Morphogenesis of Maternal and Paternal Genomes in Fertilized Oyster Eggs (Crassostrea gigas): Effects of Cytochalasin B at Different Periods During Meiotic Maturation.

Fertilized oyster (Crassostrea gigas) eggs, treated with and without cytochalasin B (CB) at varying periods during meiotic maturation, were examined following 7-aminoactinomycin D (7-AAD) and anti-{beta} tubulin staining for DNA and microtubular patterns, respectively. Electron microscopic observations of untreated fertilized eggs revealed the development of an extensive network of endoplasmic reticulum during germinal vesicle breakdown. Germinal vesicle breakdown was not accompanied by the disappearance of the nucleolus; the latter persisted morphologically unchanged throughout fertilization. The first meiotic spindle [12.4 +/- 1.0 {mu}m (1) x 5.4 +/- 0.55 {mu}m (diameter (d); metaphase plate)] was oriented with its long axis perpendicular to the egg's surface. In contrast, the second meiotic spindle was approximately one half the size of the first [7.4 +/- 0.65 {mu}m (1) x 2.91 +/- 0.29 {mu}m (d)] and initially oriented with its long axis parallel to the egg's surface. Just prior to anaphase II, the spindle rotated so that its long axis became perpendicular to the egg's surface. Following its incorporation into the egg cytoplasm, the sperm nucleus dispersed but did not form a nuclear envelope until the completion of polar body formation. Just prior to pronuclear migration, an array of microtubules assembled around the female pronucleus, and then regressed; concomitantly, a sperm aster formed in conjunction with the centrosome associated with the developing male pronucleus. Following their migration and apposition with one another, both pronuclei underwent prophase as independent structures. Chromosomes from the male and female pronuclei became organized on a mitotic spindle in preparation for first cleavage. In zygotes treated with CB continuously or for just meiosis I, anaphase I occurred; however, a polar body failed to form. Consequently, all the diads were retained within the zygote and became situated on a tripolar spindle. In such cases, anaphase II resulted in the production of a variable number of chromosomal aggregations that developed into pronuclei (3 to 6). In contrast, zygotes treated with CB during meiosis II developed spindles characteristic of untreated specimens; however, polar body formation was blocked, resulting in the development of usually two maternal pronuclei. Morphogenesis of C. gigas fertilized eggs is discussed in reference to similar processes in other species and with respect to strategies of triploid embryo production using CB.