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Fig. 2. SNAP25
20 releases oocyte meiotic arrest in
Xenopus. (A) Percentage of oocytes reaching the GVBD stage
following progesterone treatment, or injection of SNAP2520 or wild-type
SNAP25 mRNA (mean±s.e.; n=4-7 experiments with >50 oocytes
in each treatment). The percentages reported are the maximal levels of GVBD
reached. (B) Photographs showing the absence of a white spot on the
animal hemisphere, and the presence of the germinal vesicle (nucleus) (arrow)
in untreated oocytes (Ooc) and oocytes injected with wild-type SNAP25 mRNA
(WT). By contrast, progesterone (Prog) or SNAP2520 (20)
injection results in the appearance of a white spot on the animal pole and
GVBD. Top row, white spot; bottom row, GVBD. (C) Spindle structure
(top) and polar body (bottom) in progesterone-treated and
SNAP2520-injected oocytes. Spindle structure was visualized by indirect
immunofluorescence using an anti-tubulin antibody and chromosomes were stained
with Sytox Orange (scale bars: 5 µm for spindles and 10 µm for polar
body). (D) Time required for 50% of the oocytes in the population to
reach the GVBD stage of maturation (GVBD50) following progesterone
treatment or SNAP2520 mRNA injection (mean±s.e.; n=7
experiments from different female donors). (E) Western blot analysis of
cells treated with progesterone or injected with either SNAP2520 or
SNAP25 wild-type mRNA. Lysates were prepared at different time points during
oocyte maturation: (1) untreated oocytes; (2) when oocytes first reached the
GVBD stage (GVBD); (3) when 50% of the cells reach GVBD (G50). In
this case, lysates were prepared from cells with (w) and without (nw) a white
spot; (4) when 100% of the cells reached the GVBD stage (G100).
Because oocytes injected with wild-type SNAP25 mRNA do not undergo GVBD,
lysates were prepared at the same time points when SNAP2520-injected
cells reached the G50 and G100 milestones, indicated as
G50eq and G100eq, respectively. Blots were probed with
anti-phospho-MAPK, anti-phospho-cdc2 and anti-SNAP25 antibodies. (F)
Lysates from oocytes that have undergone GVBD at the G50 time point
were prepared and western blot analysis performed to assess Cdc25C activation
and SNAP25 expression. Cdc25C activation was detected as a supershift on the
gel due to hyperphosphorylation (arrowheads) from the basal state (arrow)
observed in oocytes. In contrast to progesterone or SNAP2520, no shift
is detected in oocytes or SNAP25 wild-type injected cells. The middle band is
a non-specific band. Blots were stripped and re-probed with anti-SNAP25
antibody (lower panel).
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