A Possible Role for the Pentose Phosphate Pathway: MATERIALS AND METHODS(2)

Sperm Preparation

Spermatozoa were obtained from the cauda epididymidis and the vas deferens of 10- to 16-wk-old OF1 males (BRL, Fullinsdorf, Switzerland). In all experiments, spermatozoa were incubated in 200 ^l of M16 under oil (light white mineral oil; Sigma) at 37°C for 3 h to achieve capacitation and to maximize the proportion of acrosome-reacted sperm. The concentration of motile sperm at the end of ca-pacitation was 20-40 X 106 cells/ml.

For insemination of zona-free oocytes, suspensions of capacitated sperm were diluted in M16 medium to obtain a final concentration of 0.1-0.5 X 106 motile sperm /ml. To remove glucose, 20 ^l of capacitated sperm was mixed with 4 ml of M16 without glucose and centrifuged at 150 X g for 10 min. The pellet was then resuspended in an appropriate volume of glucose-free medium to give a final concentration of about 0.1 X 106 motile sperm/ml. Insemination droplets of 50 ^l were prepared from these suspensions and covered with oil. buy ventolin inhalers

For measurements of sperm metabolism, suspensions of capacitated sperm were diluted to the appropriate concentration (100, 500, or 1000 spermatozoa/3 ^l) in glucose-free M16 for measurement of the PPP activity or in medium containing 1 mM glucose for glycolysis measurement.

This entry was posted in Spermatozoa and tagged Gamete Fusion, Pentose Phosphate Pathway, Spermatozoa.