This method can help answer key questions in the blood-testis barrier of biology fields about gene candidates, viruses, or environmental toxins that may affect blood-testis barrier function or integrity. Three days before the surgery, treat at least three eight-week-old C57Black/6 male mice daily with a five milligrams per kilogram body weight cadmium chloride via intraperitoneal injection. On the day of the surgery, place a clean tissue over a 75%ethanol-sterilized surgical area and set a thermostatic heater to 37 degrees Celsius.
Place an anesthetized mouse onto the tissue, then confirm a lack of response to toe pinch. Shave the abdominal hair with a shaver, and disinfect the exposed skin with 75%ethanol. Make a one-centimeter skin incision above the preputial glands to expose the abdominal wall, and use small forceps to lift the skin to make a 0.5-centimeter incision in the peritoneum.
Use the forceps to search for fat pads around the epididymis and testis, carefully pulling the fat pads out to expose one attached testis. Place a nine-centimeter piece of paper under the fat pads and testis, and load a microinjection pipette with freshly prepared inulin-FITC solution. Connect the pipette to a micromanipulator unit, and use a dissecting microscope to gently insert the microinjection pipette tip under the tunica albuginea.
Slowly rotate the operating lever to deliver 20 microliters of the inulin-FITC into the interstitium of the testis. If the delivery is successful, the testis will turn a bright green-yellow color. Immediately return the testis to the abdominal cavity, and inject the contralateral testis with 20 microliters of PBS, then close the skin with a surgical suture.
And place the mouse onto the heat pad of a thermostatic heater. 40 minutes after the injection, use sharp scissors to collect the testes from each mouse, and wash the tissues in one milliliter of ice-cold PBS. Next, fix the testes in 4%paraformaldehyde at four degrees Celsius for 12 to 24 hours followed by three washes in 1.5 milliliters of 1%PBS per wash.
After 30%sucrose dehydration overnight, transfer the samples into individual embedding frames, and cover the tissues with optimal cutting temperature compound. Set the temperature of a cryostat to negative 20 degrees Celsius and place the frames in the cryostat for about 10 minutes until the OCT is frozen. Then, fill each embedding frame with fresh OCT until the whole testis is covered in each mold.
When the second layer of OCT has frozen, acquire a five-micrometer-thick cross sections of each testis, capturing the tissue sections onto glass microscope slides as they are obtained. To image the samples, first warm the slides in a humidified box at room temperature. After 10 minutes, wash the slides three times in fresh Tris-buffered saline per wash and allow the sections to dry for five minutes protected from light.
Use dust-free paper to remove any residual saline, and mount the samples with DAPI-supplemented mounting medium. Then, cover each sample with an inverted cover slip, and image the samples under the 20X objective of a confocal microscope. Three days of cadmium chloride treatment causes damage to the blood-testis barrier allowing inulin-FITC diffusion from the basal compartment to the apical compartment of the seminiferous epithelium.
In contrast, this diffusion is blocked in control PBS-treated testes. The extent of the blood-testis barrier damage is determined by calculating the ratio of the distance of inulin-FITC diffusion to the radius of the same seminiferous tubule. Further, the intact blood-testis barrier in heterozygous Rictor floxed knockout mice blocks inulin diffusion across the barrier into the apical compartment, while conditional Rictor knockout mice possess a compromised blood-testis barrier that permits inulin diffusion.
While attempting this procedure, it's important to remember to deliver inulin-FITC prepared on the day of the experiment and to keep the tissue protected from light since FITC is a light-sensitive molecule. The implications of this technique extend to the therapy of impaired spermatogenesis because the function of the blood-testis barrier is to provide an immune-privileged microenvironment for the completion of meiosis in spermatogenesis. After its development, this technique provide a way for researchers in the field of reproductive medicine to explore subfertility and infertility in mice.
Don't forget that working with paraformaldehydes can be extremely hazardous and that precautions such as wearing garments and face masks should always be taken when performing this procedure.
