Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture.
In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported. However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals. In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation. Therefore, the relationship between culture conditions and mitochondrial activity was explored. Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells. In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions. Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed. The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001). Serum starvation induced an increase in mitochondrial viability in confluent cells. These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions. Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals. Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.
Takeda
Kumiko
K
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, National Agricultural Research Organization, Ibaraki, Japan. kumiko@affrc.go.jp
Akagi
Satoshi
S
Takahashi
Seiya
S
Onishi
Akira
A
Hanada
Hirofumi
H
Pinkert
Carl A
CA
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D019976:Cloning, Organism; D016895:Culture Media, Serum-Free; D004423:Ear; D018274:Electroporation; D004847:Epithelial Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D007700:Kinetics; D008928:Mitochondria / Q000502:physiology* / Q000637:transplantation; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D012867:Skin / Q000166:cytology; D012879:Skin Physiological Phenomena
D016895:Culture Media, Serum-Free