A method for differentiating nonunique estimates of membrane transport properties: mature mouse oocytes exposed to glycerol.
Measurement of the osmotic response of a cell in the presence of cryoprotectant facilitates the determination of permeability coefficients which, in turn, can be used to design cryopreservation protocols which minimize osmotic stress. One problem encountered in determining permeability coefficients, using the Kedem-Katchalsky (K-K) model of membrane permeability, is that several combinations of the three passive coupled transport coefficients, namely, hydraulic permeability (L(p), microm min(-1) atm(-1)), solute permeability (P(gly), microm s(-1)), and the reflection coefficient (sigma), can give a similar fit to the measured data. A method for determining the "correct" set of coefficients is suggested. The osmotic response of 10 metaphase II mouse oocytes was measured on perfusion with 1.5 mol L(-1) glycerol at 24 degrees C. For 8 of 10 oocytes perfused, two combinations of L(p), P(gly), and sigma gave a predicted response which closely matched the measured osmotic response, depending upon the initial estimates supplied to the software for these parameters. For the remaining two oocytes, similar values for the permeability coefficients were generated regardless of the initial estimates. To determine the correct set of parameters, the K-K equations were used to predict experimental conditions for which volumetric histories would be distinctly different for the two sets of "best-fit parameters," and then additional experimental data were compared to these predictions. Thus a further three oocytes were perfused with 0.2 or 0.5 mol L(-1) glycerol in the absence of nonpermeating solute. In the presence of both 0.2 and 0.5 mol L(-1) glycerol, L(p) = 2.11 +/- 0.69, P(gly) = 0.0016 +/- 0.0015, and sigma = 0.44 +/- 0.11 yielded a very poor fit to the measured response while L(p) = 0.98 +/- 0.70, P(gly) = 0. 0031 +/- 0.0021, and sigma = 0.91 +/- 0.15 yielded a close fit to the measured response. Thus the latter combination of coefficients was taken to be correct.
Paynter
S J
SJ
Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom.
McGrath
J J
JJ
Fuller
B J
BJ
Shaw
R W
RW
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003080:Cold Temperature; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005260:Female; D005990:Glycerol / Q000494:pharmacology*; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism*; D009995:Osmosis / Q000187:drug effects
D003451:Cryoprotective Agents; D005990:Glycerol