The overall objective is to improve a chemically-defined culture medium (HECM) to support normal development in vitro of oocytes and of in vitro fertilized embryos, from hamsters and rhesus monkeys. A strong underlying premise is that improved ability to sustain preimplantation development in vitro will reveal new information about the epigenetic regulation of embryogenesis. These two goals are interdependent. A key strategy is the use of protein-free culture media, avoiding unknown factors associated with most commercial protein (serum albumin) preparations. This allows examination of effects of particular regulators (hormones, energy substrates, etc.) on embryo development without extraneous factors compromising responses. AS well as increasing the proportions of developing embryos, reduction of experimental variance is a goal, in order to make studies more efficient and cost-effective, including smaller numbers of animals required. The experimental strategy is rigorous and systematic, with each experiment building on preceding ones to form a firm conceptual framework. A variety of technical approaches will be tested on oocytes and embryos, including: measurement of intracellular pH [pHi], respirometry, fatty acid uptake, and the novel """"""""simplex optimization"""""""" technology using computer-designed media formulations. Developmental endpoints include embryo transfers to validate culture medium improvements. Discrete developmental events will be recorded by time-lapse video and image-processing technology, providing a baseline to reveal temporal changes in embryo development responses, and to correlate morphological events with changes in [pHi]. Knowledge of the normal [pHi] of the preimplantation embryo is important for enhancing development in vitro; at present, it is now known if conventional culture media maintain normal [pHi]. A model for amino acid buffering of [pHi] will be tested that could provide insights into the mode of [pHi] control in vivo, as well as leading to further improvements in culture medium technology, including the possibility of dispensing with the need for gaseous CO2 for embryo culture. A major experiment will examine different energy substrates for embryos, in light of growing evidence that carbohydrate substrates are inadequate. Respirometry applied to embryos will be more efficient than embryo culture in determining effectiveness of different substrates. Other potentially important factors to be examined include vitamins, trace metals and hormones. The outcome of this research will be increased knowledge of the regulation of primate, including human, oocyte maturation and preimplantation embryo development, that could be applied towards the treatment of infertility as well as fertility regulation; and an improved, chemically-defined culture medium suitable for human and other primate oocytes and embryos. This medium will (i) permit considerable numbers of germinal vesicle stage oocytes harvested for human in vitro fertilization to be fully utilized in this infertility treatment; (ii) provide a means to recruit large numbers of non-human primate oocytes for production of preimplantation embryos by IVF, thus breaking down the principal barrier to progress in research on primate early development. This advance will have significant indirect benefits to human reproductive health problems.
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