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Loss of Nectin-2 at Sertoli-Spermatid: MATERIALS AND METHODS(6)

Zona-Free Hamster Oocyte Sperm Penetration Assay

Epididymal spermatozoa of nectin-2wt/LacZ and nectin-2LacZ/LacZ males (age, 24 wk) were recovered by slashing the cauda epididymis several times with a hypodermic needle and allowing the sperm to swim out for 30 min at 37°C under 5% CO2 in a dish containing 2 ml of preequilibrated human tubal fluid (Specialty Media). Sperm penetration assay (SPA) was performed as described previously with modifications. Briefly, mouse sperm were recovered as described above, except that the medium was Biggers Whitten Whittingham (BWW) containing 30 mg/ml of BSA (Sigma-Aldrich) and preequilibrated at 37°C under 5% CO2. Spermatozoa at concentrations between 25 X 106 and 30 X 106 per milliliter were capacitated by incubation at 37°C under 5% CO2 for 2 h.

Oocytes of superovulated golden hamster females were collected in BWW containing 5 mg/ml of human serum albumin (HSA) and released from cumulus masses by treatment with 1 mg/ml of hyaluronidase. Zonae pellucidae were removed by incubation in 1 mg/ml of trypsin (Sigma). Oocytes were inseminated with spermatozoa at a final concentration of 105 per milliliter in 1 ml BWW with 5 mg/ml of HSA. After 45 and 90 min of incubation at 37°C under 5% CO2, oocytes were removed and stained with acridine orange to visualize penetrated, decondensing sperm nuclei. Flovent oral aerosol