MICROTUBULE CONFIGURATIONS IN OOCYTES, ZYGOTES, AND EARLY EMBRYOS OF A MARSUPIAL, MONODELPHIS-DOMESTICA

The marsupials represent a separate evolutionary lineage from eutheria ns from which they diverged over 100 million years ago. In order to ex plore the origin and mode of centrosome inheritance amongst this group of mammals, this study investigates the microtubule organization duri ng fertilization, parthenogenesis, and polyspermy in the didelphid, Mo nodelphis domestica. Microtubules and DNA were visualized in maturing ovarian oocytes, parthenogenetically activated oocytes, monospermic an d polyspermic zygotes, and early embryos. Ovarian oocytes had a centra l region of yolky cytoplasm that, after fertilization, became polarize d; much of the yolk was then extruded into the perivitelline space as an enucleated cytoplasmic mass. Immunofluorescence microscopy, using a monoclonal antibody to beta-tubulin, demonstrated microtubules in the meiotic spindle in unfertilized oocytes, but cytasters were not detec ted. After fertilization, a cluster of microtubules forming into a spe rm aster was evident around the male pronucleus. The sperm aster remai ned largely restricted to the nonyolky region of the egg cytoplasm, re sulting in a cytoplasmic heterogeneity between a microtubule-rich regi on and one in which microtubules were largely absent. Once the two pro nuclei came close together, abundant microtubules were found surroundi ng both pronuclei. In the early embryo, microtubules were found in the outer cortical region of the blastomeres and, in addition, there was an extensive and elaborate network of microtubules throughout the yolk mass. Disruption of the meiotic spindle microtubules with nocodazole or cold treatment did not result in chromosome dispersion in the corte x and recovery from drug or cold depolymerization demonstrated that mi crotubules might not be as dynamic as those in eutherian mammals. Taxo l stabilization resulted in an increase in cortical microtubules. In t his marsupial species, therefore, the centrosome appears to be of pate rnal origin, and the radiating microtubules that form may well be invo lved both in bringing the pronuclei together and in the cytoplasmic po larization that results in extrusion of the yolk mass. (C) 1994 Academ ic Press, Inc.