Abstract
The effect of bovine serum albumin (BSA), calcium, and ionophore A23187 on in vitro capacitation and the induction of the acrosome reaction in bovine spermatozoa was examined using light, fluorescent, and electron microscopy and spectrofluorometry. Transmission electron microscopy of fixed spermatozoa labeled with the plant lectin concanavalin A indicated that a significant redistribution of lectin binding sites requires at least three hours of incubation in a capacitating medium that contains BSA and calcium. Spermatozoa underwent capacitation and the acrosome reaction following a minimum incubation time of 2โ3 hours in capacitating medium in the continuous presence of calcium ionophore A23187 (1โ10 ฮผm). The induction of the acrosome reaction was determined by light microscopy of fixedโstained cells as well as transmission electron microscopy. These data suggest that a minimum of 2โ3 hours is required for in vitro capacitation of bovine spermatozoa. Capacitation was also examined using the tetracyclineโHCl (TโHCl) binding assay of Ericsson ('67). Measurement of fluorescence by spectrofluorometry and by fluorescence microscopy demonstrated that the level of fluorescent TโHCl bound to spermatozoa was dependent upon the concentration of BSA, the presence of calcium, and time of incubation. However, the loss of bound TโHCl does not coincide with the development of the capacitated state determined by lectin binding and induction of the acrosome reaction. It is suggested that this loss of fluorescence may represent either a preliminary step in capacitation or result from nonspecific binding of fluorescent label to BSA.