Analysis of cat oocyte activation methods for the generation of feline disease models by nuclear transfer.
BACKGROUND
Somatic cell nuclear transfer in cats offers a useful tool for the generation of valuable research models. However, low birth rates after nuclear transfer hamper exploitation of the full potential of the technology. Poor embryo development after activation of the reconstructed oocytes seems to be responsible, at least in part, for the low efficiency. The objective of this study was to characterize the response of cat oocytes to various stimuli in order to fine-tune existing and possibly develop new activation methods for the generation of cat disease models by somatic cell nuclear transfer.


METHODS
First, changes in the intracellular free calcium concentration [Ca2+]i in the oocytes induced by a number of artificial stimuli were characterized. The stimuli included electroporation, ethanol, ionomycin, thimerosal, strontium-chloride and sodium (Na+)-free medium. The potential of the most promising treatments (with or without subsequent incubation in the presence of cycloheximide and cytochalasin B) to stimulate oocyte activation and support development of the resultant parthenogenetic embryos was then evaluated. Finally, the most effective methods were selected to activate oocytes reconstructed during nuclear transfer with fibroblasts from mucopolysaccharidosis I- and alpha-mannosidosis-affected cats.


RESULTS
All treatments were able to elicit a [Ca2+]i elevation in the ooplasm with various characteristics. Pronuclear formation and development up to the blastocyst stage was most efficiently triggered by electroporation (60.5 +/- 2.9 and 11.5 +/- 1.7%) and the combined thimerosal/DTT treatment (67.7 +/- 1.8 and 10.6 +/- 1.9%); incubation of the stimulated oocytes with cycloheximide and cytochalasin B had a positive effect on embryo development. When these two methods were used to activate oocytes reconstructed during nuclear transfer, up to 84.9% of the reconstructed oocytes cleaved. When the 2 to 4-cell embryos (a total of 220) were transferred into 19 recipient females, 4 animals became pregnant. All of the fetuses developed from oocytes activated by electroporation followed by cycloheximide and cytochalasin B incubation; no fetal development was detected as a result of thimerosal/DTT activation. Although heartbeats were detected in two of the cloned fetuses, no term development occurred.


CONCLUSIONS
Electroporation proved to be the most effective method for the activation of cat oocytes reconstructed by nuclear transfer. The combined thimerosal/DTT treatment followed by cycloheximide and cytochalasin B incubation triggered development effectively to the blastocyst stage; whether it is a viable option to stimulate term development of cloned cat embryos needs further investigations.
Wang
Chunmin
C
Department of Animal Sciences, Purdue University, West Lafayette, IN 47907, USA.
Swanson
William F
WF
Herrick
Jason R
JR
Lee
Kiho
K
Machaty
Zoltan
Z
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002371:Cat Diseases / Q000235:genetics*; D002415:Cats; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004195:Disease Models, Animal; D018274:Electroporation; D046149:Embryo Culture Techniques; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D008059:Mucopolysaccharidosis I / Q000235:genetics / Q000662:veterinary; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010312:Parthenogenesis; D011247:Pregnancy; D011310:Preservatives, Pharmaceutical; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013268:Stimulation, Chemical; D013849:Thimerosal / Q000494:pharmacology; D008363:alpha-Mannosidosis / Q000235:genetics / Q000662:veterinary
D011310:Preservatives, Pharmaceutical; D011500:Protein Synthesis Inhibitors; D013849:Thimerosal; D003571:Cytochalasin B; D003513:Cycloheximide; D002118:Calcium