Sperm Nuclei Preparation

We have generally followed the standard protocol of Murray (4), but have omitted the protease inhibitors leupeptin and phenylmethylsulfonyl fluoride from many steps to avoid transfer into the final mixture, which is diluted for egg injections.

1. Dissect and isolate the testes from a male:

a. Anesthetize a male in a bucket containing a liter of 0.1% Tricaine (MS222, aminobenzoic acid ethyl ester, Sigma A-5040) and 0.1% sodium bicarbonate for at least 20 min (immersion of the animal in ice water for 20 min may also be used), and pith it.

b. Cut through the ventral body wall and musculature, and lift the yellow fat bodies to isolate the two testes, which are attached to the base of the fat bodies, one on each side of the midline.

Table 1

Part for Transplantation Apparatusa

Company

Product

Catalog number

Price

Phone/address

Newport Corp. Air regulator/filter (This may not be necessary; if your house air pressure is fairly low, you can attach the tubing directly to house air.) Western Analytical

Products, Inc. (They are the US distributor for Omnifit Ltd. Phone: 01223-69841 Fax: 01023-61106 51 Norfolk St. Cambridge CB12LE) Fisher Hoffman open-side tubing clamps T-shaped connectors

Three-way valve Trivalve caps (mixed colors)

Fine Science Tools, Inc.

001102 001310

Tygon tubing 1/32 in. Tygon tubing 3/16 in. Precision micromanipulator MM33

Magnetic base

05-875A

15-319C

12-169-1A 14-169-3B 25033-10

25810-00 $84.95

1-800-222-6440 1791 Deere Ave. Irvine, CA 92714

1-800-541-8421 25407 Blackthorn Murrieta, CA 92563

1-800-521-2109 373-G Vintage Park Dr. Foster City, CA 94404

aA base somewhat heavier and more stable than the magnetic model available from Fine Science Tools was previoulsy available from Brinkman. To our knowledge, however, a comparable product is no longer available either from Brinkman or from other manufacturers; we have had a copy built by the local machine shop.

c. Remove the testes with dissecting scissors, and place them in a 35-mm tissue-culture dish containing cold 1X MMR.

Rinse the testes in three changes of cold 1X MMR and two times in cold 1X NPB, removing any attached pieces of fat body or debris with a fine forceps. Take care not to puncture the tissue pouches, since this releases the sperm. 2. Move the cleaned testes to a dry 35-mm tissue-culture dish, and macerate the tissue well (until clumps are no longer visible to the naked eye) with a pair of clean forceps.

Fig. 3. Diagram of alternative injection apparatus. A line connects the needle to a 30-mL Hamilton syringe that is held by a clamp to a ring stand. The glass syringe plunger is pushed in using a microburet that is also clamped to the ring stand. A slow, controlled flow of liquid through the needle can be obtained by leaving a large cushion of air inside the syringe between the glass plunger and the end attached to the tubing. The circular inset shows how the point of the needle should appear after it is clipped.

Fig. 3. Diagram of alternative injection apparatus. A line connects the needle to a 30-mL Hamilton syringe that is held by a clamp to a ring stand. The glass syringe plunger is pushed in using a microburet that is also clamped to the ring stand. A slow, controlled flow of liquid through the needle can be obtained by leaving a large cushion of air inside the syringe between the glass plunger and the end attached to the tubing. The circular inset shows how the point of the needle should appear after it is clipped.

3. Resuspend the macerated testes in 2 mL of 1X NPB by gently pipeting the solution up and down through a fire-polished, truncated Pasteur pipet with an opening of about 3 mm in diameter.

4. Squirt the sperm suspension through two to four thicknesses of cheesecloth placed into a funnel, and collect the solution into a 15-mL tube (we use round bottom polypropylene tubes; Fisher, cat. #: 14-956-1J). Rinse the forceps and dish with 8 mL of 1X NPB, and force this through the cheesecloth into the 15-mL tube. With a gloved hand, fold the cheesecloth and squeeze any remaining liquid through the funnel into the 15-mL tube.

5. Pellet the sperm by centrifugation at 1500g for 10 min at 4°C (we use a Sorvall HB-6 or similar swinging bucket rotor fitted with the appropriate adapters). Resuspend sperm in 8 mL NPB and repellet by centrifugation at 1500g for 10 min at 4°C.

6. Resuspend pellet in 1 mL NPB with a cut plastic pipet tip, warm the suspension to room temperature, and add 50 ||L of 10 mg/mL lysolecithin. Mix gently and incubate for 5 min at room temperature.

7. Add 10 mL cold 1X NPB + 3% BSA (with protease inhibitors; 1:1000 dilution of leupeptin and PMSF stock solutions) to the suspension, mix gently by inversion, and centrifuge at 1500g for 10 min at 4°C. Decant the supernatant.

8. Resuspend the pellet in 5 ml cold 1X NPB + 0.3%BSA (no protease inhibitors), mix gently by inversion, and repellet as before.

9. Resuspend the pellet in 500 pL of sperm dilution buffer, and transfer suspension into a 1.5-mL Eppendorf tube. Count the sperm density using a hemacytometer (Fisher, cat. #: 02-671-5): dilute a small amount of the concentrated sperm 1:100 in sperm dilution buffer, and add 1pL of 1:100 Hoechst stock to visualize the sperm heads under a fluorescence microscope. For a 1:100 dilution of our sperm stock, we typically obtain counts of 75-125 (x104 cells/mL). At this concentration, the undiluted stock contains 75-125 sperm/nL. If your sperm stock is substantially less concentrated (i.e., a count of <50 for a 1:100 dilution), repellet the sperm and resuspend in a smaller volume of sperm storage buffer. Sperm can be stored at 4°C and used for transplantations for up to 48 h.

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