Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes

(A) Brightfield images of fully grown oocytes from the Myo10 full knockout strain (left panel) and the Myo10 oocyte knockout strain (right panel). For each panel, controls are on the left, and Myo10^−/− oocytes, on the right. Scale bar = 10 μm. (B) Scatter plot of the equatorial plane area of control and Myo10^−/− full oocytes (in dark gray and green, respectively) and control and Myo10^−/− oo oocytes (in light gray and blue, respectively). (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from four to 13 independent experiments. Statistical significance of differences was assessed by an ANOVA or a Kruskal–Wallis test depending on whether the data followed a Gaussian distribution, P = 0.4459 (n.s, Ctrl full versus Myo10^−/− full), P = 0.7355 (n.s, Ctrl full versus Ctrl oo), P > 0.9999 (n.s, Ctrl full versus Myo10^−/− oo), P = 0.9905 (n.s, Myo10^−/− full versus Ctrl oo), P > 0.9999 (n.s, Myo10^−/− full versus Myo10^−/− oo), and P > 0.9999 (n.s, Ctrl oo versus Myo10^−/− oo). n.s, not significant. (C) Scatter plot of the zona pellucida texture of control and Myo10^−/− full oocytes (in dark gray and green, respectively) and control and Myo10^−/− oo oocytes (in light gray and blue, respectively). (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from four to 13 independent experiments. Statistical significance of differences was assessed by an ANOVA or a Kruskal–Wallis test depending on whether the data followed a Gaussian distribution, P = 0.0280 (*, Ctrl full versus Myo10^−/− full), P = 0.3355 (n.s, Ctrl full versus Ctrl oo), P > 0.9999 (n.s, Ctrl full versus Myo10^−/− oo), P < 0.0001 (****, Myo10^−/− full versus Ctrl oo), P = 0.0083 (**, Myo10^−/− full versus Myo10^−/− oo), and P = 0.3895 (n.s, Ctrl oo versus Myo10^−/− oo). n.s, not significant. (D) Brightfield images of a fully grown control (upper panel) and Myo10^−/− full oocyte (bottom panel). For each panel, the right image displays the automatic segmentation of the oocyte perivitelline space (perivitelline space, yellow). Scale bar = 10 μm. (E) Scatter plot of the coefficient of variation of the perivitelline space thickness, as represented by the schemes on the left. Control and Myo10^−/− full oocytes are in dark gray and green, respectively. Control and Myo10^−/− oo oocytes are in light gray and blue, respectively. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from four to 13 independent experiments. Statistical significance of differences was assessed by an ANOVA or a Kruskal–Wallis test depending on whether the data followed a Gaussian distribution, P < 0.0001 (****, Ctrl full versus Myo10^−/− full), P > 0.9999 (n.s, Ctrl full versus Ctrl oo), P = 0.3139 (n.s, Ctrl full versus Myo10^−/− oo), P < 0.0001 (****, Myo10^−/− full versus Ctrl oo), P = 0.0103 (*, Myo10^−/− full versus Myo10^−/− oo), and P = 0.0811 (n.s, Ctrl oo versus Myo10^−/− oo). n.s, not significant. (F) Images of the flattened zona pellucida obtained from oocyte segmentation. The top panel shows a control zona pellucida, and the lower one, a Myo10^−/− full zona pellucida. For each panel, the bottom image is a magnification of the brightfield image of the zona pellucida at the top. Scale bar = 10 μm. Below each flattened zona pellucida are examples of the zona pellucida from control and Myo10^−/− full oocytes. Brightfield images are on the left panels, SiR-actin (white)–labeled TZPs are on the middle panels, and merge images (SiR-actin in magenta) are on the right panels. Scale bar = 9 μm. (G) Scatter plot of the presence of vertical TZP-like structures in the zona pellucida (ZP), as represented by the schemes on the left. Control and Myo10^−/− full oocytes are in dark gray and green, respectively. Control and Myo10^−/− oo oocytes are in light gray and blue, respectively. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from four to 13 independent experiments. Statistical significance of differences was assessed by an ANOVA or a Kruskal–Wallis test depending on whether the data followed a Gaussian distribution, P < 0.0001 (****, Ctrl full versus Myo10^−/− full), P = 0.7183 (n.s, Ctrl full versus Ctrl oo), P = 0.7291 (n.s, Ctrl full versus Myo10^−/− oo), P < 0.0001 (****, Myo10^−/− full versus Ctrl oo), P < 0.0001 (****, Myo10^−/− full versus Myo10^−/− oo), and P = 0.9999 (n.s, Ctrl oo versus Myo10^−/− oo). n.s, not significant.

(A) Volcano plot of differential gene expression between fully grown Myo10^+/− and Myo10^−/− full oocytes. Differential transcriptomic analysis was performed by RNA-Seq. Up-regulated transcripts are shown in red (up, 605 transcripts); down-regulated transcripts, in blue (down, 80 transcripts, including Myo10); and non-deregulated transcripts, in gray (not significant, 15,702 transcripts). Significance was set at P-adj < 0.05. Myo10^−/− full oocytes were collected from two Myo10^flox/flox; Cre⁺ mice and Myo10^+/− oocytes from two Myo10^wt/flox; Cre⁺ mice. For each condition, three biological replicates (each containing 10 oocytes) and three technical replicates were performed. (B) Pie chart of deregulated transcript biotypes from the RNA-Seq analysis. lincRNA: long intergenic non-coding RNAs; miRNA: microRNA precursors; Mt-tRNA: transfer RNA located in the mitochondrial genome; and TEC: to be experimentally confirmed. (C) Bar graph of the relative mRNA quantity in fully grown Myo10^−/− full oocytes (colored bars) normalized to fully grown Myo10^+/− oocytes (gray bars) performed by RT–qPCR. (A) Selected mRNAs were chosen based on their biological relevance and their deregulatory strength (log₂[FC] and P-adj) in the RNA-Seq analysis described in (A). mRNAs indicated as up- or down-regulated by RNA-Seq analysis are shown in red and blue, respectively. Myo10^−/− full oocytes were collected from Myo10^flox/flox; Cre⁻ mice and Myo10^+/− oocytes from Myo10^wt/flox; Cre⁺ mice. For each condition, two biological replicates were performed (one containing 30 oocytes and the other containing 24 oocytes) from two different mice each time and two technical replicates were performed. The SEM is shown. For each mRNA, the mean of Myo10^+/− oocytes was normalized to that of Myo10^+/− oocytes. The mean and the SEM for Myo10^−/− full oocytes = 0.08 ± 0.004 (Myo10); 0.59 ± 0.171 (Stxbp5l); 0.99 ± 0.079 (Akr1b3); 1.19 ± 0.059 (Oser1); 1.48 ± 0.222 (Hsp90aa1); 1.48 ± 0.034 (Oaz1); 1.49 ± 0.333 (Eif4e); 1.27 ± 0.037 (Ccnb1); 1.44 ± 0.034 (Rps4x); and 1.53 ± 0.179 (Rpl19). Statistical significance of differences was assessed by two-tailed unpaired t tests, P < 0.0001 (Myo10), P = 0.1394 (Stxbp5l), P = 0.9001 (Akr1b3), P = 0.0864 (Oser1), P = 0.1612 (Hsp90aa1), P = 0.0050 (Oaz1), P = 0.2765 (Eif4e), P = 0.0180 (Ccnb1), P = 0.0060 (Rps4x), and P = 0.0987 (Rpl19). (D) Sum of Z projection images of oocytes stained with DAPI to label DNA (blue, top images of each panel) and incubated or not with 5-ethynyl uridine to label global RNA transcription (EU, bottom images of each panel). For each panel, control oocytes are on the left, and Myo10^−/− full oocytes, on the right. Growing and fully grown oocytes are on the left and right panels, respectively. Oocytes incubated with or without EU are on the upper and lower panels, respectively. For EU staining, contrast adjustment is similar between all conditions except for growing oocytes incubated with EU for which the signal intensity is too high to be shown with the same adjustment as the others. Scale bar = 10 μm. (E) Scatter plot of the relative EU signal intensity normalized to the DAPI signal intensity in control (dark gray) and Myo10^−/− full (green) growing or fully grown oocytes incubated or not with EU. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from three to five independent experiments. Statistical significance of differences was assessed by two-tailed Mann–Whitney’s tests, P = 0.7688 (growing Myo10^−/− full oocytes with EU compared to growing control oocytes with EU), P = 0.7564 (fully grown Myo10^−/− full oocytes with EU compared to fully grown control oocytes with EU), P = 0.0326 (growing Myo10^−/− full oocytes without EU compared to growing control oocytes without EU), P < 0.0001 (growing control oocytes with EU compared to growing control oocytes without EU), P < 0.0001 (growing Myo10^−/− full oocytes with EU compared to growing Myo10^−/− full oocytes without EU), P < 0.0001 (fully grown control oocytes with EU compared to fully grown control oocytes without EU), and P < 0.0001 (fully grown Myo10^−/− full oocytes with EU compared to fully grown Myo10^−/− full oocytes without EU). Statistical significance of differences was assessed by a two-tailed unpaired t test to compare fully grown Myo10^−/− full oocytes without EU to fully grown control oocytes without EU, P = 0.2647. n.s, not significant.

(A) Brightfield images from spinning disk videos from 3 h to 18 h after NEBD. The top images show a control oocyte; the middle ones, a Myo10^−/− full oocyte that extruded a polar body (PBE); and the bottom ones, a Myo10^−/− full oocyte that did not extrude a polar body (no PBE). The blue asterisk indicates a lysed polar body. Scale bar = 10 μm. (B) Stacked bars of first polar body extrusion rate as a percentage of control and Myo10^−/− full oocytes. The gray bars represent the percentage of oocytes that extruded a polar body (PBE), and the white bars, the percentage of oocytes that did not extrude a polar body (no PBE). (n) is the number of oocytes analyzed. Data are from three independent experiments. Statistical significance of differences was assessed by a two-sided Fisher exact test, P = 0.0113. (C) Scatter plot of the timing in hours after NEBD of first polar body extrusion of control (dark gray) and Myo10^−/− (green) full oocytes. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from six independent experiments. Statistical significance of differences was assessed by a two-tailed Mann–Whitney test, P = 0.3194. n.s, not significant.

(A) Maximum-intensity Z projection images of oocytes stained with SiR-tubulin to label microtubules (SiR-tub, yellow) merged with corresponding brightfield images. Images are extracted from spinning disk videos from 30 min to 14 h 30 min after NEBD. The top images show a control oocyte; the middle ones, a Myo10^−/− full oocyte that extruded a polar body (PBE); and the bottom ones, a Myo10^−/− full oocyte that did not extrude a polar body (no PBE). Scale bar = 10 μm. (B) Maximum-intensity Z projection images of oocytes stained with SiR-tub (yellow) 7 h after NEBD. The top image shows the spindle of a control oocyte; the middle one, the spindle of a Myo10^−/− full oocyte that extruded a polar body (PBE); and the bottom one, the spindle of a Myo10^−/− full oocyte that did not extrude a polar body (no PBE). Scale bar = 5 μm. (C) Graph of the percentage of control (dark gray) and Myo10^−/− (green) full oocytes with a bipolarized spindle as a function of time after NEBD. Data are from four independent experiments. For each time point, statistical significance of differences was assessed by a two-sided Fisher exact test, P > 0.9999 for 1 h after NEBD, P = 0.3433 for 2 h after NEBD, P = 0.7765 for 3 h after NEBD, P > 0.9999 for 4 h after NEBD, P = 0.6937 for 5 h after NEBD, and P > 0.9999 for 6 h after NEBD. (D) Scatter plots of spindle interpolar length (left) and central width (right) 7 h after NEBD in control (dark gray) and Myo10^−/− (green) full oocytes. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from four independent experiments. Statistical significance of differences was assessed by a two-tailed unpaired t test, P = 0.0989 for the length, and by a two-tailed unpaired t test with Welch’s correction, P = 0.0753 for the width. n.s, not significant. (E) Stacked bars of spindle cell localization 30 min before anaphase for oocytes that extruded a polar body or 12 h after NEBD for those that did not, as a percentage of control and Myo10^−/− full oocytes. Gray bars represent the percentage of oocytes with an off-centered spindle, and orange bars, the percentage of oocytes with a central spindle. (n) is the number of oocytes analyzed. Data are from four independent experiments. Statistical significance of differences was assessed by a two-sided Fisher exact test, P = 0.3832. n.s, not significant. (F) Maximum-intensity Z projection images from spinning disk videos of oocytes injected with H2B-GFP to label chromosomes (H2B, cyan) in a control oocyte (top images), a Myo10^−/− full oocyte that extruded a polar body (PBE, middle images), and a Myo10^−/− full oocyte that did not extrude a polar body (no PBE, bottom image). Left images show chromosomes 30 min before anaphase for polar body–extruding oocytes or 12 h after NEBD for the Myo10^−/− full oocyte that did not extrude a polar body. Right images show chromosomes after anaphase. Scale bar = 5 μm. (G) Scatter plots of metaphase plate height (left) and width (right), as shown in the lower schemes, 30 min before anaphase for oocytes that extruded a polar body or 12 h after NEBD for those that did not. Control and Myo10^−/− full oocytes are in dark gray and green, respectively. (n) is the number of oocytes analyzed. Data are the mean ± s.d. with individual data points plotted. Data are from three independent experiments. Statistical significance of differences was assessed by two-tailed unpaired t tests. P = 0.5331 for the height and P = 0.0395 for the width. n.s, not significant.