Our assays can be used to test compounds for their effects on human sperm cell function. Specifically, these methods can be used to quickly and easily screen large numbers of compounds for their effects on calcium signaling and acrosome reaction in human sperm cells. Visual demonstrations of this method will make it easier for others to set up similar experiments in their own labs.
Begin by placing one 50 milliliter tube per one milliliter of raw semen sample into a tube rack at a 45 degree angle and adding four milliliters of 37 degree Celsius Human Tubal Fluid or HTF positive medium to each tube. Next, carefully pipette one milliliter of liquified semen sample to the bottom of each tube and place the tubes at 37 degrees Celsius for one hour. At the end of the incubation, use a modified pipette tip held at a 45 degree angle to gently transfer as much of the upper fraction of motile sperm cell containing HTF positive medium as possible into a new 15 milliliter plastic tube.
To count the collected sperm cells, dilute a 20 microliter aliquot to a factor of 10, 20, 30, 50, or 90 in S100 buffer in a round bottom two milliliter tube according to the expected concentration. Vortex the diluted sperm cells for 10 seconds at 700 rotations per minute. Next, immerse an SP1 cassette into the sample and fully depress the white plunger to aspirate an aliquot of the sample into the cassette.
Then place the cassette in the image cytometer tray and run the PI stained human sperm cell assay according to the applied dilution factor. To measure changes in the free intracellular calcium concentration, gently pipette the sperm sample up and down one time and use an automatic repeater pipette to aliquot 50 microliters of fluorophore-stained sperm cells to 24 wells of one row of a 384 micro well plate. Place the micro well plate in the fluorescence plate reader at 30 degrees Celsius and select the appropriate protocol for fluorophore.
Select a well in the row of sperm cells and adjust the gain to a target value of 20%Then start the measurement. After 10 baseline cycles, pause the experiment and eject the drawer with the micro well plate. Using a 12 channel pipette, quickly add 25 microliters of the prepared solutions of compounds and controls of interest into the 12 duplicate wells in the row and immediately return the plate to the reader to continue the measurement as quickly as possible.
Any changes in fluorescence in response to the addition of the compounds or controls will be captured by the instrument. For acrosome reaction analysis, after incubating the capacitated sperm cells with the appropriate fluorescent dyes, compounds or controls, mix the samples by pipetting and add 50 microliters of each test sample to 100 microliters of immobilizing solution. Mix the samples again and load the chambers of one A2 slide per sample with approximately 30 microliters of sample per chamber.
Then place the first loaded slide onto the tray of the image cytometer. Select the FlexiCyte Protocol and press Run. Here representative results from an experiment testing the effect of four compounds, a positive progesterone control and a negative buffer control using the medium throughput calcium signaling assay as demonstrated can be observed.
In this graph, a dose response curve of progesterone derived from peak percents changes in the intracellular free calcium concentration induced by serially diluted concentration of progesterone is shown. These data illustrate the effects of one negative or one of two positive controls on the induction of an acrosome reaction in capacitated human sperm cells in a medium throughput acrosome reaction assay. To avoid missing the peaks of the induced signals, it is critical to add the chemicals quickly to the replicate wells and to immediately continue the measurement thereafter.
By changing the type of fluorophores and dyes, our assays can easily be modified to answer different scientific questions. These assays have already been used in several studies to test large groups of compounds for their effects on human sperm function.
