Synchronous sperm penetration of zona-free mouse eggs in vitro.
To synchronize sperm penetration of zona-free eggs immediately after insemination, zona-free eggs preloaded with Hoechst-33342 were inseminated under various conditions. Insemination was mostly conducted at 10 sperm/microliter. In preliminary experiments, fatty acid-free BSA (FAF) was more satisfactory for sperm penetration than fraction V BSA, and FAF was used in the following experiments. Only 26% of zona-free eggs were fertilized at 10 min after insemination when the eggs were inseminated immediately after zona removal and preloading. However, egg preincubation significantly improved the penetration rate (1 h preincubation: 63%, 2 h preincubation: 82% penetrated 10 min after insemination). Some eggs preincubated for 2 h were already penetrated at 3 min (7%), and the rate gradually increased in a time-dependent manner (3 min: 7%, 5 min: 30%, 10 min: 80%). The rate further improved as the sperm concentration was increased; the maximal level was obtained at 160 sperm/microliters. At 160 sperm/microliters, 54% of the eggs were penetrated at 3 min and 78% were at 5 min. These results indicate that it is possible to synchronize the sperm entry and that not only sperm but zona-free eggs should be preincubated before insemination. These data are considered valuable for investigating early events in fertilization.
D000818:Animals; D001562:Benzimidazoles; D002918:Chymotrypsin / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D005456:Fluorescent Dyes; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes / Q000502:physiology; D011402:Pronase / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000502:physiology; D013997:Time Factors; D015044:Zona Pellucida / Q000502:physiology*
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D002918:Chymotrypsin; D011402:Pronase; C017807:bisbenzimide ethoxide trihydrochloride