The overall goal of this protocol is prepare meiotic chromosome spreads from mouse spermatocytes to allow the study of male meiosis. This method can help answer key questions in the reproductive biology field about the mechanisms that regulate meiotic re-combination and chromosome segregation. The main advantage of this technique is that it yields an abundance of spermatocyte nuclei at different stages of meiosis, especially those cells undergoing the sub-stages of prophase 1.
Visual demonstration of this method is critical because a technique used to spread the spermatocyte nuclei onto slides is difficult to convey in writing. Begin by weighing paired testes harvested from post-natal to adult aged male mice and placing the weighed specimens in a Petri dish containing a few drops of cold hypotonic extraction buffer. To harvest the tubules, use the tips of a curved forceps to hold one testis against the bottom of the dish and use a straight-edge razor blade to make a small, vertical mid-line incision through the capsule of the testis.
Use a second set of curved forceps or a straight-edge razor blade to gently squeeze the seminiferous tubules from the testis and transfer the seminiferous tubules into a 15 milliliter conical tube containing 10 milliliters of cold hypotonic extraction buffer, taking care to avoid collecting pieces of testis capsule with the tubules. When all of the tubules have been collected, placed the conical cubical on ice for 30 to 60 minutes, depending on the size of the testes. While the tubules are incubating, label the frosted ends of pre-cleaned slides with the mouse number, slide number, and date, and place the labeled slides in a Coplin jar containing fixative solution.
At the end of the incubation, pour the tubules into a new Petri dish and separate the tubules into approximately three millimeter diameter clumps. Next, place 23 microliters of the 100 minimal or sucrose solution into one ring of a Teflon printed three ring slide and add one clump of tubules to the ring. Using curved tipped forceps to hold the clump, use a size 11 razor blade to gently mince the tubules until the solution becomes cloudy.
When the solution becomes cloudy, remove the debris and another 23 microliters of sucrose solution to the ring. Use a micro-pipette to gently pipette the mixture a few times to help separate the cells in the suspension. Then remove a slide from the fixative solution and tilt the slide over the jar so that one generous droplet collects at the bottom corner.
Take care that the droplet is large enough that coupled with additional cell suspension the slide will be very wet and be completely covered when the mixture is spread. Add 20 microliters of cells to the droplet and mix the suspension two to three times by pipetting. Next spread the cell suspension along the length of the slide and slowly tilt the slide to the side opposite the droplet.
Then slowly tilt the slide back to spread the cell suspension along the width of the slide to cover it completely. To avoid overlapping of the cells take care not to spread the cell suspension over the same area twice. After spreading, immediately place the slide in a humidified chamber and spread the next slide.
When the desired number of slides have been prepared cover the chamber and allow the slides to dry at room temperature for two and a half hours. At the end of the covered incubation, remove the cover to allow the slides to air dry completely for an additional 30 minutes. When the slides are completely dry, place them in a Coplin jar containing 0.4%Photo-Flo 200 in PBS for two two five minute incubations with gentle agitation followed by one five minute wash in 0.4%Photo-Flo 200 in water with agitation.
After the last wash carefully wipe the edges and bottom of each slide to remove the excess liquid and dry the slides for 15 to 20 minutes in an angled, almost upright position. Then store the slides at minus 80 degrees Celsius in a tightly closed slide box protected from light for up to three weeks before immunostaining. In this image well-spread pachitean spermotocyte nuclei can be observed.
These nuclei contain unevenly immunostained homologues that appear tattered due to too along in incubation of the seminiferous tubules in the hypotonic extraction buffer. These nuclei were poorly spread resulting in overlapping cells, while these nuclei contained fragmented homologues, resulting from over-mincing of the seminiferous tubules. If this technique is performed correctly however, a variety of nuclei representing the main five sub-stages of prophase 1 can be observed.
Once mastered this protocol including the solution preparation including the solution preparation, drying, and washing steps can be completed in approximately five and a half hours, if it is performed properly. While attempting this procedure it's important to incubate the seminiferous tubules in the hypotonic buffer for the appropriate amount of time, and to tilt the slides at one direction at a time when spreading the cell suspension. Following this procedure, other methods including immunostaining and microscopy are often performed to evaluate fundamental meiotic processes, such as DNA double-stranded break repair and crossover formation.
After watching this video you should have a good understanding of how to prepare male meiotic chromosome spreads. A straightforward procedure that requires some practice to gain competence and optimal results. Thanks for watching this video and good luck with this protocol.
