Bicarbonate/CO2 is not required for zona pellucida- or progesterone-induced acrosomal exocytosis of mouse spermatozoa but is essential for capacitation.

We investigated whether bicarbonate/CO2 is required for capacitation or for acrosomal exocytosis triggered with zona pellucida or progesterone. Mouse spermatozoa, incubated for 90 min in a modified Tyrode's medium with bicarbonate and equilibrated with 5% CO2 in air, were washed in medium without bicarbonate but with Hepes, resuspended in media with or without bicarbonate, and then stimulated with 1 zona pellucida/microliter or 15 microM progesterone. Spermatozoa were able to undergo exocytosis, regardless of the medium in which they were resuspended, as ascertained via the chlortetracycline assay. If, however, spermatozoa were first incubated in a medium in which bicarbonate was replaced by Hepes and were then washed and resuspended in various media, they were unable to undergo exocytosis in response to zona pellucida or progesterone even when resuspended in bicarbonate-containing medium. This indicated that spermatozoa were not capacitated. Furthermore, the proportion of cells exhibiting the "B" pattern (characteristic of capacitated cells) after incubation in medium without bicarbonate was lower than that in cells incubated with the anion and the appropriate gas phase. Extended incubation in medium without bicarbonate (up to 3 h) did not increase the proportion of cells that exhibited the "B" pattern. These results demonstrate that bicarbonate is not required for acrosomal exocytosis but that it is essential for capacitation, exerting roles beyond its action as pH buffer.