Synchronous sperm penetration of zona-free mouse eggs in vitro

Y. Takahashi, C. Meno, E. Sato, Y. Toyoda

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)


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/μl. 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/μl. At 160 sperm/μl, 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.

Original languageEnglish
Pages (from-to)424-430
Number of pages7
JournalBiology of reproduction
Issue number2
Publication statusPublished - 1995
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Reproductive Medicine
  • Cell Biology


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