CHARACTERIZATION OF OXYGEN & CALCIUM FLUXES FROM EARLY MOUSE EMBRYOS & OOCYTES

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Despite the importance of the mammalian embryo to clinical and biomedical sciences, the physiology of pre-implantation embryos and oocytes is largely unexplored. For example, although calcium is known to participate in the early events of fertilization (1) and also plays a brief but critical role at each cleavage (2, 3), the regulation of transmembrane calcium flux during the interim between cleavages or during blastocoel formation is unknown. One reason for this gap in our knowledge lies in the difficulty in studying a single oocyte or embryo. The goal of the project reported here is to take advantage of new techniques for monitoring physiological parameters from individual cells and begin to characterize changes in embryo physiology during development. This work is part of larger study on the viability of the pre-implantation embryo. Measurements taken using calcium and oxygen selective, self-referencing electrodes demonstrate that developing embryos exhibit measurable net oxygen influx and calcium efflux at all stages examined. In particular, oxygen influx measured from blastocysts is 3 times that measured from earlier stages. These data are consistent with oxygen consumption measurements obtained from groups of embryos by using ultra-microfluorescence techniques (4). The increase in oxygen consumption coincident with blastocyst formation is thought to reflect an increased metabolic demand generated by the pumping of sodium and potassium ions that drives the enlargement of the blastocoel (5). In contrast to oxygen influx, calcium efflux was relatively constant (~22 fmol . cm-2 . sec-1) throughout the developmental series. Mouse embryos are known to be able to develop from embryos through the blastocyst stage in the absence of external calcium (6). We have not yet determined the physiological mechanism underlying this net efflux. We could be measuring a steady-state loss of calcium from the internal stores or, as is more likely, an efflux linked to calcium uptake from the medium via channels. As the probe technique is based on considerable signal averaging, with both high and low pass filters (7) rapid channel events occurring over periods of a second or less may not be recorded. The increase in oxygen influx in the absence of a change in calcium efflux that we observed in blastocyst-stage embryos demonstrates that the physiological state of the embryo changes markedly between the 16- cell stage and blastocyst formation. Characterization of the transmembrane movement of other ion species will continue to improve our understanding of the physiological mechanisms underlying early embryo development. This study demonstrates the utility of the noninvasive probes for the characterization of the early embryo. References 1. Jaffe, L.A.1996. Pp. 367-378 in molecular Biology of Membrane Transport Disorders, S.G. Shultz et al, eds, Plenum Press, New York. 2. Stachecki, J.J. and D. R. Armant. 1996. Dev. 122: 2485-2496. 3. Stricker, S.A. 1995. Dev. Biol. 170: 496-518. 4. Houghton, F. D., J. G. Thompson, C. J. Kennedy, and H. J. Leese. 1996. Mol. Reprod. Dev. 44: 476-485. 5. Biggers, J.D., Bell, J.E. & Benos, D.J.. 1988. Am J. Physiol. 255: C419-C432. 6. Santals, J., M. Grossmann, and J. Egozcue. 1996. Hum Reprod Update 2: 257-261. 7. Smith, P. J. S., R. H. Sanger, and L. F. Jaffe. 1994. Meth. Cell Biol. 40: 115-134.