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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.
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