Synchronous Sperm Penetration of Zona-Free Mouse Eggs in Vitro1

Abstract

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.

β1 Integrin Is an Adhesion Protein for Sperm Binding to Eggs

“…Thus, removal of the ZP does not introduce an artifact into inhibition of sperm-egg binding by these mimics. Moreover, we ensured ample recovery time after removing the ZP in order that egg fertilizability was not impaired (38). …”

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

“…Sperm-egg binding and fusion assays were performed as described previously with minor modification (Takahashi et al, 1995;Chen et al, 1999a). Briefly, zona-free oocytes were prepared by acid treatment (pH 2.7) for 15-30 s followed by a 3-h incubation in M199 medium in a 37°C/5%CO 2 incubator to recover fertilizability (Takahashi et al, 1995).…”

“…Briefly, zona-free oocytes were prepared by acid treatment (pH 2.7) for 15-30 s followed by a 3-h incubation in M199 medium in a 37°C/5%CO 2 incubator to recover fertilizability (Takahashi et al, 1995). Recovered eggs were preloaded with 0.5 g/ml Hoechst-33342 for 15-30 min, washed, and then treated with either disintegrin proteins or antibody for 30 min at 37°C.…”

Comparison of Capacitation and Fertilizing Ability of BALB/c and ICR Mouse Epididymal Spermatozoa. Analysis by In Vitro Fertilization with Cumulus-intact and Zona-free Mouse Eggs.

“…Zona-free eggs were stained by using a slight modification of the method described by Takahashi et al [10]. A stock solution of Hoechst-33342 (1 µg/ml; H-33342; Sigma) was prepared in modified PBS containing 1.0 mg/ml of polyvinylalcohol (cold water soluble, Sigma); the solution was stored in the dark at -20°C prior to use.…”

“…In the case of zona-free eggs, Hoechst 33342, which was previously reported as an efficient DNA-specific fluorochrome of sperm-egg fusion [9,10], was used to ascertain the early events in fertilization.…”