Figure 6.
MRCK promotes myosin II activation in the FC and male PN centration. (A) Immunofluorescence detection of activated myosin II (P-MRLC) in fertilized oocytes. Top row: Control oocyte treated with DMSO. Bottom row: Oocyte fertilized in the presence of BDP9066 (2 µM). Note the P-MRLC ring at the base of the FC in the control oocyte and the absence in the MRCK-inhibited oocyte. Oocytes were fixed 3 h after insemination. Fluorescence intensity profiles correspond to the cortical regions delineated by the gray arrowheads in the merge images. (B) Bar graph depicting the fraction of fertilized oocytes showing a P-MRLC ring at the base of the FC, as shown in A. The fertilization medium was supplemented with DMSO (control) or BDP9066 (2 µM). (C) Scatter plot of the average curvature of the FC. (D) Schematic illustrating the experimental protocol for monitoring male PN migration after zona-intact fertilization. (E) Detection of male and female pronuclei in zygotes fixed 8 h after insemination. The left image shows a control zygote treated with DMSO after PB2 emission. The yellow line denotes the distance (d) of the male PN to the nearest cortex. The right image shows a zygote treated with BDP9066 (2 µM) after PB2 emission. Note the expanded male PN still apposed to the FC cortex. Images are z-compressions of two consecutive confocal frames. (F) Scatter plot of the male PN distance to the nearest cortex, as depicted in E. (G) Schematic illustrating the experimental protocol for monitoring male PN migration after injection and zona-free fertilization. (H) Detection of male and female pronuclei in zygotes fixed 8 h after zona-free insemination. The left image shows a control zygote injected with water. The yellow line denotes the distance (d) of the male PN to the nearest cortex. The right image shows a zygote expressing MRCKβ-K105A. (I) Scatter plot of the male PN distance to the nearest cortex, as depicted in H. Red and blue horizontal bars indicate means (C and I) or medians (F). P values were calculated using Fisher’s exact test (B), two-tailed Student’s t test (C and I), or a two-tailed Mann–Whitney U test ( F). The number of oocytes scored is indicated in parentheses above each bar/scatter plot. Scale bars are 10 µm. Chromosomes were stained with TO-PRO-3 (magenta). F-actin was labeled with Alexa Fluor 568–phalloidin. In E and H, female pronuclei were immuno-stained for H3K4me3 (green). Sp: sperm chromatin.

MRCK promotes myosin II activation in the FC and male PN centration. (A) Immunofluorescence detection of activated myosin II (P-MRLC) in fertilized oocytes. Top row: Control oocyte treated with DMSO. Bottom row: Oocyte fertilized in the presence of BDP9066 (2 µM). Note the P-MRLC ring at the base of the FC in the control oocyte and the absence in the MRCK-inhibited oocyte. Oocytes were fixed 3 h after insemination. Fluorescence intensity profiles correspond to the cortical regions delineated by the gray arrowheads in the merge images. (B) Bar graph depicting the fraction of fertilized oocytes showing a P-MRLC ring at the base of the FC, as shown in A. The fertilization medium was supplemented with DMSO (control) or BDP9066 (2 µM). (C) Scatter plot of the average curvature of the FC. (D) Schematic illustrating the experimental protocol for monitoring male PN migration after zona-intact fertilization. (E) Detection of male and female pronuclei in zygotes fixed 8 h after insemination. The left image shows a control zygote treated with DMSO after PB2 emission. The yellow line denotes the distance (d) of the male PN to the nearest cortex. The right image shows a zygote treated with BDP9066 (2 µM) after PB2 emission. Note the expanded male PN still apposed to the FC cortex. Images are z-compressions of two consecutive confocal frames. (F) Scatter plot of the male PN distance to the nearest cortex, as depicted in E. (G) Schematic illustrating the experimental protocol for monitoring male PN migration after injection and zona-free fertilization. (H) Detection of male and female pronuclei in zygotes fixed 8 h after zona-free insemination. The left image shows a control zygote injected with water. The yellow line denotes the distance (d) of the male PN to the nearest cortex. The right image shows a zygote expressing MRCKβ-K105A. (I) Scatter plot of the male PN distance to the nearest cortex, as depicted in H. Red and blue horizontal bars indicate means (C and I) or medians (F). P values were calculated using Fisher’s exact test (B), two-tailed Student’s t test (C and I), or a two-tailed Mann–Whitney U test ( F). The number of oocytes scored is indicated in parentheses above each bar/scatter plot. Scale bars are 10 µm. Chromosomes were stained with TO-PRO-3 (magenta). F-actin was labeled with Alexa Fluor 568–phalloidin. In E and H, female pronuclei were immuno-stained for H3K4me3 (green). Sp: sperm chromatin.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal