The number of bound spermatozoa was determined under phase-contrast microscopy by counting sperm tails protruding from the oocyte at a median focal plane. Penetration events were assessed under epifluorescence. All samples were counted by the same observer. itat on
In Vitro Sperm Binding Assay to Zona-Intact Mouse Oocytes
Epididymal spermatozoa of three age-matched nectin-2wt/LacZ controls and three nectin-2LacZ/LacZmales were recovered and capacitated as described above. Oocytes of 12 ovulated CD1 females, recovered 15 h post-hCG injection, were pooled and subdivided into fertilization dishes containing 800 l^l of BWW.
Then, 5 X 105 spermatozoa were added to oocyte dishes in a final volume of 1 ml of BWW. After 15 min at 37°C under 5% CO2, sperm-exposed oocytes and bound sperm were washed through subsequent drops of M2 medium to remove loosely adhered sperm. Bound sperm were fixed with 2% paraformaldehyde and stained with 100 ng/ml of Hoechst 33258 to visualize sperm nuclei. The number of spermatozoa bound per sperm-exposed oocyte was determined by counting spermatozoan nuclei associated with each oocyte under a fluorescence microscope. Bound sperm nuclei were counted by slowly focusing through the whole oocyte. No attempts were made to assess the acrosome status of the spermatozoa.