MORPHOLOGICAL AND ELECTROPHYSIOLOGICAL PROPERTIES OF CENTRIFUGED STRATIFIED XENOPUS OOCYTES

To separate and concentrate various cytoplasmic organelles in wild typ e and albino Xenopus oocytes, defolliculated cells were loaded on a Fi coll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10000 g for 5 min at 20 degrees C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100-300 mu m, and the green ish protein yolk at the bottom. To determine the cellular constituents , particularly of the transparent zone, electron microscopy was perfor med. The transparent zone was found to contain (from animal to vegetal ) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged strat ified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. C entrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The rest ing membrane potentials of centrifuged oocytes were between -35 and -6 5 mV, and the membrane resistances were in the 500 k Ohm to 1 M Ohm ra nge. Under voltage clamp conditions, the oocytes exhibited Ca2+-activa ted Cl- currents with biphasic kinetics and spontaneous oscillations o f these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suita ble preparation to study the contribution of various cellular organell es to the propagation of second messengers in the cytosol.