The acrosome reaction of epididymal spermatozoa and fertilization in vitro of mouse eggs in chemically defined media without tissue fluid were investigated. About 8 to 10% of motile spermatozoa lost their acrosome but no eggs were penetrated when the spermatozoa and eggs were incubated in a basic medium (modified Krebs-Ringer bicarbonate solution containing glucose) for 5 to 7 hr. Addition of a single metabolic intermediate, such as sodium oxaloacetate or sodium pyruvate, to the basic medium increased the proportion of motile spermatozoa without an acrosome (19 to 34%) and the proportion of eggs penetrated (3·2 to 25·5%). Incubation of spermatozoa and eggs in the basic medium containing serum albumin of various species caused a further increase in the proportion of motile spermatozoa without an acrosome (50 to 65%) and in that of penetrated eggs (60·7 to 86%). The best medium for sperm capacitation and fertilization of mouse eggs in vitro, however, was the basic medium containing bovine serum albumin, sodium lactate and sodium pyruvate. The time required for sperm capacitation was 1 hr in this medium, and 2 hr in the medium containing only serum albumin. Certain components present in the oviducal fluid and in the cumulus egg clots, probably similar to serum albumin and sodium lactate or sodium pyruvate, appeared to be beneficial for the capacitation of spermatozoa and fertilization of eggs. It was concluded that serum albumin, sodium lactate and sodium pyruvate can be substituted for tissue fluid in the induction of capacitation of spermatozoa and fertilization of mouse eggs in vitro.
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