The long-term goal of this proposal is to devise new culture media that support normal preimplantation embryo development. The developmental competence and viability of cultured embryos are compromised, showing that improvements in culture media are still needed. Improved media are urgently needed by human IVF clinics to increase the mean embryo viabili-ty, allowing fewer embryos, perhaps only one, to be transferred and support a successful pregnancy; this could eliminate the need for gonadotropin stimulation of patients. Other applications could benefit from improved embryo culture media, e.g., production of transgenic hamsters and rats for biomedical and toxicological research. Improvements are also needed to better support development of embryos stressed by cryopreservation or by micromanipulation (e.g., for preimplantation genetic diagnosis). Instead of using empirical methods for cultured medium design, the proposed research takes a mechanistic approach to find how hamster and rhesus monkey embryo development are regulated by key epigenetic factors, so these can be optimized and incorporated into new media formulations. The connecting theme of the re-search is """"""""cytoplasmic homeostasis,"""""""" targeting three critically important aspects if preimplantation embryo development the ionicregulation of intracellular protons (pHi) and calcium (pCai), oxidative metabolism and cytoplasmic organization, which are all perturbed in culture. The experimental strategy is to determine how culture conditions perturb these components, then modify the culture environment to compensate for these perturbations, and finally incorporate this new knowledge into improved culture media for preimplantation embryos. The proposed research is hypothesis-driven to provide answers to specific questions.
Specific aims are to examine mechanisms of pHi homeostasis and relationships among pHi, calcium/magnesium and phosphate, and embryo development: to determine relationships among mitochondrial metabolism, glycolysis and development; to find how cytoplasmic components, including mitochondria and the cytoskeleton, are perturbed by culture conditions; and to test new media formulations on development. Techniques include fluorescent probe measurement of pHi and pCai; measuring substrate utilization by single embryos; static confocal and dynamic two-photon microscopic analysis of cell structural changes; objective analysis of embryo development by four-dimensional videomicroscopy; and embryo transfers.
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