The overall goal of this procedure is to measure intracellular calcium fluctuations in human sperm with fluorescent dyes. This is accomplished by first preparing the sperm sample by the swim up method and if needed, promoting capacitation. The second step is to load the sperm with a calcium fluorescent dye.
Next, using conventional fluorometry stopped flow fluorometry, flow cytometry or single cell imaging, perform the experiment and record the calcium measurements. Ultimately, the results are analyzed to determine changes in intracellular calcium concentrations triggered by progesterone or during the capacitation process. The main advantage of this technique over existing methods like those involving radioactivity is that this is a very sensitive procedure.
It is safe and easy to perform. This method can help answer key questions in the SPR calcium signaling field, such as identification of compounds that induce intracellular calcium, increases with high spatial and temporal resolution and identification of different cellular sub populations in a siemen sample. The implications of using, for example, the flow cytometry technique extend towards diagnosis of fertility problems through the analysis of the physiological state of male gamuts.
Even though this method can provide insights into the study of calcium mobilization of human sperm, it can also be applied to other type of cells such as male gamuts from other species or different type of cells such as neurons or muscle cells. Generally, individuals who use this method will struggle because the sperm handling is not easy and it is important to maintain appropriate conditions to obtain viable and motor cells through the experiment. We implemented the use of these techniques by combining strategies using different fields.
Visual demonstration of this method is important as preparation and handling of the sample, as well as the addition of the compounds are difficult to learn because sperm cells can be damaged during the procedure and artifacts in the responses could be obtained during the edition of the compounds. To prepare the sperm sample, one milliliter of hams F 10 medium must be carefully layered on top of each 500 microliter liquified semen. Eloqua, touch the wall of the tube with the tip of the micro pipet and gently dispense the medium above the sample.
It is crucial to do it slowly to avoid mixing the sample and medium layers. The medium is supplemented with two millimolar calcium chloride and five milligrams per milliliter of BSA. To promote capacitation in vitro, carefully lean the tubes to approximately a 30 degree angle.
This will increase the surface area between the two liquids, thus enhancing the displacement or swim up of sperm cells from the sample to the medium during incubation. Next, place the lean test tubes inside an incubator at 37 degrees Celsius and 5%carbon dioxide. 95%air for one hour after one hour.
Use a micro pipet to carefully remove from each tube the upper 700 microliters of the hams F 10 medium that now contains motil spermatozoa. Pool all the collected samples into a single clean glass tube, avoiding bubble formation. Place 10 microliters of pooled sample on the optical flat glass of a macular counting chamber base and place the cover glass.
Make sure to avoid bubble formation inside the chamber as this would result in an inaccurate cell count. Observe under a compound microscope equipped with a 20 x objective. The cover glass of the macular counting chamber has a big square composed of 100 smaller squares.
Count the cells in any strip of 10 squares. This number represents the cell concentration in millions of cells per milliliter. Repeat the count in two additional 10 square strips and calculate the mean of the three counts to load cells with a fluorescent dye.
Combine in a 1.5 milliliter fuge tube, the required volume of sperm suspension with enough one millimolar flu oh 3:00 AM stock solution to obtain a final concentration of two micromolar flu oh 3:00 AM incubate for 30 minutes at 37 degrees Celsius and protected from light centrifuge the tube at 750 G for five minutes. The formation of a cloud rather than a pellet indicates that the cells are in good condition, aspirate and discard the supernatant and resuspend the pellet in the appropriate volume of human sperm medium or HSM. To begin this experiment, combine in a flat bottom glass tube, 570 microliters of HSM and 30 microliters of sperm cell suspension previously loaded with flu oh 3:00 AM and resuspended in HSM to obtain one times 10 to the eighth cells per milliliter.
Place a magnetic stir bar inside the glass tube and insert the tube into the reading chamber of the spectometer preheated to 37 degrees Celsius. The sample must be stirred throughout the acquisition time. Start the experiment using the oli software and proceed to acquire fluorescence values at a frequency of 0.5 hertz during 300 seconds.
After acquiring basal fluorescence for 30 seconds, use a Hamilton Micro syringe to inject the appropriate volume of test compound in this case for micromolar progesterone at 100 seconds. At 20 micromolar ion mycin as a positive control to obtain the maximum fluorescence value. In this experiment, intracellular calcium changes are measured with high temporal resolution.
Using an SFM 20 stopped flow mixer coupled to a rabid kinetics optical system, fill one of the instrument syringes with one milliliter of flu. 3:00 AM loaded sperm cells and the second syringe with one milliliter of the compound to be tested. 20 micromolar progesterone dissolved in HSM.
In this example, it is crucial to avoid bubble formation while drawing the liquids into the syringes. Lift both instrument pistons until they touch the tip of the syringe plungers. Set the total sampling time in this case to 50 seconds and the frequency to 10 milliseconds in order to minimize cell damage, set the flow rate to the minimum value that will provide a measurable response trigger.
The mixing of reagents, the trace of raw fluorescence versus thyme will be displayed on the computer screen. Repeat this procedure replacing the progesterone with HSM as a negative control and 10 micromolar ion mycin as a positive control prior to flow cytometry. Prepare the experimental samples by placing 500 microliters of cell suspension per cytometer tube under each condition to be tested.
Set up an experiment using the equipment software. First, create a new folder, experiment specimen, and number of tubes. Then select appropriate cytometer settings for flu O 3:00 AM.Use the fluorescein isothiocyanate and propidium iodide filters.
Run unstained control tubes one and two in the cytometer. Collect forward and side scatter data to verify that threshold settings are appropriate and to create the corresponding gate in order to discriminate debris from cells. Run the experimental tubes and collect fluorescence data from 10, 000 events per sample.
At the end. Export all data to the software available for analysis. Prepare round cover slips needed for this procedure by applying a five microliter drop of poly L lysine solution onto the center of each cover slip.
Leave for at least one hour prior to use rinse treated area with water. This will remove excess polylysine and allow sperm cells to adhere to the cover slip from their head while their flagella can still move. Assemble the cover slip inside the recording chamber.
Place 10 microliters of flu oh 3:00 AM loaded cells at a concentration of one times 10 to the seventh cells per milliliter. In the center of the cover slip. Cover the cells with 200 microliters of pre prewarm HSM.
Place the chamber on the stage of the microscope, preheated to 37 degrees Celsius, and view the cells using phase contrast. Select an area where cell density is appropriate for imaging. Too many cells make analysis difficult due to overlapping signals.
Cells should be firmly attached to the cover slip by their head, but exhibit flagella movement, which confirms viability. Start the experiment by activating the time series image acquisition software. Typically four images are required per second with an illumination of two milliseconds per image.
Acquire fluorescence images in live mode. To adjust the focus and brightness. Use a micro pipette to carefully add dropwise the test compound progesterone in this case and continue image acquisition as required.
Perform two sequential control additions into the same chamber. 20 micromolar ion mycin to obtain maximal fluorescence and five millimolar manganese chloride to obtain minimal fluorescence. Perform image analysis offline using IQ software.
Draw the regions of interest or ROIs around each cell or part of a cell. In addition, select a cell-free area for automatic background subtraction by the software. A time fluorescence intensity series is then obtained for each ROI and these data may be exported to Microsoft Excel for further analysis.
Progesterone is one of the known acrosome reaction inducers and as expected provokes, a transient intracellular calcium concentration increase in human sperm as measured by conventional fluorometry. The red fluorescence trace versus time shows intracellular calcium concentration changes caused by the addition of four micromolar progesterone as a negative control HSM was added, which did not cause any change in intracellular calcium concentration levels as indicated by the blue fluorescence trace as a positive control. The change induced by 10 micromolar ion mycin is shown for each trace.
Addition of this calcium ionophore causes the maximum RA cellular calcium concentration increase, which does not return to basal levels. This bar graph showed the average change in fluorescence delta F from each condition, plus or minus standard error where N equals three and the asterisk indicates a P value of less than 0.001 compared with the control. The progesterone induced intracellular calcium concentration increase was measured with greater temporal resolution by stopped flow fluorometry and representative results are shown here.
Raw traces are shown in panel A, both progesterone represented by the red line and ion mycin represented by the blue line caused a rapid intracellular calcium concentration increase. The green trace is the negative control where cells were mixed with HSM. Panel B shows corrected traces for the signals induced with progesterone or with ion mycin obtained by subtracting the control signal from their corresponding raw signals.
Progesterone caused a very fast and transient intracellular calcium concentration increase with a maximum fluorescence value occurring 2.7 seconds after addition of the inductor. On the other hand, CIN caused a rapid and sustained intracellular calcium concentration increase throughout 50 seconds. The inset in panel B shows an expanded view of the first 500 milliseconds for each response.
The absence of a delay in the progesterone induced intracellular calcium concentration increase is consistent with previous reports suggesting that progesterone directly activates the calcium channel cat spur without intermediate signaling. Intracellular calcium concentration was also measured in capacitated and non capacitated human sperm using flow cytometry. In these representative forward and side scatter plots.
Capacitated cells are in blue and non capacitated cells are in red. The selected gate is indicated with the blue line and only the cells within that area were used for further analysis. Panel B shows the fluorescence histogram in the Fitzy channel from Unstained, spermatozoa, and panel D shows the fluorescence histogram in the FZ channel from flu oh 3:00 AM stained spermatozoa as observed in Panel D.The distribution of fluorescence values for capacitated sperm represented by the blue trace is shifted to higher values compared to non capacitated sperm represented by the red trace.
Panel C shows the fluorescence histogram in the PI channel from unstained cells and panel E shows the fluorescence histogram in the PI channel from dead cells. The fluorescence values for each individual cell can be observed in the two dimensional fluorescence dot plots shown here. For unstained cells and cells double stained with flu oh 3:00 AM and pi, the percentage of cells recorded in each quadrant is indicated by the red number.
Importantly, the signal arising from dead cells can be eliminated. Lastly, the progesterone induced intracellular calcium concentration change was measured in single sperm cells by imaging these representative pseudocolor images show cells visualized at the beginning and after progesterone, CIN and manganese chloride additions. Progesterone addition causes an increment in intracellular calcium concentration, both in the sperm head and the lum manganese chloride is used to decrease the flu.
Oh 3:00 AM fluorescence by quenching representative normalized fluorescence traces of nine individual cells from the experiment are shown in this figure. As observed in population experiments, single cell analysis revealed a transient and a sustained increase for progesterone and ion mycin respectively. Once mastered, this technique can be done in three to four hours if it is performed properly.
While attempting this procedure, it is important to remember to verify the viability of the cells and to perform the appropriate controls Following this procedure. Other methods like electrophysiology could be performed in order to answer additional questions such as the identification of specific ion channels involved in the calcium increases by using a proppe solutions and simulation protocols After its development. This technique paved the way for researchers in the field of cell physiology to explore calcium dynamics in living cells with high spatial and temporal resolution.
After watching this video, you should have a good understanding of how to select the most appropriate technique to measure intracellular calcium increases using fluorescent dy in any type of cell, depending on the specific question that you would like to answer. Don't forget that working with human siemen samples can be hazardous and precautions such as the use of donors of proven health. The use of latex globes during the procedure and appropriate disposal of solution and materials should always be taken while performing this procedure.
