Supplementary Figure 2: Rab11a-positive vesicles are dispensable for oocyte viability and progression through meiosis.

(a) Live oocytes expressing H2B-mRFP (magenta, chromosomes, merged with DIC) and mEGFP-Rab11a (control) or mEGFP-Rab11a S25N (Rab11a S25N). Representative examples from 2 independent experiments (>10 oocytes total for each condition). Scale bar, 10 μm. (b) Control and mCherry-Rab11a S25N expressing oocytes from (C57BL×CBA) F1 females were fixed and stained with Hoechst (chromosomes, cyan) and fluorescent phalloidin (F-actin, pseudocoloured). The arrow heads highlight the cortical enrichment of actin in proximity of the chromosomes, which is independent of the presence of Rab11a-positive vesicles. Representative examples from 2 independent experiments (>10 oocytes total for each condition). Scale bar, 10 μm. (c) Control and mCherry-Rab11a S25N expressing oocytes from (C57BL×CBA) F1 females were fixed and stained with Hoechst (chromosomes, cyan) and fluorescent Lens culinaris agglutinin to label cortical granules (pseudocoloured). The arrow heads highlight the cortical granule free domain in proximity of the chromosomes, which is independent of the presence of Rab11a-positive vesicles. Representative examples from one experiment (>5 oocytes total for each condition). Scale bar, 10 μm. (d) The percentage of oocytes undergoing NEBD was quantified by live cell microscopy of oocytes expressing mCherry-Rab11a (Control) or mCherry-Rab11a S25N (Rab11a S25N). The number of analysed oocytes is specified in italics (aggregation over 4 independent experiments). (e) The percentage of oocytes that form a bipolar spindle was quantified by live cell microscopy of oocytes expressing mCherry-Rab11a (Control) or mCherry-Rab11a S25N (Rab11a S25N). The number of analysed oocytes is specified in italics (aggregation over 4 independent experiments). (f) The percentage of oocytes that progress into anaphase was quantified by live cell microscopy of oocytes expressing EGFP-MAP4 to label the spindle together with mCherry-Rab11a (Control) or mCherry-Rab11a S25N (Rab11a S25N). The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments). (g) The time between NEBD and bipolar spindle assembly was measured in live oocytes expressing EGFP-MAP4 to label the spindle together with mCherry-Rab11a (Control) or mCherry-Rab11a S25N (Rab11a S25N). Box plot as in Fig. 1e. The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments). (h) The time between NEBD and anaphase onset was measured in live oocytes expressing mCherry-Rab11a (Control) or mCherry-Rab11a S25N (Rab11a S25N). Box plot as in Fig. 1e. The number of analysed oocytes is specified in italics (aggregation over 2 independent experiments).