Protocol for preparing demembranated sperm nuclei

This protocol is based on Paula Deming, Sally Kornbluth, Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods Mol Biol 322, 379-393 (2006).

Buffers

Buffer X

10 mM HEPES pH7.4, 80 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM EDTA

10x Buffer X

Include 5 µg/ml aprotinin, 5 µg/ml leupeptin and 1 mM DTT in buffers #5, #6 and 5 ml of buffer #1.

Procedure

All procedures are carried out at room temperature, unless stated otherwise. The preparation works for 2-8 male Xenopus laevis. Before collecting testes make your sucrose solutions, because they take a long time to dissolve. The discussion below assumes that 8 male frogs are used.

1. Anesthetize a male frog by placing it in Tricaine solution (1 g Tricaine and 2 g sodium bicarbonate per 1 L of frog tank water), and sacrifice either by cervical dislocation or by pithing, followed by cutting through the spinal cord. Recover the testes (beige color, located along the midline of the abdominal cavity, 2 pieces per frog) and blot off the blood. Place in a small petri dish containing 2 ml buffer #1.

2. Remove excess buffer and mince the testes thoroughly into tiny pieces by chopping with razor blades.

3. Transfer the mushed up testes to a 15 ml screw cap conical centrifuge tube. Rinse the petri dish with a small volume of buffer #1 and combine with the testes. Vortex vigorously for 1 minute, and pellet the larger pieces by a mild centrifugation for 10 sec in a clinical centrifuge at 200 x g.

4. Transfer the supernatant to a new 15 ml conical tube. Add 2-3 ml of buffer #1 to the pellet, vortex 2 min, and recentrifuged for 15 sec at 200 x g. Combine the supernatants and repeat the extraction of the pellets 2-3 times until the supernatant is not very cloudy.

5. Centrifuge the combined supernatants for 50 sec at 450 x g to pellet larger pieces of tissue. Transfer the supernatant to a 14 ml round-bottom polypropylene tube (for example, Corning 352059). Avoid tissue carry-over.

   5a. Add 12 ml of buffer #1 to the pellet. Vortex vigorously for 1 min and centrifuge for 50 sec at 450 x g. Transfer the supernatant to a new 14 ml round-bottom tube. 

   5b. Centrifuge both round-bottom tubes and pellet the sperm by centrifugation in a swinging bucket rotor at 2600 x g, 4C, for 15 min.

6. Prepare sucrose step gradients in four 2.2 ml ultra-clear centrifuge tubes (Beckman #347356). In each tube, underlay 1.7 ml buffer #2 with 0.25 ml of buffer #3.

7. Resuspend the sperm pellet very well in 0.8 ml total buffer #4. Overlay the sucrose gradient with the sperm (0.2-0.4 ml per tube). Stir the interface well but carefully between the sperm and the 2.3 M sucrose with a flame-sealed Pasteur pipette tip to ~2.5x original sperm layer depth. Centrifuge with a swinging bucket rotor (Beckman TLS-55) in an ultracentrifuge at 93000 x g for 33 min, 2C.

8. The contaminating red blood cells should band on top of the 2.3 M sucrose layer. The sperm goes to the bottom. If there is still a cloudy layer above the red blood cells, remix it with the flamed Pasteur pipette and spin for another 20 minutes. When you are sure all the sperm went down, aspirate off the top half of the gradient containing the red blood cells. Remove the rest of the cushion with a P-1000 pipette and transfer to a 14 ml round-bottom tube. To resuspend the sperm, add 500 µl of buffer #1 and pipette up/down with P-1000 tip (avoid the upper walls of the tube to avoid contamination by red blood cells). Transfer the sperm to the 14 ml tube. Any gray spots on the bottom of the tube means you didn’t get all the sperm.

9. Dilute the sperm to 12 ml with buffer #1. Pellet the sperm by centrifugation at 4100 x g, 10 min, 4C, in a swinging bucket rotor.

10. Resuspend the sperm pellet in 1.7 ml of buffer #1 (plus 5 µg/ml aprotinin, leupeptin, and 1 mM DTT) and transfer to a 5 ml Falcon round-bottom polypropylene tube (Corning 352063). Resuspension should be done with 1 ml first using a pipette. From a 20% stock, add Triton X-100 to a final concentration of 0.4%. Incubate for 30 min, rotating on a wheel at 4C.

11. Prepare four 1.5 ml microtubes containing 0.5 ml buffer #5, + 3% BSA (w/v, plus 5 µg/ml of aprotinin, leupeptin, and 1 mM DTT). Overlay each sucrose cushion with 1/4 of the sperm preparation. Centrifuge in a clinical centrifuge for 10 min at room temperature at 870 x g.

12. Remove the supernatant and resuspend each pellet in 0.2 ml buffer #6, + 3% BSA (plus 5 µg/ml aprotinin, leupeptin, and 1 mM DTT). Avoid the sides of the tubes containing residual Triton X-100. Transfer the sperm chromatin to four new 1.5 ml microtubes, dilute to 0.7 ml with the same buffer mix, and centrifuge again at 870 x g for 5 min in a clinical centrifuge. Repeat once.

13. Resuspend the sperm chromatin pellet in 1.8 ml of buffer #6, + 3% BSA (plus 5 µg/ml aprotinin, leupeptin, and 1 mM DTT).

14. Determine the sperm concentration under a hemacytometer.

15. Dilute the sperm to 100000/µl and freeze in small aliquots in liquid nitrogen. Store at -80C.