Figure 3.
Figure 3. Hurp−/− oocytes show reduced MT density and improper MTOC distribution. (A) Reduced MT density early on in Hurp−/− oocytes. Fluorescence intensity in wt and Hurp−/− oocytes, expressing EB3-GFP and H2B-RFP, was assessed in the chromosome vicinity. MT density is lower in Hurp−/− oocytes (n = 9) from NEBD + 30 and + 60 min (**, P = 0.0125 and 0.0071) compared with wt (n = 10). (B) Prometaphase I monasters are smaller, with fewer MTs in Hurp−/− oocytes. Fluorescence intensity was assessed as in A in monastrol-treated oocytes at NEBD + 2 h. Mean monaster size: 29.19 ± 3.04 µm for Hurp−/− compared with 32.86 ± 2.22 µm for wt (left; **, P = 0.0143). Monaster MT density is lower in Hurp−/− than wt (right histogram; wt, n = 9; and Hurp−/−, n = 7; **, P = 0.0228). Error bars represent standard deviation. (C) Low MT density in the spindle center in Hurp−/− oocytes. Longitudinal line scans from MI spindles of wt (n = 6) and Hurp−/− (n = 10). Intensities were normalized to maximum value within the same spindle, and spindle size was interpolated. Light curves, individual spindles. Dark curves, mean values. (D) MTOCs are progressively sorted at spindle poles in MI. (top) MTOCs were labeled using pericentrin (green) and chromosomes with Hoechst (red). (bottom) Quantitative analysis of MTOC distribution along the spindle axis at 2 (n = 16), 4 (n = 18), 6 (n = 22), and 8 h (n = 17) after NEBD. The pericentrin signal was binarized to assess the individual MTOC area (a.u., arbitrary units). Coordinates of MTOCs are plotted along the spindle axis (0–1). (E) MTOCs are progressively clustered during MI. Box plot representation of individual MTOC size at selected time points. Median values are shown in red. (F) Abolished MTOC sorting in Hurp−/− oocytes. wt and Hurp−/− oocytes were observed at NEBD + 7 h and labeled as in D. (G) Quantification of MTOC distribution along the spindle axis in late MI in wt (n = 5) versus Hurp−/− (n = 10). Pericentrin signal was binarized as in D. Individual MTOC size is plotted against its coordinates on the spindle axis (0–1). Bars, 10 µm.

Hurp−/− oocytes show reduced MT density and improper MTOC distribution. (A) Reduced MT density early on in Hurp−/− oocytes. Fluorescence intensity in wt and Hurp−/− oocytes, expressing EB3-GFP and H2B-RFP, was assessed in the chromosome vicinity. MT density is lower in Hurp−/− oocytes (n = 9) from NEBD + 30 and + 60 min (**, P = 0.0125 and 0.0071) compared with wt (n = 10). (B) Prometaphase I monasters are smaller, with fewer MTs in Hurp−/− oocytes. Fluorescence intensity was assessed as in A in monastrol-treated oocytes at NEBD + 2 h. Mean monaster size: 29.19 ± 3.04 µm for Hurp−/− compared with 32.86 ± 2.22 µm for wt (left; **, P = 0.0143). Monaster MT density is lower in Hurp−/− than wt (right histogram; wt, n = 9; and Hurp−/−, n = 7; **, P = 0.0228). Error bars represent standard deviation. (C) Low MT density in the spindle center in Hurp−/− oocytes. Longitudinal line scans from MI spindles of wt (n = 6) and Hurp−/− (n = 10). Intensities were normalized to maximum value within the same spindle, and spindle size was interpolated. Light curves, individual spindles. Dark curves, mean values. (D) MTOCs are progressively sorted at spindle poles in MI. (top) MTOCs were labeled using pericentrin (green) and chromosomes with Hoechst (red). (bottom) Quantitative analysis of MTOC distribution along the spindle axis at 2 (n = 16), 4 (n = 18), 6 (n = 22), and 8 h (n = 17) after NEBD. The pericentrin signal was binarized to assess the individual MTOC area (a.u., arbitrary units). Coordinates of MTOCs are plotted along the spindle axis (0–1). (E) MTOCs are progressively clustered during MI. Box plot representation of individual MTOC size at selected time points. Median values are shown in red. (F) Abolished MTOC sorting in Hurp−/− oocytes. wt and Hurp−/− oocytes were observed at NEBD + 7 h and labeled as in D. (G) Quantification of MTOC distribution along the spindle axis in late MI in wt (n = 5) versus Hurp−/− (n = 10). Pericentrin signal was binarized as in D. Individual MTOC size is plotted against its coordinates on the spindle axis (0–1). Bars, 10 µm.

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