Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

The maintenance of laboratory mice and all experiments were performed in accordance with the relevant guidelines and regulations for the care and use of laboratory animals.

1. Equipment and Reagent Set-up

1. Set the water bath to 37 °C.
2. Set the cell culture incubator to 34 °C, 5% CO₂, 95% humidity. Put the tube rotator inside the incubator.
   NOTE: Incubators require a long time to change internal temperature. If an incubator constantly set at 34 °C is not available, set one up 1 day before the experiment.
3. Prepare and label the appropriate amount of microscopy glass slides. Draw a ring of ~1 cm in diameter with a grease pen and let the grease dry.
4. Prepare 1x Krebs buffer, pH 7.8 (Table 1). Put two conical tubes with 50 mL of 1x Krebs to 34 °C to pre-warm for steps 2.5−2.8. Store the rest of the 1x Krebs at 4 °C or on ice.
5. Prepare BSA solutions. First prepare 25 mL of 10% (w/v) BSA solution in Krebs by dissolving 2.5 g of BSA in 1x Krebs buffer to a final volume of 25 mL. Dilute the 10% BSA solution with 1x Krebs buffer to obtain different BSA concentrations (Table 2). Keep all BSA solutions at 4 °C.
   NOTE: Prepare solutions the same day of the procedure and store at 4 °C until use.
6. Prepare the digestion enzymes by weighing the correct amount of trypsin and collagenase to 50 mL conical tubes (Table 3).

2. Animal Dissection and Preparation of Germ Cell Suspension

NOTE: This takes approximately 1 h to complete.

1. Sacrifice an adult male mouse (aged 7 weeks or older, testis weighing 80−120 mg depending on the strain and age) via cervical dislocation or CO₂ asphyxiation.
2. Spray the ventral abdomen of the mouse with 70% ethanol. Open the abdominopelvic cavity using scissors, making a V-shaped opening.
3. Pulling on the epididymal fat pad with forceps, locate the testes and remove them with scissors. Avoid disturbing the tunica albuginea. Place the testes on a 6 cm Petri dish containing 1x Krebs.
4. Decapsulate the testes and discard the tunica albuginea. Slightly disperse the seminiferous tubules by gently teasing them apart with forceps.
5. Use forceps to transfer the seminiferous tubules into a 50 mL conical tube containing 2 mL of freshly prepared collagenase solution (Table 3).
6. Incubate the tubules in the 37 °C water bath for 3 min. Agitate gently by rocking the tube.
   NOTE: Freely floating tubules should occur within 3 min due to the removal of interstitial cells. The physiological temperature for testicular cells is 34 °C; therefore, long digestions are usually done at this temperature. However, the short 3 min digestion can be conducted at 37 °C (recommended temperature by the manufacturer). Note that if 34 °C is used, the digestion time should be re-optimized.
7. Add at least 40 mL of warm 1x Krebs and allow the tubules to sediment (~1 min) at room temperature (RT). Remove the supernatant and repeat 1x.
8. Add 25 mL of freshly prepared trypsin solution (Table 3), place the tube on the tube rotator inside the 34 °C incubator, and incubate for 15−20 min (~15 rpm). Sporadically check the status of the tubules. Once the solution becomes cloudy and only small pieces of tubules remain, place the tube on ice and immediately proceed to the next step.
   NOTE: To avoid overdigestion and cell lysis, move swiftly to the following washing steps. Some protocols include the deactivation of trypsin by fetal bovine serum (FBS). In this protocol, FBS treatment is omitted, and instead, trypsin is removed by immediate centrifugation and subsequent washes with cold 1x Krebs.
9. Filter the solution through a 40 µm cell strainer into a new 50 mL conical tube on ice.
10. Centrifuge 600 x g for 5 min at 4 °C to pellet the cells.
    NOTE: Centrifugation with too strong of a force may harm the cells.
11. Remove supernatant by carefully pouring it out.
12. Tap the cell pellet to resuspend the cells in the remainder of 1x Krebs.
13. Add at least 40 mL of cold 1x Krebs to the resuspended cells.
14. Repeat steps 2.10 and 2.11.
15. Tap the tube with the cell pellet to resuspend the cells. Add 1 mL of 0.5% BSA in 1x Krebs and with a cut pipette tip resuspend the cells by pipetting the solution up and down. Avoid making bubbles.
16. Finally, add 1−3 mL of 0.5% BSA in 1x Krebs so that the final volume is ~3 mL. Filtrate the germ cell suspension through a 40 µm cell strainer and proceed immediately to loading the cells on the BSA gradient.

3. Separation of Germ Cells Through the Discontinuous BSA Gradient

NOTE: This section takes approximately 2 h to complete. Start preparing the discontinuous BSA gradient during the wash steps (steps 2.10−2.14) to load the cells as soon as the pretreatment is ready.

1. Accommodate a 50 mL tube vertically on ice so that it is possible to see one side of the tube. Alternatively, run the protocol in a cold room at 4 °C, in which case no ice is needed.
2. Cut the tip of a 10 mL serological pipette about 5−10 mm from the tip to obtain a larger aperture (Figure 1A), and mount it on the pipette controller (Figure 1C).
   NOTE: This will decrease the velocity during pipetting, which facilitates stacking of the different BSA solutions. Alternatively, use a 1 mL mechanical pipette with a smooth piston and cut the pipette tips with an aperture of about 3 mm in diameter (Figure 1B).
3. Start by pipetting 5 mL of 5% BSA solution to the bottom of the 50 mL tube.
4. Slowly pipet 5 mL of 4% BSA solution on top of the 5% solution. Start by gently touching the surface of the 5% solution with the cut tip of the pipette and layer the two solutions while carefully maintaining contact with the surface, ensuring to not allow the pipet tip to become immersed.
   NOTE: A clear line should be visible between the two layers at the end of this step.
5. Repeat step 3.4 with the other BSA solutions obtain a discontinuous gradient from 5% up to 1% (Figure 1D).
6. Carefully load the single-cell suspension on top of the gradient without disturbing. Let the cells sediment through the gradient for 1.5 h at 4 °C or on ice.

4. Collection of Enriched Germ Cell Fractions

NOTE: This section takes approximately 30 min to complete.

1. Mount a 1 mL pipette tip onto a 1 mL mechanical pipette and cut the tip so that the aperture is ~3 mm in diameter (Figure 1B).
2. Carefully collect ~1 mL fractions in separate 1.5 mL tubes, starting from the top of the BSA gradient, and store them on ice. Number the tubes in the same order as the fractions will be collected.
   NOTE: At this point, there should be a series of ~28 tubes containing cell suspensions. Use a 1 mL mechanical pipette with a smooth piston and cut the pipette tips to minimize the risk of disturbing the BSA gradient.
3. Centrifuge the germ cell fractions at 600 x g for 10 min at 4 °C.
4. Careful not to disturb the pellet, discard most of the supernatant and resuspend the cell pellet by flicking the tube. Add 1 mL of ice-cold 1x Krebs buffer to each tube and repeat centrifugation.
   NOTE: Repeat the washing step if BSA interferes with downstream analysis.
5. Discard most of the supernatant but leave ~100 µL after the final wash. Resuspend the cell pellet carefully.
   NOTE: Resuspending the cells carefully ensures that the sample taken from this solution represents all cells of the given fraction. Keep the cell suspensions on ice while preparing the slides.

5. Analysis of Cell Fractions

NOTE: Analysis takes 2 h to complete.

1. One at a time, pipet 20 µL of 4% paraformaldehyde (PFA) inside each grease pen ring on a numbered microscopy slide.
2. Immediately add 2 µL of the resuspended cell suspension from the corresponding fraction. Repeat for each fraction.
   NOTE: While preparing slides, keep all cell suspensions on ice.
3. Dry the slides at RT for 1 h to overnight (O/N).
   NOTE: During this step, after taking a sample from each fraction, the cells can be processed for downstream analyses or storage (section 6).
4. Rinse the slides once with 1x PBS and mount with mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Table of Materials).
5. Analyze the slides under a fluorescence microscope to estimate which particular germ cell type is enriched in each fraction.
   NOTE: Different cell types can be recognized by their characteristic nuclear morphology visualized by DAPI staining. Figure 2A shows representative fractions enriched in elongating spermatids, round spermatids and pachytene spermatocytes. The round spermatid nucleus is small (6−7 µm in diameter) and round (Figure 2A). Mouse round spermatid nuclei are also characterized by a single, round heterochromatin structure called the chromocenter. Nuclei of elongating spermatids have a typical elongated shape and reduced nuclear size due to chromatin condensation (Figure 2A). Pachytene spermatocyte nuclei are much larger (diameter more than 10 µm) and more irregular in shape, with areas of densely packed chromatin distributed throughout (Figure 2A).
6. If necessary, perform immunostaining in addition to the DAPI staining to support the recognition of the different cell types.
   1. In this case, spread 2 µL of cell suspension with 20 µL of a fixing solution (4% PFA and 0.1% nonionic surfactant in PBS) inside a grease pen ring. After drying the slides, postfix the cells with 4% PFA for 10 min at RT.
   2. Rinse with PBS, then permeabilize with 0.1% nonionic surfactant in PBS for 5 min. Rinse with PBS and deactivate any remaining PFA by incubating slides in 100 mM NH₄Cl for 5 min.
   3. Rinse with PBS and proceed to a standard immunofluorescence protocol consisting of blocking and incubations with specific primary antibodies and fluorochrome-labeled secondary antibodies. Mount the slides with antifade mountant with DAPI (Table of Materials) and allow them to set for 24 h.
      NOTE: Examples of useful markers for primary antibody incubations include peanut agglutinin (PNA; 1:2,000 in blocking solution) that stains the acrosome in round and elongating spermatids, anti-DDX4 antibody (1:200 in blocking solution) that labels a single cytoplasmic granule in round spermatids, and anti-γH2AX antibody (1:100 in blocking solution) that recognizes sex bodies in pachytene spermatocytes. See the representative results and Figure 2C,D for examples of immunofluorescence analysis of the fractions.

6. Processing the Remaining Samples for Storage

NOTE: Section 6 takes approximately 20 min to complete.

1. Once a sample from each fraction has been taken for a microscopy slide, add 1 mL of ice-cold 1x Krebs to each fraction and centrifuge the cells down at 600−13,000 x g for 10 min at 4 °C.
   NOTE: A low-speed centrifugation results in a loose pellet, which makes it difficult to completely remove 1x Krebs, while a high-speed centrifugation may harm the cells. Choose the centrifugation speed according to specific requirements of the downstream protocol.
2. Remove and discard the supernatant and continue with preferred downstream analysis.
   NOTE: At this point, the cells can be snap-frozen in liquid nitrogen and stored at -70 °C. Once familiar with this technique, it is possible to continue with the downstream protocol of preference directly, but it is always advisable to take a sample and ensure the purity of each fraction. Total RNA can be extracted, quantified, and analysed by methods such as reverse transcription polymerase chain reaction (PCR) and RNA sequencing. Total protein extracts can also be prepared from which immunoprecipitation or western blotting analysis can be performed (Figure 3).