Effects of cytochalasins B and D on the fertilization of zebrafish (Brachydanio) eggs

The effects of selected concentrations of cytochalasins B (1-10 micrograms/ml; CB) and D (10, 50 micrograms/ml; CD) on the morphology and fertilization of zebra danio (Brachydanio) eggs were studied primarily with light and scanning electron microscopy. Eggs pretreated with either CB (10 micrograms/ml) or CD (10, 50 micrograms/ml) prepared in Fish Ringer's solution-0.5% DMSO showed a flattened shape, alterations in the form of surface microplicae and microvilli, and occasional spontaneous exocytosis of cortical granules. All eggs preincubated in either CB or CD were activated upon transfer to tap water, showing cortical granule exocytosis, elevation of the chorion, and formation of a fertilization cone. When eggs were pretreated for 5 minutes with 1-5 micrograms/ml CB or 10 micrograms/ml CD and inseminated, they incorporated the fertilizing sperm and typically developed to the two-cell stage. A single sperm cell attached to and fused with the sperm entry site microvilli but failed to enter the cytoplasm in eggs preincubated with 10 micrograms/ml CB. Eggs that were immersed continuously in either CB (10 micrograms/ml) or CD (50 micrograms/ml) 15 seconds after insemination also failed to incorporate the fertilizing sperm. Treatment of eggs after insemination with CD (10 micrograms/ml), however, did not prevent sperm cell incorporation or fertilization cone formation. Our drug data suggest the presence of actin-containing filaments in the danio egg before and following fertilization. These filaments appear to play a role in maintaining the shape of the egg cell and its surface specializations and in the incorporation of the fertilizing sperm. The fertilization cone appears to form independently of actin polymerization.