This method provides a tool for scientists to study environmental genetic factors that affect sperm migration and behavior in their native environments inside the female reproductive tract. The main advantage of this technique stems from the transparent epidermis of the C.elegans, which allows experimenters to easily visualize and record sperm movement in live animals. To begin, use a worm pick made from a Pasteur pipette and platinum wire flattened on one end to pick 20 to 30 L4 stage hermaphrodites, and gently place them onto a six-centimeter seeded NGM plate.
Incubate the hermaphrodites at 20 degrees Celsius for 28 to 30 hours. Next, make a male staining plate. Use the end of a glass stirring rod to scrape E.coli from the bacteria lawn of a seeded plate, and deposit it onto the center of an unseeded NGM plate, making a five-to seven-millimeter-diameter food dot.
Mix together two microliters of one-millimolar mito-dye in DMSO and 10 microliters of M9 buffer in a microcentrifuge tube. Pipette all of the mito-dye solution onto the food dot on the male staining plate. Place the plate in the dark to dry for 30 minutes.
Under a microscope, on a plate containing one-to three-day-old adult C.elegans, identify the males by their fan-shaped tail. Pick approximately 100 males to the mito-dye-stained food dot on the male staining plate. Wrap the plate in aluminum foil, and incubate overnight at 16 degrees Celsius.
On the second day, transfer the stained males onto a new seeded NGM plate to clean off excess mito-dye-stained bacteria. Keep the plate in the dark until mating. To make a mating plate, on an unseeded NGM plate, drip two microliters of thick E.coli mixture.
Wait approximately 10 to 15 minutes for the thick bacteria to dry to form a mating dot. While waiting for the mating plate to dry, mix together 300 microliters of 1%weight by volume tricaine, 300 microliters of 0.1%weight by volume tetramisole, and 900 microliters of M9.Transfer 600 microliters of the tetramisole/tricaine solution to a watch glass. Transfer 12 to 15 hermaphrodites picked on day one into the tetramisole/tricaine solution on the watch glass.
Place the bottom half of a Petri dish over the watch glass to keep it covered from evaporation, and incubate for 30 minutes to immobilize the hermaphrodites. Next, retrieve the seeded NGM plate that contains the stained males from the dark, and pick 50 to 60 stained males onto the dried mating dot on the mating plate. Store the plate in the dark.
After the hermaphrodites are immobilized, use a glass Pasteur pipette to pick up the immobilized hermaphrodites from the watch glass. Avoid sucking the hermaphrodites beyond the narrow part of the pipette. Transfer the hermaphrodites onto an unseeded NGM plate.
Remove as much liquid as possible, and let the excess liquid dry. As soon as all visible liquid has evaporated, transfer the anesthetized hermaphrodites onto the mating dot with the stained males. Incubate in the dark for 30 minutes to allow mating.
If sperm distribution assessment is desired, transfer the mated hermaphrodites to a seeded NGM plate and allow them to rest for one hour before mounting for visualization. To mount worms for visualization, first drip 10 to 15 microliters of the tetramisole/tricaine solution onto the prepared 2%agarose pad, and transfer the mated hermaphrodites into the solution on the pad. Place a coverslip over the worms.
Mount the slide onto an upright microscope equipped with epifluorescence, and position the worm so that both the vulva and one spermatheca are in view. Focus the image by focusing on the center of the spermatheca. Check the exposure for both DIC and TRITC channels.
In DIC, internal worm structures are clearly visible. In TRITC, individual sperm are visible as distinct puncta. Acquire DIC and fluorescence images for each uterus.
To capture time-lapse videos, first locate the hermaphrodites that contain labeled sperm within the uterus. On the Acquisition panel, adjust the image acquire intervals to 15 to 30 seconds and the duration to 10 to 20 minutes per uterus. Then, click Run to acquire time-lapse images in DIC and TRITC channels.
Starting with the vulva on one end and the spermatheca on the other, divide the uterus into three zones, with zone containing the vulva and zone three containing the spermatheca. Manually count the number of sperm within each third of the uterus, and report the number in each zone as a percent of the total sperm in the entire uterus. If necessary, use the lookup table to adjust the signal intensity of the TRITC channel images so that every sperm can be visible and quantified.
To track sperm in time-lapse images, use Fiji software for analysis. Use the Bio-Formats Import function to import the images from one time-lapse series as one hyperstack. Then, click Plugins, Tracking, and then Manual Tracking.
In the opened dialog box, select the TrackMate tool in the Fiji toolbar. Double-click on the sperm that will be tracked. Click inside the appeared green circle with dashed lines, and drag to the desired position.
Click on the circle again. The dashed green line turns into a solid green line. Simultaneously hit the Shift and L keys to turn on tracking mode.
Move to the next frame in the time-lapse series. To set the new location of the tracked sperm in the new frame, hover the mouse over the new point and press the A key. The tracker appears at the new location, and a line appears, connecting the location where the tracker has been placed in the previous frame.
Repeat the same procedure for the rest of the frames. After the traces have been completed, click Analyze in the TrackMate dialog box to generate the data needed. To calculate the speed, divide the total path length of the sperm by the elapsed time.
To calculate the vectorial velocity, draw a line through the uterus starting from the vulva, pointing toward the spermatheca. Use the Line tool to measure the distance the sperm has migrated along this line from the beginning to the end of the trace. Divide this distance by the elapsed time.
Negative values indicate the sperm has migrated away from the spermatheca. To record reversal frequency, count the number of times during which the sperm trace has generated an angle less than 90 degrees during three consecutive time-lapse frames. In this protocol, fog-2 q71 male worms were stained with mito-dye and mated to wild-type N2 hermaphrodites.
The adult hermaphrodite reproductive tract has two arms that are mirror images of each other. Upon mating, labeled sperm are deposited in the hermaphrodite uterus through the vulva. The sperm move around the developing embryos within the uterus, toward the spermatheca, where they are stored until fertilization.
The hermaphrodite uterus was divided into three zones for quantifying sperm distribution and migration. To assess sperm velocity and reversal frequency, only sperm in zone two were tracked through time-lapse images. Sperm in zone one, starting from the vulva, and zone three, including the spermatheca, tend to move in a circular pattern.
Sperm marked by the red and blue dots had sperm velocity and reversal frequency quantified. When quantifying sperm distribution in the uterus, proper sperm guidance using wild-type N2 hermaphrodites and fog-2 q71 males resulted in approximately 90%of the labeled sperm reaching the spermatheca. Data should not be counted if mating results in too few sperm in the uterus or too many sperm in the uterus, filling each crevice.
It is important that the animals are handled gently during each transfer step, that the hermaphrodites are immobilized completely, and that the plates and worms containing the myo-dye are shielded from light. This method may be combined with gene knockdown or knockout experiments, environmental or chemical exposure experiments, or other manipulations to identify factors that may regulate sperm migration. This technique has enabled us to identify specific prostaglandins that are important for guiding the sperm to the oocyte.
These prostaglandins are synthesized via an unconventional mechanism that may be conserved in higher mammals. Tetramisole, tricaine, DMSO are skin and eye irritants and may have toxic effects at high doses. It is important to wear the appropriate protective gear when handling any chemicals.
