Herein.Two different methods for evaluating sperm chemo attraction are described for the two chamber assay. Activated frog sperm are first dispensed into inserts with porous membrane bottoms set into the wells of a 24 well plate. Then once the sperm are in place, a chemo attractant is carefully added to the bottom of the well to initiate a chemo attractive gradient.
Next, the sperm are incubated to allow the cells time to cross the porous membrane in response to the chemotactic gradient. Finally, the insert is removed and the sperm that have migrated to the bottom of the well in the two chamber assay are enumerated by hemo cytometer. Ultimately, the activity of sperm chemo attractants can be compared and quantified, for example, by a dose response curve analysis for the zigman chamber tracking assay.
A cover glass is first applied to the zigman chamber with silicon oil and placed upside down over the circular opening of a microscope. Stage one side of the trth of the chamber is then filled with activated frog sperm. Next, the other trth is filled with the chemo attractant solution.
After five minutes, the chemo tactic gradient forms, and at this time, the sperm can be videotaped swimming along the gradient. Ultimately, the footage acquired from the zigmund chamber tracking assay can be analyzed to determine the velocity of sperm and the chemo kinetic patterns and trajectories of sperm travel in response to a chemo attractant gradient. These two methods for detecting sperm chemotaxis are complimentary and they produce different types of data.
The two chamber assay allows sperm to pass through a porous membrane, thereby allowing quantitation of the sperm response to a chemo attractant. Visual demonstration of the two chamber assay is critical because the sperm and chemo attractant addition steps in the beginning as well as the removal of sperm and inserts steps at the end require precise precisioning of the micro pipette. The zigmund chamber assay uses video to record sperm swimming in a chemo tact gradient.
This allows determination of dynamic parameters such as velocity and direction. The main advantage of the zigmund chamber tracking assay over the two chamber assay is that the sperm swimming can be visualized and analyzed with a number of dynamic parameters. Flush the sperm from a frog testis by poking holes at one end and injecting buffer with syringe and needle at the opposite end.
After flushing the sperm from the frog testes, estimate the number of cells collected by hemo. Then dilute the stock sperm suspension with oocyte ringer's buffer to obtain a sperm density of two times 10 to the seven cells per milliliter. Assess sperm motility by diluting five microliters of sperm one to 10 with F1 buffer and visualizing movement using phase contrast optics.
Next micro pipette, 700 microliters of F1 buffer into each well of a plastic 24 well tissue culture plate. Then dilute 100 microliters of the sperm suspension with 900 microliters of F1 buffer at room temperature and a micro centrifuge tube to activate sperm motility by osmotic shock for each assay to be performed. Place a 12 millimeter OD 12 micrometer porosity insert into a buffer filled well, making certain that the insert placement is off center, leaving a space to one side.
Then using a micro pipette with a cutoff tip immediately transfer 400 microliters of motility activated sperm into the, well apply the sperm suspension to the wall of the insert, allowing it to run down onto the filter at the bottom of the insert. Next, carefully micro pipette. 50 microliters of the chemo attraction agent into each well of the plate in the space between the well and the off-center insert.
Being careful to deposit the drop where the side and the bottom of the well meet. Withdraw the pipette with no disturbance to the system. After all the assays have been set up.
Leave the plate at room temperature until the first assay started has incubated for 50 minutes. Then after first, carefully steadying the insert with one hand. Stop the assay by removing most or all of the sperm suspension in the upper chamber by micro pipette.
Once the sperm has been transferred from the upper chamber, immediately pull out the insert and discard it. Now mix the sperm suspensions in each well to resuspend any sperm that have settled on the bottom. Then transfer the entire sperm suspension from each well into individual micro centrifuge tubes.
Add formaldehyde to a final concentration of 0.5%volume per volume. Pellet the fixed sperm in each tube. Using the ten second push button, spin on a personal micro centrifuge.
Then remove all but 100 microliters of the supernatant from each tube and resuspend the pellet in that volume. Next, dilute 20 microliters of the sperm suspension, one to 10 with distilled water and count the sperm on a hemo cytometer. Using the 40 x objective of an upright microscope, the two chamber assay provides quantitative results that are appropriate to dose response studies or to comparisons between attractants.
In example, frog egg water contains the sperm chemo attractant alluring as shown with the red symbols. Egg water produces an increase and then a decrease in the number of sperm crossing the porous membrane as the dose of sperm chemo attractant is increased. In contrast, bovine serum albumin represented by the white symbols has little effect over the same dose range.
Before starting the zigman chamber assay first, confirm that the inverted microscope workstation is prepared for videotaping. Set up the microscope. Initiate the camera control software to acquire seven frames per second.
Check that the camera is focused on the observation platform and that the output is being sent to the computer monitor. Next, check that the microscopic field includes most or all of the observation platform width. Then prepare a frog sperm suspension in oocyte ringer's buffer, and a chemo attractant diluted to the proper concentration with F1 buffer and store both on ice until use.
Then starting with a clean, dry zigmund chamber. Use a micro pipette to place a line of approximately four microliters of silicone oil, about five milliliters from and parallel to the outer edge of each trth. Then place a 22 by 40 millimeter cover glass onto the chamber, allowing the silicone oil to evenly spread to the outer edge of each trth.
Now invert the chamber and place it over the large circular cutout in the microscope stage. Being careful that the cover glass does not make contact with the stage activate 20 microliters of the xap sperm in oocyte ringer's buffer by mixing one to 10 with F1 buffer at room temperature. Using a micro pipette with a cut tip immediately draw up 70 microliters of the motility activated sperm.
Then holding the micro pipette at a low angle and placing the tip at the side opening. Fill the trth and bridge by capillary action. Then fill the opposite trth in the same manner using a chemo attractant solution as xap sperm have limited motility lifetime.
Begin videotaping within three minutes and continue for five minutes. The zigman chamber tracking assay allows video microscopy of sperm swimming in a chemo attractant gradient. This movie shows a recording of sperm swimming on the observation platform with their trajectories being traced in red as shown in this diagram.
Sperm trajectories consist of segments with each segment indicated by an arrow and representing sperm travel between successive movie frames. The trajectory segments trace out swimming behavior of individual sperm and highlight patterns such as circles and turns. From these data, one can determine the velocity and direction of each segment or the properties of all the segments in a trajectory or a group of trajectories.
For example, net linear distance. The vector joining the start of a trajectory with its end has an x axis component and lies at an angle theta from the x axis as represented here in red theta should decrease and x axis travel should increase when a chemo attractant gradient is present. These changes mean that sperm are showing a preference for swimming up the gradient towards the chemo attractant trth, While performing both of these assays is important to remember that attention to the proper loading of sperm and chemo attractant in each device is critical to obtaining reproducible chemotactic gradients in sperm performance.
