LEUCINE TRANSPORT IN XENOPUS-LAEVIS OOCYTES - FUNCTIONAL AND MORPHOLOGICAL ANALYSIS OF DIFFERENT DEFOLLICULATION PROCEDURES

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence o f 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defo lliculation was performed enzymatically by collagenase treatment, wher eas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na+-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol ooc yte(-1) 40 min(-1) in folliculated oocytes and 5.6 +/- 1.9 in collagen ase defolliculated oocytes (means +/- SE). L-leucine uptake was not af fected by the removal of the follicular layer if defolliculation occur red after the transport period; radiolabeled L-leucine is therefore no t taken up into a compartment that is removed by the defolliculation p rocess. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine bind ing to membranes. L-leucine kinetics :showed that the L-leucine V-max and K-m values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The V-m ax and K-m values of Na+-dependent L-leucine transport, calculated fro m data obtained the day after defolliculation by collagenase treatment , were: 16+/-1.5 pmol oocyte(-1) 40 min(-1) and 57 +/- 21 mu mol (mean +/- SD). The Na+ -activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experi ments showed that after defolliculation, the fibers forming the vitell ine membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase tre atment. Mechanical defolliculation did not appreciably affect the oocy te microvilli, however this procedure did not completely remove all fo llicle cells. The damage to collagenase treated oocytes was reversible , and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to in volve protein synthesis in view of the increased value of L-leucine V- max, and microscopic observation showing recovery of the microvillar a pparatus. (C) 1998 Elsevier Science Inc. All rights reserved.