Collection of Ovulated Oocytes and In Vitro Fertilization
Sexually mature female Wistar rats (2-3 mo old) were maintained under controlled lighting conditions (14L:10D; lights-on at 0600 h). For collection of ovulated oocytes, rats were killed between 0500 h and 0600 h on the day of estrus, which was identified by examination of vaginal smears. The oviducts were isolated and placed in a dish containing paraffin oil and the preincubated-diluted sperm suspension. The cumulus-oocyte complexes were dissected out of the oviducts, introduced into the sperm suspension, and cultured for 10 h in an atmosphere of 5% CO2 in air at 37°C. After the coculture with spermatozoa, the oocytes were stripped of cumulus cells by pipetting with 0.1% hyaluron-idase (Sigma Chemical Co., St. Louis, MO) in mR1ECM. antibiotics levaquin
Assessment of Sperm Penetration
The denuded oocytes were mounted, fixed, dehydrated, stained with 0.25% lacmoid, and examined under a phase-contrast microscope as described previously. Oocytes were designated as penetrated when they had at least one male pronucleus and corresponding sperm tail in the vitel-lus. Oocytes with spermatozoa in their perivitelline space were not considered penetrated.