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Loss of Nectin-2 at Sertoli-Spermatid: MATERIALS AND METHODS(5)

METHODS(5)

Scanning Electron Microscopy

Epididymal spermatozoa were fixed for 1 h in 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM cacodylate buffer (pH 7.4) containing 2.5 mM CaCl2. A drop of sperm suspension was then adhered to poly-L-lysine-coated coverslips, and fixed spermatozoa were rinsed three times in caco-dylate buffer. The coverslips were then postfixed in a freshly prepared solution of 1% OsO4 in cacodylate buffer for 30 min at room temperature. After three more rinses in cacodylate buffer, the samples were dehydrated through a series of increasing concentrations of acetone in water and twice in 100% acetone. Finally, the samples were subjected to critical point drying. Images were acquired on a Jeol 5300 scanning electron microscope.

In Vivo Fertilization and Assessment of Oviductal Spermatozoa

CD1 female mice (age, 6 wk) were injected with 6 IU of eCG, followed by 6 IU of hCG 50 h thereafter to induce ovulation. Two females were caged with one male for one night following the hCG administration. Copulation plugs were observed 16 h post-hCG, at which time plugged females were killed and the sperm-exposed oocytes were collected from the ampullae in M2 medium (Specialty Media, Phillipsburg, NJ). Egg clusters were briefly treated with hyaluronidase (Sigma, St. Louis, MO) to disperse oocytes. The oocytes were then transferred to M16 medium (Specialty Media) and cultured for 6 h at 37°C under 5% CO2. At this point, the presence of a male pronuclei was determined by microscopic observation under Nomarski optics. After 24 h in culture, embryos were transferred to KSOM medium (Specialty Media) and allowed to develop for an additional 48 h to assess the formation of blastomeres. To assess the ability of spermatozoa to enter the oviducts following overnight mating, the spermatozoa were flushed out of the oviducts, collected in 1 ml of M2 medium, and briefly subjected to hyaluronidase treatment. Spermatozoa were then concentrated by centrifugation at 500 g for 5 min, resuspended in 20 |xl of M2 medium, fixed with 2% paraformaldehyde, and counted in a hemocytometer. In the present study, P values were determined by the two-tailed, unpaired /-test.