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First published online 15 August 2007
doi: 10.1242/dev.005454

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Vesicular traffic at the cell membrane regulates oocyte meiotic arrest

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR 72205, USA.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Dominant-negative SNAP2520 blocks exocytosis. Xenopus ocytes were injected with either wild-type or SNAP2520 mRNA (5-10 ng) and membrane capacitance was measured [Cm(nF)] using two electrode-voltage clamp over time, as indicated (mean±s.e.; n=8-20). Western blot analysis using SNAP25 antibody shows similar levels of expression of wild-type and SNAP2520, and no band was detected in uninjected oocytes. SNAP2520 runs faster than wild-type full-length SNAP25 because of the deletion of the last 20 residues. Ooc, uninjected oocytes; WT, wild type.

View larger version (29K): [in this window] [in a new window]   Fig. 2. SNAP2520 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).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Timecourse analysis of SNAP2520-induced oocyte maturation in Xenopus. (A) Extent of GVBD (black) and membrane capacitance (blue) in progesterone- (left panel) and SNAP2520- (right panel) treated cells. The inset (below left) shows no change in capacitance or maturation in untreated oocytes over a similar timecourse. (B) MAPK and MPF activation and SNAP25 expression over the maturation timecourse following SNAP2520 mRNA injection (left panel) and progesterone treatment (right panel). (C) Oocytes were either left untreated and matured with maximal levels of progesterone (16 µM), or injected with the light chain of BoNT A (200 nM) or BSA as the carrier control and matured with sub-threshold levels of progesterone (100 nM). The maturation timecourses for the three treatments show that BoNT A injection potentiates the effects of sub-threshold levels of progesterone on maturation. This experiment was repeated four times on oocytes from different donor females, and the potentiation effects of BoNT A were observed in 2/4 experiments. (D) Membrane capacitance (Cm) measurements at different times, as indicated, after BSA or BoNT A (200 nM) injection, showing that BoNT A is ineffective at reducing Cm.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Activation of adenylate cyclase inhibits SNAP2520-induced maturation. (A,B) Extent of maturation of Xenopus oocytes treated with progesterone or injected with SNAP2520 mRNA in the presence or absence of 100 µM forskolin (A) or 5x10-9 M cholera toxin (B). Timecourses of maturation were carried out and the plateau levels of GVBD at the end of the experiments are reported (above). The activation of MAPK and MPF and the expression level of SNAP2520 are also shown (below). Lysates for western blot analysis were prepared from cells at the GVBD50 time point. (C) Percent GVBD of Mos mRNA- (10 ng) injected oocytes in the presence or absence of 100 µM forskolin or 5x10-9 M cholera toxin. The inset (below) shows the time to GVBD50 for Mos-injected cells±forskolin. CT, cholera toxin; Forsk, forskolin.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Inhibition of clathrin-mediated endocytosis negatively regulates oocyte maturation. (A) Membrane capacitance [Cm(nF)] of control Xenopus oocytes and oocytes injected with C3 exoenzyme (1 ng/oocyte) 2 hours earlier. (B) GVBD timecourse from a representative experiment after C3 exoenzyme injection. Cells were injected with C3 exoenzyme 1 hour before progesterone treatment or left untreated. The histograms indicate percent GVBD and normalized time to GVBD50 (mean±s.e.; n=3). GVBD levels are those reached at the end of the experiment, typically no longer than 18 hours. (C) Transferrin endocytosis assay. Examples of confocal cross-sectional images through oocytes used to quantify Alexa-633-labeled transferrin internalization after overnight incubation with either the carrier control DMSO or 332 µM MDC. The number of labeled vesicular structures and their equivalent sphere volumes were quantified, as detailed in Materials and methods. (D) Membrane capacitance of oocytes incubated in the DMSO carrier control and oocytes treated with 332 µM monodansyl cadaverine (MDC) overnight. (E) GVBD time course from a representative experiment after MDC treatment. Cells were incubated with MDC overnight or left untreated before progesterone treatment. The histograms indicate percent GVBD and normalized time to GVBD50 (mean±s.e.; n=5). Scale bar: 100 µm. Con, control oocytes.