Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Our research goal is to better understand the molecular mechanisms that regulate sperm function, including the processes such as capacitation and fertilization. Specifically, we're exploring how membrane lipids activate calcium signaling and acrosome accessibility. This protocol addresses the gap in our understanding of the interplay between capacitation, sperm activation, calcium signaling, and acrosome exocytosis.

The advantages of this protocol include imaging sperm under physiological conditions, as well as its ability to simultaneously monitor both calcium signaling and acrosome exocystosis without the need for complicated preloading or standing procedures. Begin by collecting sperm from the epididymus isolated from a transgenic model mouse expressing AcroSensE. Perform the sperm collection in a 3.5 centimeter tissue culture dish containing 0.5 milliliters of MW medium by allowing a 15 minute swim out procedure at 37 degrees Celsius.

Then using fine forceps, carefully remove any epididymal tissue from the swim out collection dish. Transfer the medium containing the sperm into a 15 milliliter conical tube, and using a swinging bucket rotor, centrifuge the suspension at 100 G for one minute at room temperature. With the help of a plastic pipette, collect the MW supernatant containing the sperm while discarding any pellet of gross tissue debris and transfer it to a round-bottom tube.

Adjust the supernatant volume to one milliliter by adding 500 microliters of MW medium, pre-warmed to 37 degrees Celsius. Centrifuge this mixture at 400 G for eight minutes at room temperature in a round-bottom tube. Then, carefully remove the supernatant while leaving behind the loosely pelleted sperm.

Using a large bore transfer pipet, resuspend the sperm by gently tapping the tube manually, known as finger flicking. Once the sperm are evenly resuspended, transfer them into a new round bottom tube. To begin, pull borosilicate glass capillaries with an outer diameter of two millimeters and an inner diameter of 1.56 millimeters, following appropriate settings.

Prior to filling the capillary before each experiment, use fine tweezers to break open the tip of the capillary. For loading the capillary, use the stimulating solution at three to five times the normal concentration of the stimulant required. With a thin plastic transfer pipette, slowly inject the stimulating solution into the capillary until three fourths of its length is filled.

Eliminate any air bubbles that may form during the filling process by gently flicking the capillary. Then mount the filled capillary on the micro manipulator. Configure the single cell delivery system settings to provide a ten second pulse at 5 PSI.

Connect the Borosilicate capillary mounted on the micro manipulator to the single cell delivery system pipette holder. Apply a brief puff lasting two seconds at a pressure of 20 PSI while observing the capillary tip to confirm that the solution is being dispensed through the tip's end. The setup is now ready for use with microscopy for real-time imaging of a single mouse sperm.

Begin by coating the 35 millimeter cover slip dishes with Poly-D-Lysine or PDL. To do so, dispense a 0.5 microliter droplet of PDL at the center of a cover slip dish. Using a 10 microliter graduated pipette, smear the PDL droplet across the cover slip.

Allow the coated cover slip dish to dry at 37 degrees Celsius for 10 minutes. For imaging the freshly collected sperm from a mouse model expressing AcroSensE, add 80 microliters of the pre-prepared sperm suspension to the center of the PDL-coated cover slip dish. Then slowly add three milliliters of supplemented modified Whitten's base media, prewarm to 37 degrees Celsius, to the dish.

Proceed to perform the imaging using a combination of a microscope with the single cell stimulant delivery setup. First mount the dish containing the sperm on the microscope. Then using the micro manipulator, position the capillary tip of the stimulant delivery system about 100 microns to the side and five to 10 microns above the plane of the cell of interest.

Position the capillary within 100 microns from the sperm head. Next, start the imaging sequence on the microscope. Image sperm cells at a high frame rate, preferably greater than 10 frames per second.

10 seconds after initiating the image acquisition, activate the single cell delivery system to deliver a ten-second puff of stimulant. Continue capturing images of cells for a duration of 10 to 15 minutes. In a similar manner, image multiple cells from a single dish according to the locations shown on the screen.