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Characterization of oxygen and calcium fluxes from early mouse embryos and oocytes.

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 to begin to characterize changes in embryo physiology during development. This work is part of larger study on the viability of the pre-implantation embryo.

The self-referencing electrode technique (4) pioneered by the BioCurrents Research Center at the MBL is ideal for exploring the physiology of the early embryo. The technique allows the derivation of flux values by measuring the concentrations of free ions or dissolved oxygen at two probe positions micrometers apart, where the electrode is moved in a square wave oscillation. One pole of this oscillation is brought close to the zona pellucida (within about 1 [[micro]meter]). With this approach, physiological measurements can be obtained from individual oocytes or embryos. Being noninvasive, the self-referencing electrode can be used to record from oocytes or developing embryos without perturbation, an assumption recently confirmed by the successful birth of implanted, post-experimental blastocysts (Trimarchi and Liu, unpub. obs.).

We employed self-referencing electrode techniques to characterize calcium and oxygen flux from a series of developmental stages of mouse embryos. The embryos were collected at stages ranging from unfertilized oocytes to expanded blastocysts and cultured in M2 medium according to standard techniques (5). Physiological experiments were conducted in M2 medium with reduced calcium (50 [[micro]molar]) at 37 [degrees] C in a Faraday box mounted on an airtable.

For the measurement of oxygen fluxes, oxygen microelectrodes, with a tip diameter of 2-3 [[micro]meter], were purchased from Diamond General Development Corp. (Model 723, Ann Arbor, MI). These were calibrated in [N.sub.2] and air-bubbled d[H.sub.2]O. The measured current was convened to a flux value as previously described (6). The ion-selective electrodes were constructed as described by Smith et al. (4), incorporating a short (30 [[micro]meter]) column of ion-selective resin (ETH 1001: Fluka). Unlike the oxygen electrode response, which is linear with concentration, these electrodes are Nernstian, with voltages being converted to flux values by the following equation:

J = -D [C.sub.av][10.sup.s[Delta]v] - [C.sub.av]/[Delta]r

where, J is flux, D is the diffusion coefficient (0.79 [multiplied by] [10.sup.-5] [multiplied by] [cm.sup.2] [multiplied by] [s.sup.-1]), [Delta]V is the differential voltage measurement (mV) between the measurement positions separated by [Delta]r (cm), S is the inverse of the Nernst slope of the electrode, and [C.sub.av] is the average free [Ca.sup.2+] concentration measured in the medium between the poles of the electrode movement. This equation is modified from Kuhtreiber and Jaffe (7). For both oxygen and calcium detection, all electrodes were oscillated at 0.3 Hz over 10 [[micro]meter]. A return electrode (Ag/AgCl) completed the circuit via a bridge of 3M KCl in 5% agar.

Measurements taken with calcium and oxygen selective, self-referencing electrodes demonstrate that developing embryos exhibit measurable net oxygen influx and calcium efflux at all stages examined (oocyte to blastocyst: [ILLUSTRATION FOR FIGURE 1 OMITTED]). In particular, oxygen influx measured from blastocysts is three times that measured from earlier stages. These data are consistent with oxygen consumption measurements obtained from groups of embryos by ultra-microfluorescence techniques (8). 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 blastocoele (9).

In contrast to oxygen influx, calcium efflux was relatively constant ([approximately]22 fmol [multiplied by] [cm.sup.-2] [multiplied by] [s.sup.-1]) throughout the developmental series [ILLUSTRATION FOR FIGURE 1 OMITTED]. Mouse embryos are known to be able to develop from embryos through the blastocyst stage in the absence of external calcium (10). We have not yet determined the physiological mechanisms 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 (4), 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 the 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.

Literature Cited

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. Development 122: 2485-2496.

3. Stricker, S. A. 1995. Dev. Biol. 170: 496-518.

4. Smith, P. J. S., R. H. Sanger, and L. F. Jaffe. 1994. Methods Cell Biol. 40: 115-134.

5. Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo. CSHL Press, Cold Spring Harbor. New York.

6. Malchow, R. P., S. C. Land, L. S. Patel, and P. J. S. Smith. 1997. Biol. Bull. 193: 231-232.

7. Kuhtreiber, W. M., and L. F. Jaffe. 1990. J. Cell Biol. 110: 1565-1573.

8. Houghton, F. D., J. G. Thompson, C. J. Kennedy, and H. J. Leese. 1996. Mol. Reprod. Dev. 44: 476-485.

9. Biggers, J. D., J. E. Bell, and D. J. Benos. 1988. Am. J. Physiol. 255: C419-C432.

10. Santalo, J., M. Grossmann, and J. Egozcue. 1996. Hum. Re-prod. Update 2: 257-261.
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Author:Porterfield, D.M.; Trimarchi, J.R.; Keefe, D.L.; Smith, P.J.S.
Publication:The Biological Bulletin
Date:Oct 1, 1998
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