The overall goal of this procedure is to visualize sperm morphology. This is accomplished by first isolating males for dissection one day in advance. The second step of the procedure is to dissect the males to release sperm.
The third step of the procedure is to visualize sperm morphology. The final step of the procedure is to prepare sperm for downstream applications. Ultimately, results can be obtained that show sperm morphology and the ability to activate through treatment with in vitro activators.
The sperm isolation and activation protocol was first developed over 30 years ago by Sam Ward, Diane Shakes, and Gregory Nelson. We currently use this procedure to characterize mutations that alter sperm function and fertility in C elegance. The overall goal of this procedure is to visualize CL elgan a OID sperm morphology.
This is accomplished by isolating males one day prior to dissection in order to keep them celibate. The second step involves dissecting the males in order to release round non modal spermatids. The third step is to visualize spermatid activation in vitro to the mature AME sperm morphology.
The procedure will be demonstrated by three postdoctoral fellows in the lab, Dr.Matt Marchello, Dr and Chatterjee, and Dr.Guna SLU To isolate sperm. First, obtain a large number of males by crossing five wild type males and one hermaphrodite on a small lawn of e coli. OP 50 bacteria seated at the center of an nematode growth medium or NGM plate.
Roughly 50%of the next generation will be wild type males to collect. L four stage males examine developmental stage and tail morphology under a dissecting microscope. An L four stage worm is picked based on tail morphology.
A male worm has a rounded tail while a hermaphrodite has a pointed tail. Using a platinum wire, pick seven to 10 L four stage males per experiment and transfer them to an NGM plate seeded with OP 50 and let them grow for a day or two. Growing celibate males in the absence of hermaphrodites prevents the loss of sperm, and thus a large number of sperms will be available during the experimental procedure.
Prior to beginning the dissection, prepare one liter of M nine buffer using six grams of DI sodium hydrogen phosphate, three grams of mono potassium phosphate, five grams of sodium chloride 0.25 grams of magnesium sulfate, hep to hydrate per liter, and five milliliters of sperm medium For sperm activation, M nine buffer and sperm media are prepared from autoclave or filter sterilized stock solutions. On the day of dissection, transfer the celibate males onto an NGM plate without bacteria and let them crawl for a few minutes. This step helps to remove the small layer of e coli adhered on the surface of the worms.
Alternatively, pipette a small volume of M nine buffer in a watch glass and transfer males into the buffer. Next, using a pat pen, Mark A.Small circle on a glass slide pipe at 30 to 50 microliters of one x sperm medium within the circle. The hydrophobic circle of the pat pen helps to retain the sperm medium within the boundary.
Transfer five to 10 males onto the sperm medium am using a dissecting scope. Hold one or two syringes depending on comfort level. Attached with 27 gauge needles on both of your hands, use one needle to hold the worm and use the other needle to cut the posterior end of the males to release sids.
The released spermatids are visible as minute granules under the stereo microscope. However, sometimes part or most of the spermatids may be retained within the carcass. In that case, gently dragging the carcass over the glass slide may facilitate the release of spermatids from the carcass.
To visualize activation of the spermatids, dissect the worms in one x sperm medium containing P pronase E.Under a dissecting microscope, let the worm sit for five minutes. Activated sperm will have pseudopods. Gently place a cover slip on the surface of the dissected sample.
Find the field of spermatids under 100 x magnification. Spermatids have a round shape. An example of a DIC image of spermatids isolated from male C elegance is shown in figure one.
Spermatids are spherical in shape and the nuclei are prominent. In vitro activated sperm are shown in figure two, spermatozoa have a prominent pseudopod. 80 to 95%of spermatids are converted to spermatozoa If needed.
The final step of the procedure can be to prepare sperm for downstream applications. For example, sperm can be fixed for immunofluorescence. Ultimately, the results obtained can show the ability to activate sperm in vitro and assay sperm morphology.
