Demembranated Sperm Chromatin

Demembranated sperm chromatin is the conventional template for DNA replication in Xenopus egg extracts and is a necessary reagent in the production of nucleo-plasmic extract described in Subheading 3.5. The preparation of demembranated sperm chromatin is described in Subheadings 3.1.1. to 3.1.4. This includes: (1) euthanization of Xenopus male frogs, (2) surgical extraction of the testes, (3) extraction and purification of sperm from the testes, and (4) determination of sperm concentration. This procedure is designed to yield approx 109 sperm and requires four to six male frogs. All steps in Subheadings 3.1.1. to 3.1.4. are performed at room temperature unless otherwise noted.

3.1.1. Euthanization of Xenopus Males

Anesthetize one frog at a time by placing into 1 L 0.05% benzocaine (5 mL 10% benzo-caine in 1 L water). After about 5 to 7 min, the frog should exhibit no movement and should not be able to right itself when turned over. The frog also should not exhibit a sucking reflex on insertion of a finger into its mouth. Remove the frog from the benzocaine solution and place on several paper towels. Euthanize by pithing.

3.1.2. Extraction of Testes

Open the peritoneal cavity by creating a midline incision through the abdominal wall. Move the organs of the intestinal tract to one side to reveal the testes. They are located in the midbody on either side of the midline. The testes appear grayish-white and almond shaped and are about 5 to 8 mm long. Remove them by cutting them with dissection scissors along the base. Blot excess blood away from the testes and place them in a Petri dish containing about 2 mL of 200 mM sucrose in buffer X. Repeat steps in Subheadings 3.1.1. and 3.1.2. for the remaining frogs.

3.1.3. Sperm Extraction and Purification

1. Mince the testes from all frogs in the Petri dish with a fresh single-edge razor blade. Use a repeated chopping motion until the tissue forms a viscous sludge.

2. Transfer the minced testes to a 15-mL screw-cap conical tube using a wide-bore disposable transfer pipet. Rinse the Petri dish with 1 to 2 mL 0.2 M sucrose in buffer X and combine with the testes in the 15-mL tube. Vortex vigorously for 1 min and pellet the larger tissue fragments by mild centrifugation for 10 s at 1000 rpm (170g) in a clinical centrifuge.

3. Transfer the supernatant to a new 15-mL tube. Add 2 to 3 mL 0.2 M sucrose in buffer X to the pellet, vortex vigorously for 1 min, and recentrifuge for 10 s at 1000 rpm (170s). Combine the supernatants and repeat the pellet extraction three or four times or until the supernatant is not cloudy.

4. Centrifuge the combined supernatants in a clinical centrifuge for 50 s at 1.5 rpm (380s) to pellet the larger tissue fragments. Transfer the supernatant to a new 15-mL tube. Add 5 mL of 0.2 M sucrose in buffer X to the pellet, vortex vigorously for 1 min, and recentrifuge for 50 s at 1500 rpm (380s). Combine the supernatants.

5. Transfer the combined supernatants into Falcon 2059 tubes and pellet the sperm by cen-trifugation for 10 min at 4°C in a Sorvall HB4 or HB6 swinging bucket rotor at 4000 rpm (2600s).

6. Prepare sucrose step gradients in four 2.5-mL Beckman ultracentrifuge tubes (cat. no. 347356). To each tube, add 0.25 mL 2.5 M sucrose in buffer X. Carefully overlay with 1.7 mL 2.3 M sucrose in buffer X.

7. Resuspend the sperm pellets thoroughly by pipeting up and down with 0.8 mL 2 M sucrose in buffer X.

8. Overlay each gradient with 0.2 to 0.25 mL resuspended sperm. Stir the interface between the sperm and the 2.3 M sucrose extensively with a flame-sealed Pasteur pipet tip. The interface should no longer be visible.

9. Centrifuge the gradients for 25 min at 33,000 rpm (93,300s) at 2°C in a Beckman Optima MAX-E ultracentrifuge using a TLS55 swinging bucket rotor.

10. The sperm pellets to the bottom of the gradient and is visible as a light gray pellet (see Note 1). Red blood cells from the testes sediment on top of the 2.3 M sucrose layer. Aspirate off the top half of the gradient containing the red blood cells. Transfer the remainder of the gradient, including the sperm pellet, to a new Falcon 2059 tube. Completely resuspend the sperm pellet with gentle pipeting with 0.5 mL 0.2 M sucrose in buffer X. Avoid contamination with the upper walls of the tube, which may contain residual red blood cells. Transfer the resuspended sperm to the Falcon 2059 tube that contains the lower half of the gradient.

11. Dilute the sperm to 12 mL with 0.2 M sucrose in buffer X and mix well. Pellet the sperm by centrifugation for 10 min at 5000 rpm (3000s) in the HB4 or HB6 swinging bucket rotor at 4°C.

12. Aspirate the supernatant and resuspend the sperm pellet in 0.8 mL 0.2 M sucrose in buffer X supplemented with 0.4% Triton X-100, 1X protease inhibitor mix, and 1 mM DTT. Incubate on a rotating wheel for 30 min at 4°C.

13. Prepare four sucrose cushions by placing 0.5 mL 0.5 M sucrose in buffer X supplemented with 3% BSA, 1 mM DTT, and 1X protease inhibitor mix in 1.5-mL microcentrifuge tubes. Overlay each cushion with 25% of the sperm prep. Centrifuge for 10 min at 2100 rpm (750s) in a clinical centrifuge at room temperature.

14. Aspirate the supernatant and resuspend each sperm pellet with 0.2 mL 0.2 M sucrose in buffer X supplemented with 3% BSA, 1X protease inhibitor mix, and 1 mM DTT. Avoid contaminating the pellet with residual Triton X-100 from the walls of the tube. Transfer the sperm to four new microcentrifuge tubes, dilute to 0.7 mL total with the same buffer, and recentrifuge for 5 min at 2100 rpm (750s) in a clinical centrifuge at room temperature. Repeat once.

15. Resuspend and combine the sperm pellets in a total of 2 mL 0.2 M sucrose in buffer X supplemented with 3% BSA, 1X protease inhibitor mix, and 1 mM DTT.

3.1.4. Determination of Sperm Concentration and Storage

1. Dilute 3 ||L of the sperm prep from Subheading 3.1.3. into 267 ||L water and 30 ||L Hoechst fix solution (see Note 2).

2. Pipet 12 |L into a standard hemocytometer and count the sperm on a fluorescence microscope with a 10x nonoil objective lens. Use the DAPI channel to visualize the sperm and simultaneous low-level bright field illumination to visualize the grid lines on the hemocytometer. Count the sperm in 0.1-|L volume (usually 16 squares) and multiply by 103 to calculate the number of sperm per microliter.

3. Dilute the sperm preparation to 200,000/|L with 0.2 M sucrose in buffer X supplemented with 3% BSA, 1X protease inhibitor mix, and 1 mM DTT. Freeze 90-|L aliquots in 1.5-mL microcentrifuge tubes in liquid N2. These aliquots will be used in the preparation of NPE, described in Subheading 3.5. Save 100 to 200 |L, dilute further to 100,000/|L, and freeze as 5-|L aliquots (in 0.65-mL or similar tubes) in liquid N2. These aliquots will be used in the assays described in Subheadings 3.6 to 3.8. Frozen sperm are stable for up to several years.

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