ENHANCEMENT OF ACROSOME REACTION AND SUBZONAL INSEMINATION OF A SINGLE SPERMATOZOON IN MOUSE EGGS

The acrosome reaction in mouse spermatozoa was induced by various means. These were 1) varying incubation time in T6 medium, 2) incubation in T6 medium with added A23187, 3) incubation in T6 medium with added dbcGMP and imidazole, 4) exposure to an electric field, and 5) a combination of incubation in a medium with dbcGMP and imidazole and electroporation. The mean percentages of acrosome-free spermatozoa obtained by these various methods and assessed on the basis of both Bryan's stain and immunolocalization by FITC-labeled monoclonal antibodies increased by steps from 36% to 67%, 73%, 86%, and 92%. Individual spermatozoa from the various treatments were afterwards microinjected under the zona pellucida of a mouse oocyte. The fertilization rate for eggs microinjected with a spermatozoon treated with A23187, dbcGMP, and imidazole, by electroporation and by a combination of the last two methods also increased by steps from 17% to 34%, 36%, and 70%, respectively. Ninety-five percent of the fertilized oocytes reached the early blastocyst stage, thirty-eight percent of these blastocysts implanted in pseudopregnant mice, and twenty-eight percent developed to term. These results indicated the varying degrees of success of different ways of inducing acrosomal loss in spermatozoa and their subsequent success rates in fertilization and further in vitro and in vivo development.