The overall goal of this experiment is to evaluate the intracellular location of reactive-oxygen species within Solea senegalensis spermatozoa by confocal microscopy. Reactive-oxygen species have in uses of safety to the effective sperm function, causing cellular damage at different levels. Spermatozoa could be particularly sensitive to oxidative stress, since lipid peroxidation can produce a negative effect on motility and fertilization.
Reactive-oxygen species can even trigger an intrinsic opoptotic escate. Peroxide is released from the mitochondria, and it's able to penetrate the nucleus, affecting the DNA, because it's a small antartless molecule. In this protocol we use a fluorophore that after being incorporated into the cell can be modified by oxidative reaction, emitting green fluorescence.
Therefore, you can know exactly the localization of reactive-oxygen species. In this video we work with Solea senegalensis spermatozoa, but you can use this protocol with related species with commercial interest, or with mothers like Sibrifis or Mirica. Some examples of the use of this technique in physically-reactive biology, or apiculture fields are.
The evaluation of cryprecipitation following protocols. The comparison of reactive-oxygen species lables before and after this sperm activation. Or you can correlate reactive-oxygen species labels with good and bad breathers.
First of all, prepare the stocks of your fluorochromes. Take into account that you must perform fluorchrome manipulation according to safety-data-sheet recommendations. Special care should be taken while weighing and preparing the stocks.
Keep the stock solutions in aliquots at minus 20 degrees Centigrade until needed. In this protocol we use three fluorochromes. The far red fluorescent dye stains mitochondria in live cells, and can be used for mitochondrial localization.
DAPI, which is a widely-used probe in fluorescent microscopy for DNA staining. It binds strongly to adenine-thiamine-rich regions in DNA. And, 27-dichlorofluorescein diacetate in a cell-permeable non-fluorescent probe.
It is de-esterified intracellularly, and turns to highly fluorescent 27-dichlorofluorescein upon oxidation. This protocol can be started with fresh or cryopreserved spermatozoa. In this case, we used a cryopreserved sample.
Prepare a bath for thawing at 40 degrees Centigrade. Clean the material. With the adequate corporal protection, select your cryopreserved straw.
Immerse the straw in the bath for seven seconds. Using scissors, empty the thawed sample into a microcentrifuge tube. Always work at four degrees Centigrade.
Centrifuge the sample for a minute. Carefully remove the cryoprotectants without affecting the pelleted sowels. Resuspend in a small volume of 50 or 100 microliters of RINGA solution.
Load into a new bower or similar counting chamber a dilution of your cell suspension, and count the cells. Calculate this concentration of the sample. Dilute the cell suspension up to one to two million cells per millimeter in a final volume of 0.5 milliliters in pre-cooled RINGA solution.
The spermatozoa must be gently handled when pipetting and resuspending. Add 3.125 microliters of dichlorofluorescein diacetate stock solution to the working sample solution. The final concentration in the sample is 25 micromolar.
Incubate at four to seven degrees Centigrade for 40 minutes. After 30 minutes, add to the sample 0.5 microliters of the DAPI stock solution and 0.5 microliters of the mitochondria stained stock solution. Both fluorochromes have an incubation time of 10 minutes.
Place a five-microliter concentrated cell-suspension drop on a slide. Carefully put a cover slide on the preparation, and seal it. Switch on the equipment, and select 100 magnification objective in the control panel.
Locate the sample in the microscope, and turn on the lamp. The system software enables the fluorochromes that you need to be selected directly from a list. Choose the set.
DAPI, with animation maximum of 465 nanometers, MitoTracker Deep Red with animation maximum of 662 nanometers, and DCF with an emission maximum of 525 nanometers. Redirect the beam to the oculars. Focus a field of interest.
Back to the software, select the frame size. Establish the bidirectional scanning mode for a better resolution. Choose the desired scan speed.
Open and center the scan area to begin. Take a snap of the field. Using the crop tool, select the region of interest, and press Live.
Then you will have selected your area of work. With the help of the range-indicator tool, adjust the digital offset to reduce the background. Do this for each channel, and take your snap.
This is an example of the results. For a merge image, individual channels for each fluorochrome, and their combinations. This is a label scheme of a Solea senegalensis sperm cell.
After seeing the acquired images, you can observe that DAPI and MitoTracker are, as expected, located within the nucleus and mitochondrial ring, respectively. The first step is to perform a zed-stack experiment. Go to the zed-stack window, and with the help of the fine focus, select the first position and the last position of acquisition.
Choose the optimum acquisition parameters, and run the experiment. You will get images for each slide. This is an example of a zed-stack result.
You can split the channels, and observe the location of each signal within the cells. Moving to the 3D window, you can select different graph options. You can customize many parameters, and split and merge channels.
This is an example of 3D views showing different patterns of reactive-oxygen species location within Solea senegalensis sperm. In the upper field, DCF is colocalized mainly with mitochondria;whereas, in the field below, the signal is extended to the nucleus. After watching this video, you know how to evaluate peroxide intracellular localization in these spermatozoa using confocal microscopy.
You can find troubleshooting advices and other information in the text.
