Microtubule configurations in oocytes, zygotes, and early embryos of a marsupial, Monodelphis domestica

The marsupials represent a separate evolutionary lineage from eutherians from which they diverged over 100 million years ago. In order to explore the origin and mode of centrosome inheritance amongst this group of mammals, this study investigates the microtubule organization during fertilization, parthenogenesis, and polyspermy in the didelphid, Monodelphis domestica. Microtubules and DNA were visualized in maturing ovarian oocytes, parthenogenetically activated oocytes, monospermic and polyspermic zygotes, and early embryos. Ovarian oocytes had a central region of yolky cytoplasm that, after fertilization, became polarized; much of the yolk was then extruded into the perivitelline space as an enucleated cytoplasmic mass. Immunofluorescence microscopy, using a monoclonal antibody to β-tubulin, demonstrated microtubules in the meiotic spindle in unfertilized oocytes, but cytasters were not detected. After fertilization, a cluster of microtubules forming into a sperm aster was evident around the male pronucleus. The sperm aster remained largely restricted to the nonyolky region of the egg cytoplasm, resulting in a cytoplasmic heterogeneity between a microtubule- rich region and one in which microtubules were largely absent. Once the two pronuclei came close together, abundant microtubules were found surrounding 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 cortex and recovery from drug or cold depolymerization demonstrated that microtubules might not be as dynamic as those in eutherian mammals. Taxol stabilization resulted in an increase in cortical microtubules. In this marsupial species, therefore, the centrosome appears to be of paternal origin, and the radiating microtubules that form may well be involved both in bringing the pronuclei together and in the cytoplasmic polarization that results in extrusion of the yolk mass.