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Conditions for In Vitro Maturation and Artificial Activation of Ferret Oocytes¹

^a Departments of Anatomy & Cell Biology and ^b Internal Medicine ^c the Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, College of Medicine, University of Iowa, Iowa City, Iowa 52242

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 µg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%–36%, n = 67–73). At 48 h, similar maturation rates (56%–69%, n = 76–87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean ± SEM, 10.3 ± 1.1; n = 11) and trophectoderm (60.8 ± 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.

First decision: 1 October 2001.

¹ Funded by the Cystic Fibrosis Foundation (JIANG00G0) and the National Institutes of Health DK47967 (J.F.E.) and HL61234 (M.J.W.).

² Correspondence: John F. Engelhardt, Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, 51 Newton Road, Room 1-111 BSB, Iowa City, IA 52242. FAX: 319 335 6581; john-engelhardt{at}uiowa.edu

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				Z. Li, M. Rezaei Sabet, Q. Zhou, X. Liu, W. Ding, Y. Zhang, J.-P. Renard, and J. F. Engelhardt Developmental Capacity of Ferret Embryos by Nuclear Transfer Using G0/G1-Phase Fetal Fibroblasts Biol Reprod, June 1, 2003; 68(6): 2297 - 2303. [Abstract] [Full Text] [PDF]