This method facilitates the identification and dissection of stage-specific seminiferous tubal segments allowing the study of very specific steps in spermatogenesis. The stages of the seminiferous epithelial cycle can be recognized in fresh, non-fixed tubules, and, therefore, we can select only the stages of interest for downstream analyses. It requires some practice to learn to recognize the stages on the basis of their transillumination patterns, but this protocol should ensure your success.
Demonstrating the procedure will be Opeyemi Olotu, who is a Ph.D candidate in my laboratory. Begin by placing an at least 8-week-old adult male mouse in the supine position and disinfecting the ventral abdomen with 70%ethanol. Use sterile scissors to make a v-shaped incision in the abdominal pelvic region, and use sterile forceps to pull on the epididymal fat pad to locate the testes.
Use scissors to dissect the testes, placing the tissues in a sterile 100-millimeter Petri dish with PBS as they are harvested. Use fine-tipped scissors to cut a slit in the tunica albuginea, the thick fibrous sheet encapsulating the testes, and use forceps to tear the tunica open and to force the tubules out. Place the decapsulated seminiferous tubules into a new Petri dish, and add enough sterile PBS to cover the bottom of the dish.
Then, use the forceps to gently pull the tubules apart without damaging the tissues. For dissection of the tubules, tape the dish onto a dissecting microscope stage and turn on the transillumination. Use fine forceps to carefully move the bundles of tubules to get acquainted with the light absorption and scatter patterns of the different stages.
Then, use forceps with a hooked tip to lift a tubule of interest and use microdissection scissors to cut a segment of an appropriate length. After cutting the stage-specific tubule segments, use a pipette to collect a segment of interest in a 10-microliters volume of PBS and place the segment onto a microscope slide. Carefully press a 20 by 20 millimeter cover glass onto the tubule without squashing the cells too much and place the slide onto the stage of a phase-contrast microscope.
Place a filter paper onto the edge of the cover glass to facilitate the spreading of the cells into a monolayer and use the 40 times objective to verify the cell stages. Once the cells have formed a round monolayer from both ends of the tubule, dip the slide into a container of liquid nitrogen for 10 seconds. Use a scalpel to flip the cover glass off of the cells and immediately place the slide in 90%ethanol for 2 to 5 minutes.
Then, air dry and store the slide at room temperature for up to several days. For whole-mount immunostaining of the seminiferous tubules, use a pipette to transfer segments of interest into a 15-milliliter conical tube of ice-cold PBS on ice. After allowing the segments to sediment, carefully remove the PBS.
Add 10 milliliters of fresh ice-cold PBS and mix by inversion. Allow the tubules to settle for a few minutes before removing the supernatant again as demonstrated. When all of the supernatant has been removed, fix the tubules in 5 milliliters of paraformaldehyde for five hours at 20 to 30 revolutions per minute at 4 degrees Celsius.
At the end of the incubation, allow the tubules to settle for a few minutes before washing the sample three times in fresh PBS at 4 degrees Celsius for 10 minutes per wash with rotation. After the last wash, use a 1-milliliter pipette to transfer 10 to 20 fixed tubule segments into a 2-milliliter round bottom tube. When the segments have settled, replace the supernatant with 1 milliliter of 2%BSA and 10%FBS in PBSX for one hour incubation at room temperature and 20 to 30 revolutions per minute.
At the end of the blocking incubation, wash the samples with 1 milliliter of PBSX. Before labeling the tubules with 250 microliters of primary antibody, dilute it in 1%BSA in PBSX for two hours at room temperature at 20 to 30 revolutions per minute. At the end of the incubation, wash the tubules three times in PBSX as demonstrated before labeling the samples with 250 microliters of the appropriate secondary antibodies for one hour, protected from light at room temperature and 20 to 30 revolutions per minute.
At the end of the incubation, wash the tubules three times before decanting the tubules onto a microscope slide in a small volume of supernatant. Then, use gel-loading tips to arrange the tubules into linear strips and drain the excess buffer. Then, add 1 to 2 drops of mounting medium before mounting the slide with a coverslip.
Under transillumination, stages 7 through 8 appear homogeneously dark because they contain a high number of fully condensed elongating spermatids that are aligned at the apical surface of the epithelium. After mature spermatozoa are released into the lumen during spermiation, the tubule appears very pale at stages 9 through 10 due to the absence of condensed elongating spermatids within the epithelium. This pale zone is followed by the weak spot zone, the spotty appearance of which originates from the organization of elongating spermatids with condensed chromatin in bundles.
These bundles become very prominent in the subsequent strong spot zone. Spermatid bundles migrate towards Sertoli cell nuclei that are located close to the basal lamina, which is reflected as a striped appearance of stage 2 to 5 tubules when transilluminated. The bundles finally disperse at stage 6, and condensing elongating spermatids move close to the lumen to be released from the epithelium at stage 8.
The exact stage of the tubule segment can be accurately verified by phase-contrast microscopy of the squash preparations. Immunostaining of staged squash preparations can be used to study the expression and localization of proteins of interest in the seminiferous epithelium. Seminiferous tubule whole-mount staining is typically used to study the cell types that are in contact with the basement membrane of the seminiferous epithelium, either on the tubular or interstitial side.
The method can also be used to study cells or structures that are located deeper within the epithelium, such as the blood-testis barrier or post-meiotic germ cells. In addition to immunostaining, we can use stage tubal segments for many other downstream analyses, including biochemical RNA and protein assays, flow cytometry and ex vivo tubal culture. Because this method allows a very detailed analysis of spermatogenesis, it really provides a useful tool to study all important processes required for the sperm production and male fertility.
