCl₂ was added to the medium at a final concentration of 20 mm. Changes in the intracellular free calcium levels were then monitored using an InCyt Im2™ fluorescence imaging system (Intracellular Imaging, Cincinnati, OH, USA). Preimplantation embryonic development was also evaluated by incubating the oocytes with 20 mm SrCl₂ in calcium-free HEPES medium supplemented with 7.5 µg mL^–1 cytochalasin B for 6 h. Control oocytes were activated by two 20-µs-long, 100 kV cm^–1 direct current pulses and incubated in the presence of 7.5 µg mL^–1 cytochalasin B for 6 h. After activation, the oocytes were cultured in FOCM for 6 days. At the end of the culture period, embryonic development was recorded; the nuclear number of the embryos was also determined after staining with Hoechst 33342. Data were subjected to one-way ANOVA, and differences between treatments were analyzed using the Tukey test. We found that strontium triggered a transient rise in the intracellular free calcium concentration in all oocytes tested (N = 20). Strontium treatment also induced cleavage in 49.7% (92/185) of the oocytes, while 4.9% (9/185) of the activated oocytes developed to the blastocyst stage. In the electroporated group, cleavage frequency was 57.1% (104/182) and blastocyst formation was 8.8% (16/182). Data analysis showed that there was no significant difference between the two groups in terms of cleavage frequency and blastocyst formation. This is the first study to demonstrate that strontium can induce cytoplasmic calcium increase in cat oocytes and trigger development up to the blastocyst stage. The results also indicate that SrCl₂ may be useful for oocyte activation during cat nuclear transfer. Additional studies are needed to determine whether SrCl₂ can trigger development more effectively than current activation techniques.