Acrosome Biogenesis in the Hamster: MATERIALS AND METHODS(1)
Care and use of animals conformed to NIH guidelines for humane animal care and use in research. All protocols were approved by the institutional Animal Care Committee, and animals were housed under the supervision of University Veterinarians in an AAALAC-approved Central Animal Care Facility. Hamsters and mice were killed by C02 asphyxiation, and guinea pigs were killed with methoxy-flurane.
Preparation of Sperm Suspensions
The caput and cauda epididymides were separated and minced in Hank’s saline or Tyrode’s solution (Sigma Chemical Co., St. Louis, MO) at 37°C. Cauda sperm were pelleted by centrifugation for 10 min at 500 X g. Caput sperm were purified by centrifugation for 5 min at 650 X g on a step gradient of 40%, 60%, and 80% Percoll (Pharmacia Biotech, Piscataway, NJ); the spermatozoa at the 60%-80% interface were diluted with Tyrode’s solution and pelleted by centrifugation at 500 X g for 10 min. buy ortho tri-cyclen
Isolation of Acrosomal Fraction
The ALM complex was isolated from cauda epididymal spermatozoa as described previously . Sperm from the cauda epididymides of 4 animals were resuspended into 10 volumes of ice-cold extraction solution (T-TNI) composed of 0.1% Triton X-100 in TNI (TNI = 150 mM NaCl, 25 mM Tris-HCl [pH 7.0], 2 mM benzamidine, 1 |xg/ml leupeptin, and 1 (xg/ml pepstatin) and immediately centrifuged at 500 X g for 10 min. The pellet was extracted for 30 min at 4°C with T-TNI and homogenized with 10-20 strokes of a glass silicone-coated homogenizer to detach the ALM complex from the sperm heads. Twenty milliliters of the sperm suspension was mixed with 100 ml of 45% Percoll, 0.25 M sucrose, 0.1% Triton X-100, and 25 mM Tris-HCl (pH 7.0), and then centrifuged at 60 000 X g for 35 min in a Beckman 60Ti rotor (Beckman Instruments, Palo Alto, CA). The ALM fraction formed a band at the top of the gradient which was collected, diluted with TNI, and pelleted at 100 000 X g for 20 min in a Beckman TL55 rotor.