Fig. 2. Fertilization-associated events in F. serratus. (A) The first indicator of fertilization was the secretion of cell wall components, imaged using 2-photon microscopy of the cellulose stain CFW. CFW staining was evident within 15 minutes of sperm and egg mixing, and propagated across the egg within 5 minutes. (B) Sperm pronuclear ('sp') motion towards the egg pronucleus ('ep'), visualized using 2-photon microscopy of Hoechst 33342-labelled sperm. This representative series of images shows the sperm pronucleus as a light blue dot against a green autofluorescent background. The final image was just before pronuclear fusion. (C) Time courses during the first 3 hours after fertilization for cell wall secretion (upper plot, grey circles, n=8), sperm pronuclear migration (upper plot, white circles, n=10), cytosolic [Ca²⁺] (lower plot, white circles, n=8) and nuclear [Ca²⁺] (lower plot, black circles, n=8). Cell wall secretion is given in degrees of coverage of zygote circumference. Migration of the sperm pronucleus is given as distance into the zygote. [Ca²⁺] was measured using Texas Red/fura dextran and divided into cytosolic and nuclear components using the regions of interest drawn in D, below. An initial sustained elevation of both cytosolic and nuclear [Ca²⁺] was followed by a nuclear [Ca²⁺] elevation at the time of pronuclear fusion. Data are mean±s.e.m. (D) Representative pseudocolour-ratiometric Texas Red/fura dextran images of post-fertilization [Ca²⁺] changes. Regions `c' and `n' on the left image define, respectively, the mutually exclusive cytosolic and nuclear regions of interest. The `gp' region is an artefact where the gold pellets (biolistic microcarriers) have gathered and can be ignored.