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. 2016 Dec 19:7:13726.
doi: 10.1038/ncomms13726.

Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes

Liam P Cheeseman  1 , Jérôme Boulanger  1 , Lisa M Bond  1 , Melina Schuh  1   2
Affiliations

Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes

Liam P Cheeseman et al. Nat Commun. .

Abstract

An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

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Figures

Figure 1
Figure 1. Rab27a is a marker for cortical granules.
(a) Oocyte expressing GFP-Rab27a, fixed and stained with lens culinaris agglutinin, a cortical granule marker. Scale bar, 20 μm. (b) Quantification of the colocalization of Rab27a puncta with lens culinaris agglutinin puncta (mean±s.d. from three experiments). Total number of Rab27a puncta quantified are indicated. (c) Cortical granules visualized with GFP-Rab27a translocate from the centre of oocytes to the cortex, where they become enriched. Scale bars, 20 μm (overview) and 5 μm (enlarged). (d) Schematic diagram of the quantification methods for cortical granule translocation and enrichment at the cortex. Scale bars, 20 μm. (e) Cortical granule dynamics in the oocyte centre during meiotic maturation. Three separate phases can be identified in the cell centre, which are colour-coded in all subsequent graphs. The start and end of each phase was determined by calculating the derivative of the data. (f) Cortical granule enrichment at the oocyte cortex during meiotic maturation. The three separate phases identified in the cell centre are colour-coded.
Figure 2
Figure 2. Rab27a is required for cortical granule translocation.
(a) Maximum intensity projections of confocal micrographs of Rab27a+/Ash (control) or Rab27aAsh/Ash (mutant) oocytes at NEBD or released for 5 h, then fixed and stained with lens culinaris agglutinin. Scale bar, 20 μm. (b) Quantification of cortical granules in the cell centre for the conditions in a. (c) Quantification of cortical granules at the cell cortex for the conditions in a. Tukey box plots in b,c show the median (line), mean (small square), interquartile range, and the 5th and 95th (whiskers). Significance levels: ns non-significant, *P<0.05, ***P<0.001, analysed using Kruskal–Wallis' ANOVA test from three experiments.
Figure 3
Figure 3. Cortical granule translocation is dependent on the cytoplasmic actin network.
Quantification of cortical granule translocation in the oocyte centre or cortex (mean±s.e.m.) in cells treated with nocodazole or DMSO (a), cytochalasin D or DMSO (c), in Fmn2−/− (which lack an important actin nucleator) or Fmn2+/−oocytes (e). Representative still images (all maximum intensity projections of confocal Z sections) for each condition are presented in b,d and f. Control images are included in Supplementary Fig. 2. Scale bars, 20 μm (overview) and 5 μm (enlarged). (g-h) Still confocal images of a cortical granule being transported along actin (g), or interacting with an actin node (h). Scale bars, 2 μm.
Figure 4
Figure 4. Cortical granules are translocated to the cortex by hitchhiking on Rab11a vesicles.
(a) Maximum intensity projections of confocal micrographs of an oocyte expressing GFP-Rab27a and mCherry-Rab11a. Note the close association between both types of vesicles (insets). Scale bar, 20 μm. (b) Example still images of a cortical granule (Rab27a, green; indicated by arrows) hitchhiking on a Rab11a vesicle (magenta) to reach the cortex (dotted line), with corresponding kymograph. Scale bar, 2 μm. (c) Quantification of the number of cortical granules associated to four example Rab11a vesicles. (d) Number of cortical granule hitchhiking ‘on' and ‘off' events per Rab11a vesicle per minute (n=23 trajectories from three oocytes). Vesicle speed (e) and vesicle displacement speed (f) were quantified for cortical granules while associated to Rab11a vesicles (Rab27a+Rab11a), cortical granules while not associated to Rab11a vesicles (Rab27a alone), and for Rab11a vesicles. Number of tracks used for quantification are indicated in e, from six oocytes. Tukey box plots in d,e and f show the median (line), mean (small square), interquartile range, and the 5th and 95th (whiskers). Significance levels: *P<0.05, ***P<0.001, analysed using Kruskal–Wallis' ANOVA test from two experiments. (g) Representative example trajectories of the particle categories indicated in e and f (six trajectories per condition), projected in two dimensions. Duration of each trajectory is 52 s. (h) Quantification of cortical granule translocation in the oocyte centre or cortex (mean±s.e.m.) in oocytes expressing Rab11aS25N or wild-type Rab11a. (i) Representative still images (maximum intensity projections of confocal Z sections) of oocytes expressing Rab11aS25N. Control images are included in Supplementary Fig. 2. Scale bars, 20 μm (overview) and 5 μm (enlarged).
Figure 5
Figure 5. A myosin Va-dependent pathway powers cortical granule translocation.
Quantification of cortical granule translocation in the oocyte centre or cortex (mean±s.e.m) in cells expressing either wild-type myosin Va (MyoVaWT), a dominant-negative mutant (MyoVaLT), or control (a), with representative example still images (maximum intensity projections of confocal Z sections) in b. Still images from MyoVaWT-expressing cells are included in Supplementary Fig. 2. Scale bars, 20 μm (overview) and 5 μm (enlarged). (c) Example still images of a cortical granule (Rab27a) translocating to the cortex in a Rab11a-independent manner (indicated by arrows), with corresponding kymograph. Scale bar, 2 μm. (d) Schematic diagram of the local mean squared displacement analysis of Rab27a trajectories that contained Rab11a-dependent transport (hitchhiking) or Rab11a-independent transport, and excluding purely diffusive trajectories. Each track was analysed by MSD with a short lag time to distinguish between active (magenta) and diffusive (blue) states on a step-by-step basis. (e) Combined Rab27a trajectories that contained Rab11a-dependent transport (hitchhiking) or Rab11a-independent transport in oocytes expressing MyoVaLT, MyoVbLT, or control, after local MSD analysis. Active states are coloured in magenta, while diffusive states are in blue. Proportion of each state is indicated (%). (f–g) Total number of active steps from Rab27a trajectories (as determined by MSD analysis; % of total steps) containing either Rab11a-independent transport (f), or Rab11a-dependent transport (hitchhiking; g) in oocytes expressing MyoVaLT, MyoVbLT, or control. Total number of steps in each category is indicated. Compared using Fischer's exact test. (h–i) Mean squared displacement velocity parameters of the active steps from Rab27a trajectories containing Rab11a-independent (h) or Rab11a-dependent transport (i) in oocytes expressing MyoVaLT, MyoVbLT, or control. Number of active steps in each category is indicated. Tukey box plots in h and i show the median (line), mean (small square), interquartile range, and the 5th and 95th (whiskers), compared using Mann-Whitney's U test. Significance levels: ns, non-significant, ***P<0.001, from three experiments.
Figure 6
Figure 6. Disruption of cortical granule translocation severely impairs the zona pellucida block to polyspermy.
(a) Maximum intensity Z projections of in vitro fertilized oocytes from Rab27a+/Ash (control) or Rab27aAsh/Ash (mutant) oocytes, fixed and stained with Hoechst. Male and female pronuclei and polar body (PB) are indicated. Note the extra sperm heads present inside the zona pellucida in mutant oocytes (arrows). Scale bar, 20 μm. (b) Quantification of the number of fertilized oocytes which had extra sperm heads in the perivitelline space in Rab27a+/Ash or Rab27aAsh/Ash oocytes (mean±s.d.; Student's t test) (c) Quantification of the number of extra sperm inside the perivitelline space of fertilized Rab27a+/Ash or Rab27aAsh/Ash oocytes. (d) Quantification of remaining cortical granules in the centre of Rab27a+/Ash or Rab27aAsh/Ash oocytes after fertilization (mean±s.d.; Student's t test). (e) Mechanistic model of the two cortical granule translocation mechanisms in mammalian oocytes. See text for details. Significance levels: ***P<0.001, from three experiments.
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