This protocol can be used to apply an extracellular flux analyzer to monitor changes in energy metabolism during sperm capacitation. We developed this protocol to compare changes in glycolysis and oxidative phosphorylation in real time between non-capacitated and capacitated mouse sperm. On the day before the assay, remove the sensor cartridge from an extracellular flux assay kit and place the cartridge upside down next to the utility plate.
Fill a solution reservoir with 25 milliliters of double distilled water and add 200 microliters of water to each well of the utility plate, then place the sensor cartridge back into the utility plate. Place the plate into a 37 degree Celsius non-carbon dioxide incubator. Next, aliquot 25 milliliters of extracellular flux analyzer calibrant into a 50 milliliter conical tube and place the tube into the 37 degree Celsius non-carbon dioxide incubator.
To prepare ConA coated micro plates, dissolve 2.5 milligrams of ConA in five milliliters of double distilled water and use a multichannel pipette to fill each well of an extracellular flux analyzer 96-well plate with 50 microliters of the ConA solution, then leave the lid open to let the plate dry overnight. To prepare the sperm buffer, heat 250 milliliters of extracellular flux analyzer TYH buffer to 37 degrees Celsius and adjust the pH to 7.4. On the day of the assay, replace the water in each well of the utility plate with 200 microliters of XF calibrant per well and place the plate back into the non-carbon dioxide incubator for at least one hour.
To generate a wave template, turn on the extracellular flux analyzer. While the temperature is stabilizing to 37 degrees Celsius, open the wave software and open a blank template. Under the group definitions tab, define the assay medium as TYH and the cell type as mouse sperm, leaving the injection strategies and pretreatments blank.
Create different groups for each assay condition and open the plate map to assign each group to specific wells. Under the protocol tab, unhighlight equilibrate, add each of four different measurement cycles, select the respective port, and edit the measurement details so that the measure after injection is highlighted, then save the assay template, fill out the project summary and save the results. Before harvesting the sperm, prewarm 50 milliliters of TYH buffer at 37 degrees Celsius.
After harvesting caudas from adult male mice, place each cauda in 500 microliters of the pre-warmed TYH buffer in individual wells of a 24-well plate. Use forceps to immobilize each cauda individually at the bottom of each well and quickly make five to seven small incisions in each tissue using feather scissors. When all of the caudas have been incised, immediately place the plate into the non-carbon dioxide incubator to allow the sperm to disperse for 15 minutes.
To load the sensor cartridge, remove the lid from the sensor cartridge and align the letter of the respective port loading guide with the upper left corner of the cartridge. Holding the port loading guide in place, insert the pipette tips vertically into the port loading guide holes of port A and inject 20 microliters of TYH buffer into every column. For port B, inject 22 microliters of TYH buffer into columns one through four, 22 microliters of 500 millimolar 2-deoxyglucose into columns five through eight, and 25 microliters of five micromolar antimycin A rotenone into columns nine through 12.
For port C, inject 25 microliters of TYH buffer into every column with odd numbers and 25 microliters of 10 millimolar of dibutyryl-cyclic AMP 500 micromolar IBMX into every column with even numbers, then place the sensor cartridge with a calibration plate into the extracellular flux analyzer and start the assay. The calibration will take 10 to 15 minutes. To prepare the sperm plates, add three milliliters of three milligrams per milliliter bovine serum albumin in TYH buffer to each of three five milliliter centrifuge tubes.
Next, pool two wells of sperm into each of three 1.5 milliliter centrifuge tubes. After counting, dilute the sperm to a two times 10 to the seven sperm per milliliter concentration per sample in fresh TYH supplemented with bovine serum albumin. Sediment the sperm by centrifugation and resuspend the sperm pellets in one milliliter of TYH buffer.
Centrifuge the sperm again and remove the supernatants taking care not to disturb the pellets. Transfer each sperm suspension into one of the prepared five milliliter centrifuge tubes and add 180 microliters of TYH buffer into each corner well of a ConA coated plate. Add 180 microliters of sperm into each empty well of the ConA coated plate and centrifuge the plate two times rotating the plate 180 degrees between centrifugations, then place the calibration plate with the sperm plate and continue the assay.
In this representative experiment, sperm were capacitated in the presence of glucose as the only energy substrate and 2-deoxyglucose and antimycin and rotenone as the pharmacological modulators. The arrow indicates induction of capacitation. In the presence of glucose, capacitation was accompanied by a seven-fold increase in the extracellular acidification rate which is inhibited by blocking glycolysis with 2-deoxyglucose.
Capacitated sperm demonstrated a 20-fold increase in the oxygen consumption rate compared to non-capacitated sperm demonstrating that mouse sperm enhance both glycolysis and oxidative phosphorylation to support the increasing energy demand during capacitation. The rise in the extracellular acidification rate during sperm capacitation is not affected by the oxidative phosphorylation inhibitors antimycin A and rotenone indicating that the change in this rate is mainly driven by hydrogen release from glycolysis. The increase in oxygen consumption rate, however, is blocked by antimycin A and rotenone as well as by 2-deoxyglucose revealing that the increase in OXPHOS during sperm capacitation is dependent on glycolytic activity.
The energy source and pharmacological activators and inhibitors can be varied depending on the goal of the experiment and up to 12 different conditions can be measured in parallel. The protocol can be easily adapted to other species like human or bovine where the changes in energy metabolism during capacitation are equally enigmatic.
