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Mitochondrial Distribution in Mouse Oocytes and Embryonic Stem Cells Judith A. Newmark and Carol M. Warner Bernard M. Gordon Center for Subsurface Sensing and Imaging Systems; Department of Biology Northeastern University, Boston, MA 02115 This work was supported in part by CenSSIS, the Bernard M. Gordon Center for Subsurface Sensing and Imaging Systems, under the Engineering Research Centers Program of the National Science Foundation (Award Number EEC-9986821) and the W.M. Keck Foundation. ABSTRACT SIGNIFICANCE STATE OF THE ART MITOCHONDRIAL STORM FUTURE PLANS PUBLICATIONS/ REFERENCES CONTACTS Dr. Carol Warner (617) 373-4036 [email protected] Biology Judith Newmark (617) 373-3973 [email protected] Biology Mitochondria are the organelles that generate the energy for the cell. They are also critical regulators of apoptosis (programmed cell death). The activity and localization of mitochondria influence whether a cell lives or dies. We are using imaging techniques in order to gain insight into how mitochondria influence cellular health in two biological systems: mouse oocytes and embryonic stem cells. Post-ovulatory oocytes undergo apoptosis if they are not fertilized. We have characterized the pattern of mitochondrial distribution in mouse oocytes in relation to post-ovulatory age, and discovered that the mitochondria do not simply clump as an oocyte ages, but they swirl around the oocyte cytoplasm like a storm. Analysis of the mitochondrial storm may lead to a better understanding of oocyte viability. A long range goal is to extend the window during which an oocyte can be fertilized, perhaps by inhibiting the mitochondrial storm, a feat which would be of enormous interest to infertility clinics around the world. After fertilization, oocytes become embryos that are a source of embryonic stem (ES) cells. ES cells have the capacity to form any tissue type in the body. Selective agents may be used in order to encourage the growth of a particular type of cell such as neurons or cardiomyocytes. However, scientists are just beginning to understand the process of ES cell growth and differentiation. We are using a combination of imaging by the Keck 3DFM and FACscan analysis in order to correlate mitochondrial activity and localization with cellular differentiation. The results would help clinicians learn to maximize the therapeutic potential of ES cells. Warner, C.M., Newmark, J.A., Comiskey, M., DeFazio, S.R., O’Malley, D.M., Rajadhyaksha, M., Townsend, D.J., McKnight, S., Roysam, B., Dwyer, P.J., and DiMarzio, C.A. (2004). Genetics and imaging to assess oocyte and preimplantation embryo health. Reproduction, Fertility and Development. 16, 729-741. Review: The Future of Stem Cells. Financial Times and Scientific American special report (2005) ♦ The Keck 3DFM is a State-of-the-Art microscope with DIC, Confocal, and Two- Photon capabilities ♦ Live-cell imaging is the State-of-the-Art in microscopy ♦ This is the first official report of a mitochondrial storm ♦ This is the first investigation of mitochondrial activity and distribution in ES cells ♦ Oocytes were collected at the beginning of the window of fertilization ♦ Oocytes were stained with Mitotracker GreenFM (a live cell dye) ♦ Oocytes were imaged by confocal microscopy every 15 min. in a Bioptechs live- cell culture chamber on the Keck 3DFM ♦ Images were viewed as movies and are being analyzed by k-means clustering, entropy, and flow analysis (Badri Roysam and Andy Cohen, RPI) ♦ Analyze the motion of the mitochondrial storm: velocity, direction, etc. ♦ Look for a mitochondrial storm in other examples of aging and/or dying cells ♦ Correlate the cytoskeleton and markers of apoptosis with the mitochondrial storm ♦ Determine whether we can inhibit the storm and perhaps prolong oocyte health ♦ Analyze changes in mitochondrial distribution and activity in aging ES cells ♦ Predict ES cell differentiation by using mitochondrial distribution and activity DIC Epifluorescence- overlay Still images show mitochondrial clumping, but the live video shows a more complex pattern of mitochondrial movement around the chromosomes (metaphase plate) ♦ The mitochondrial storm first moves towards the chromosomes, splits, and then swirls away from the chromosomes Please see www.keck3dfm.neu.edu for the full video of the mitochondrial storm . ♦ Mitochondria are different in each cell type: ex. Oocyte, ES cell ♦ Morphology ♦ Distribution ♦ Activity ♦ Mitochondria reflect the health of a cell Zona pellucida Polar body Chromosomes Oocyte body with arrows representing mitochondrial movement TECHNOLOGY TRANSFER ES CELLS ♦ Infertility clinics are interested in expanding the window of fertilization of oocytes ♦ Inhibition of the mitochondrial storm may extend fertilizability ♦ Predicting differentiation is key to ES cell therapy ES cells stained with JC-1 CCCP Treated Untreated Activity of Mitochondria in ES Cells: Imaging Blue=nuclei, Green=JC-1 green (inactive mitochondria), Red=JC-1 red (active mitochondria) ♦ ES are maintained on fibroblasts with LIF to keep them undifferentiated ♦ ES are removed from fibroblasts and LIF to cause random differentiation ♦ We are looking for a correlation between cell fate and mitochondrial distribution Blue=Hoechst stained chromosomes, JC-1 Green=inactive mitochondria, JC-1 Red=active mitochondria DIC Epifluorescence- overlay Blue=Hoechst stained chromosomes, Green=mitochondria Diagram of the Mitochondrial Storm The majority of mitochondria in oocytes are inactive Active mitochondria become inactive when treated with CCCP, an uncoupling agent ES cells contain active and inactive mitochondria in different locations and with different morphologies
