Ventolin Inhalers

- Sperm Factor Induces Intracellular Free Calcium: MATERIALS AND METHODS(1)

Egg and Oocyte Recovery

Eggs were obtained from the oviducts of CD-1 female mice (6-12 wk old) superovulated by i.p. injection of 5 IU of eCG (Sigma, St. Louis, MO) followed 48 h later by injection of 5 IU of hCG (Sigma) to induce ovulation. Eggs were recovered 14 h post-hCG into a HEPES-buffered solution (TL-Hepes) supplemented with 10% heat-treated fetal calf serum (FCS; Gibco, Grand Island, NY). Cumulus cells were removed with bovine testes hyaluronidase (Sigma). For this study, eggs with the first polar body and no signs of degeneration were chosen. These eggs were placed in 50-^l drops of KSOM (Specialty Media, Phil-lipsburg, NJ) under paraffin oil at 36.5°C in a humidified atmosphere containing 7% CO2 until the time of injection.

Stage V and stage VI oocytes were obtained from Xenopus laevis as previously described. Oocytes were cultured in NaCl (96 mM), KCl (2 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), Hepes (5 mM, pH 7.6), and sodium pyruvate (2.5 mM) at 18°C until used in experiments.

Preparation of SF and Testis Extracts

Cytosolic SFs were prepared from boar semen as previously described. After washing, the sperm suspension was sonicated and the lysate was ultracentrifuged. The clear supernatant was concentrated with ultrafiltration membranes (Centriprep-30; Amicon, Beverly, MA) to final concentrations of 20-30 mg/ml protein. These extracts were then mixed for 30 min at 4°C with ammonium sulfate at 50% final saturation, the precipitates were collected by centrifugation (10 000 X g, 15 min, 4°C), and the pellets were stored at -20°C until use. Pellets were resuspended in injection buffer (75 mM KCL and 20 mM Hepes, pH 7.0), washed in the same buffer at least three times to remove all traces of ammonium sulfate, and concentrated with ultrafiltration membranes. Samples were aliquoted and frozen at -80°C.

September 10, 2013 Sperm
Tags: calcium developmental biology fertilization