The research proposed in this application is concerned with the intracellular pH (pHi) and metabolism of the oocyte and preimplantation stages of the mouse embryo. pH of the cells of single embryos will be measured by a method developed in our laboratories which uses one of the pH-sensitive fluorescent dyes. Detailed attention will be given to the absolute intracellular pH at different stages of development and its regulation. Specific attention will be paid to whether the pHi changes upon reinitiation of meiosis, fertilization, compaction, and/or exposure of the blastocyst to growth factors. The possibility of developing a non- invasive test of viability based on measurement of the acidification of the bathing medium when healthy embryos export protons to regulate their pHi will be investigated. The research is also concerned with the uptake of glutamine by single mouse preimplantation embryos. A new ultramicrofluorescence technique developed in our laboratories measures uptake of glutamine in the pmole range. The method will be used to (1) characterize the mechanisms that transport glutamine into and out of the preimplantation embryo, (2) study the action of insulin and related growth factors on the uptake of glutamine, and (3) investigate the possibility of measuring glutamine uptake by preimplantation embryos as a non-invasive test of viability.
