The lab aims to better understand the mechanisms involved in the acquisition of female gamete quantity, which is essential for the reproduction of sexual species using the mouse oocyte model system. Recently we identified a novel mechanical transduction process that promotes agitation of the nucleus and its content leading to the fine regulation of maternal RNA in mouse oocytes. The imaging and image analysis pipeline presented in this protocol allows us to highlight the transmission of cytoskeletal forces as a continuum across scales from the cytoplasm to the nucleus and its components, including nuclear RNA processing bodies in mouse oocytes.
This protocol provides simple tools that allow us to address first transmission across multiple cellular scales in a single pipeline for the first time. Complemented with biophysical modeling, this protocol constitutes a non-invasive technique to address changes in nuclear mechanics and the dissipation of energy across cellular compartments. Begin by arranging all the materials required for the experiment on the working platform.
After extracting ovaries from the mouse, place them into pre-warmed M2+bovine serum albumin medium supplemented with one micromolar milrinone. Puncture ovarian follicles with surgical needles to release growing oocytes from the antral follicles. Using a micropipet, collect oocytes of the size needed for experiments, then transfer the oocytes into a dish with fresh medium.
Transfer oocytes from drop to drop using the micropipette. This allows the mechanical dissociation of oocytes from antral follicular cells. Stabilize the oocyte for one hour at 37 degrees Celsius.
Centrifuge the cRNA aliquots coating for YFP-Rango and SSF2 GFP. Using a micro injector, inject cRNA into the cytoplasm of oocytes in a warm M2+bovine serum albumin medium, supplemented with milrinone. Incubate oocytes for two hours in a culture medium at 37 degrees Celsius for cRNA translation.
Next, deposit oocytes into culture medium droplets on a 35 millimeter tissue culture dish with a cover glass bottom chamber covered with mineral oil. To begin, turn on the imaging software. Open the Acquire window, set the Exposure Time to 500 milliseconds, Camera Area to Full Chip, and Binning to 1.
Then set the illumination for the required channel. For cytoplasmic stirring, use transmitted light to focus on the oocyte nucleolus. For nuclear speckle experiments, focus on SRSF2 GFP droplets.
To optimize acquisition speed in the Special tab, set the Camera Shutter to Open for Exposure and Clear Mode to CLEAR PRE SEQUENCE. Next, open the Stream Acquisition window. In the Acquired tab, set the Acquisition Mode to Stream to RAM and the Number of frames to 480.
Set Camera Parameters to Acquire images at frame rate, and Number of frames to skip to 0. In the Digital Camera Controller Parameters tab, set the Camera State to HALT, Shutter Mode to OPEN NEVER, Clear Mode to CLEAR PRE SEQUENCE, and Number of frames to average to 1. Next, adjust a region of interest around the object.
In the Stream Acquisition window, click on Acquire to launch the movie. After acquisition, save the movie as a tiff file. For image analysis, open image J or Fiji.
In the plugins, click on CIRB and open Verlhac menu, then select Oocyte Nucleus Shape. In the dialogue box, select the folder containing the movies to analyze. Then select the Theta angle and the margins for cropping.
Now select the XY calibration and the Time interval. Then click on OK.To plot the mean shape over time, in a spreadsheet calculate the mean radius R over all the time points T for each defined theta angle. For each T and theta, subtract the mean radius R over time from the radius lowercase r.
Finally, calculate the mean of the fluctuations for all time points T and all angles theta for each nucleus and all nuclei from one condition. In control oocytes, the nucleus displayed significant peripheral fluctuations as visualized with the nuclear probe YFP-Rango. In contrast, the nucleus in oocytes with disrupted cytoskeletal forces, remain stable over time.
The variance of the distance from the nucleus centroid to its periphery revealed a sixfold increase in control oocytes compared to those with disrupted cytoskeletal forces. Nuclear speckles in control oocytes showed greater fluctuation in droplet surfaces compared to the disrupted cytoplasmic forces.
