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(177) PARTHENOGENETIC DEVELOPMENT OF FERRET OOCYTES MATURED IN VITRO FOLLOWING ELECTRICAL AND CHEMICAL STIMULATION. Li, Ziyi1, Jiang, Qinshi2, Rezaei Sabet, Maryam3, Zhang, Yulong4, Ritchie, Teresa5, Engelhardt, John6, 1 Departments of Anatomy & Cell Biology, Iowa City, IA2 Departments of Anatomy & Cell Biology, Iowa City, IA3 Departments of Anatomy & Cell Biology, Iowa City, IA4 Departments of Anatomy & Cell Biology, Iowa City, IA5 Departments of Anatomy & Cell Biology, Iowa City, IA6 Departments of Anatomy & Cell Biology, Iowa City, IA ABSTRACT- The domestic ferret, Mustela Putorius Furos, has proven to be a useful model for lung diseases due to the similarity between ferret and human in lung biology. In an effort to establish protocols necessary for cloning ferrets, optimized conditions for artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes (COCs) were harvested from ovaries of superovulated ferrets and cultured in the medium containing TCM-199 + 10% FBS + eCG [10 IU /ml] + hCG [5 IU/ml]. After 24 hrs of maturation in vitro, the COCs were removed and placed in Ca2+- and Mg2+-free Dulbecco PBS containing 0.1% BSA and 0.2% hyaluronidase for 1-3 min. Upon removing cumulus cells by pipetting, only oocytes with normal morphology, uniform cytoplasm and a first polar body were selected for parthenogenetic activation. The oocytes were stimulated by either electrical (3V AC for 5 sec followed by 1 DC pulse of 180V/mm for 30 us) and chemical (5 ug/ml cyclohexamide for 5 min or 2 mM/ml 6-DMAP for 4 hrs) treatments individually or in combination. After stimulation, the oocytes were cultured in medium containing TCM-199 + 10% FBS at 38.5°C in 5% CO2, 95% air for 1-6 days. Treatment with cyclohexamide and 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 (P>0.01) over those obtainable under other tested conditions including individual treatment with electrical pulses (10%, n=41), cyclohexamide (3%, n=58) or 6-DMAP (5%, n=59). Selected blastocysts were differentially stained with 10 KEY WORDS: activation, oocyte, ferret |
g/ml each of hoechst 33342 and propidium iodide (PI) and examined by fluorescent microscopy. The blastocysts derived from in vitro activation appeared to have normal morphology and were composed of appropriate numbers of both inner cell mass (10.3±1.1, n=11) and trophectoderm (60.8±2.9, n=11) cells as compared to normal blastocysts developed in vitro from in vivo fertilized 1-cell stage embryos. Our results indicate that both electrical stimulation and chemical treatment are needed for relatively efficient parthenogenetic activation of ferret oocytes matured in vitro. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.