Membrane transport properties of mammalian oocytes: a micropipette perfusion technique.

ABSTRACT

A perfusion technique using micropipette methodology was developed to determine quantitatively the membrane transport properties of mammalian oocytes. This method eliminates modelling ambiguities inherent in microdiffusion, a closely related technology, and should prove to be especially valuable for study of the coupled transport of water and cryoprotectant through mammalian oocytes and embryos. The method is described and evidence given for validity of the method for the simple case of uncoupled flow of water through the mouse oocyte membrane. The zona pellucida of a mouse oocyte was held by a micropipette with an 8-10 microns diameter tip opening and perfused by hyperosmotic media. The kinetic volume change of the cell was videotaped and quantified by image analysis. Experimental data and mathematical modelling were used to determine the hydraulic conductivity of the oocyte membrane (Lp) found to be 1.05, 0.45 and 0.26 microns min-1 atm-1 at 30 degrees C, 22 degrees C and 12 degrees C, respectively. The corresponding activation energy, Ea, for Lp was calculated to be 13.0 kcal mol-1. These values are in agreement with data obtained by other techniques. One of the major advantages of this technique is that the extracellular osmotic condition can be changed readily by perfusing a single cell with a prepared medium. To study the response of the same cell to different osmotic conditions, the old perfusion medium can be removed easily and the cell reperfused with a different medium.(ABSTRACT TRUNCATED AT 250 WORDS)