The purpose of this research is to explore mitochondrial distribution as markers for developmental potential of cloned pig embryos. Cloning of mammalian embryos has become attractive in recent years because of the high potential for biomedical and agricultural applications. Cloning of pigs as tissue and organ donors is of high interest because of the exceptional physiological compatibility with humans. However, practical applications are not yet feasible because of the low cloning efficiency (ca. 0.2% in pigs) for which causes are only little understood. One reason may be an asymmetrical mitochondria distribution that can result in reduced ATP generating capacity and an inability to support normal cell functions. We propose that specific patterns of mitochondria aggregation and microtubule organization will allow us to predict developmental potential of cloned embryos and increase cloning efficiency.
Our specific aims are to analyze: 1a) whether differences in mitochondrial distribution occur among individual blastomeres in cohorts of morphologically normal cleavage stage embryos; 1b) whether changes in intracellular pH are associated with disruption of mitochondrial organization and reduced development in vitro; 2a) whether microtubule organization plays a role in mitochondrial distribution after nuclear transfer in cloned embryos; and 2b) whether unequal centrosome separation after nuclear transfer plays a role in mitochondrial distribution. The distribution of mitochondria will be examined by scanning laser confocal microscopy in fixed and live oocytes, in cleavage stage embryos, and development to the blastocyst stages. MitoTracker Green FM and Mitotracker-X-Rosamine will be used to stain mitochondria. Double and triple immunofluorescence staining with anti-tubulin and anti-centrosome antibodies will determine the relationship between mitochondria and microtubule organization. We will correlate survival to the blastocyst stages with characteristic mitochondria fluorescence patterns. These experiments will provide new information on mitochondria distribution and microtubule organization after nuclear cloning and allow future research aimed at selecting embryos that are most likely to survive and increase live birth of cloned animals. Pilot data from this RO3 small grants program research will be used to apply for funding through the RO1 mechanism. ? ?

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Small Research Grants (R03)
Project #
5R03HD043829-02
Application #
6832809
Study Section
Pediatrics Subcommittee (CHHD)
Program Officer
Tasca, Richard J
Project Start
2003-12-08
Project End
2006-11-30
Budget Start
2004-12-01
Budget End
2006-11-30
Support Year
2
Fiscal Year
2005
Total Cost
$72,500
Indirect Cost
Name
University of Missouri-Columbia
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
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Schatten, Heide; Sun, Qing-Yuan (2009) The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol Hum Reprod 15:531-8
Sun, Qing-Yuan; Schatten, Heide (2007) Centrosome inheritance after fertilization and nuclear transfer in mammals. Adv Exp Med Biol 591:58-71
Katayama, Mika; Zhong, Zhisheng; Lai, Liangxue et al. (2006) Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential. Dev Biol 299:206-20
Sun, Qing-Yuan; Schatten, Heide (2006) Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction 131:193-205
Sun, Qing-Yuan; Schatten, Heide (2006) Role of NuMA in vertebrate cells: review of an intriguing multifunctional protein. Front Biosci 11:1137-46
Liu, Zhonghua; Schatten, Heide; Hao, Yanhong et al. (2006) The Nuclear Mitotic Apparatus (NuMA) protein is contributed by the donor cell nucleus in cloned porcine embryos. Front Biosci 11:1945-57
Schatten, Heide; Prather, Randall S; Sun, Qing-Yuan (2005) The significance of mitochondria for embryo development in cloned farm animals. Mitochondrion 5:303-21
Zhong, Zhi-Sheng; Zhang, Gang; Meng, Xiao-Qian et al. (2005) Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos. Exp Cell Res 306:35-46