Sperm Penetration Of Zona-free Eggs Research Articles

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.

Effect of glycerol on the penetrating ability of fresh ram spermatozoa with zona-free hamster eggs.

The presence of 10% glycerol in diluted semen for 30-90 min before preincubation stimulated sperm penetration of zona-free eggs. Prolongation of semen storage in medium with glycerol did not further improve penetration ability. The penetrating activity of spermatozoa depended on the concentration of glycerol and incubation time. These results indicate that the presence of glycerol accelerates induction of the acrosome reaction which could be one of the causes of lower conception rate after insemination with deep-frozen ram semen.