Using light microscopy techniques, we have studied the movements that follow fertilization in the denuded egg of the ascidian Phallusia mammillata. In particular, our observations show that, as a result of a series of movements described below, the mitochondria-rich subcortical myoplasm is split in two parts during the second phase of ooplasmic segregation. This offers a potential explanation for the origin of larval muscle cells from both posterior and anterior blastomeres. The first visible event at fertilization is a bulging at the animal pole of the egg, which is immediately followed by a wave of contraction, travelling towards the vegetal pole with a surface velocity of 1.4 microns s-1. This wave accompanies the first phase of ooplasmic segregation of the mitochondria-rich subcortical myoplasm. After this contraction wave has reached the vegetal pole after about 2 min, a transient cytoplasmic lobe remains there until 6 min after fertilization. Several new features of the morphogenetic movements were then observed: between the extrusion of the first and second polar body (at 5 and 24–29 min, respectively), a series of transient animal protrusions form at regular intervals. Each animal protrusion involves a flow of the centrally located cytoplasm in the animal direction. Shortly before the second polar body is extruded, a second transient vegetal lobe (‘the vegetal button’) forms, which, like the first, resembles a protostome polar lobe. Immediately after the second polar body is extruded, three events occur almost simultaneously: first, the sperm aster moves from the vegetal hemisphere to the equator. Second, the bulk of the vegetally located myoplasm moves with the sperm aster towards the future posterior pole, but interestingly about 20% remains behind at the anterior side of the embryo. This second phase of myoplasmic movement shows two distinct subphases: a first, oscillatory subphase with an average velocity of about 6 microns min-1, and a second steady subphase with a velocity of about 26 microns min-1. The myoplasm reaches its final position as the male pronucleus with its surrounding aster moves towards the centre of the egg. Third, the female pronucleus moves towards the centre of the egg to meet with the male pronucleus. Like the myoplasm, the migrations of both the sperm aster and the female pronucleus shows two subphases with distinctly different velocities. Finally, the pronuclear membranes dissolve, a small mitotic spindle is formed with very large asters, and at about 60–65 min after fertilization, the egg cleaves.
Microtubule-Based Mechanisms of Pronuclear Positioning