Bicarbonate is Essential for Fertilization of Mouse Eggs: Mouse Sperm Require it to Undergo the Acrosome Reaction1

Fertilization of mouse eggs by capacitated mouse sperm occurs readily in vitro in a defined culture medium designated medium CM, derived from Krebs-Ringer bicarbonate medium by supplementation with glucose, lactate, pyruvate, and albumin. Medium CM contains 25 mM HCO-3 equilibrated with CO2 to pH 7.4. Replacement of the CO2/HCO-3 buffer with 25 mM N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) while otherwise maintaining pH, ionic strength, and composition identical to that of medium CM, yielded medium HM in which the percentage of in vitro fertilization observed was consistently zero. Addition of 10 mM HCO-3 to medium HM yielded medium HMB, in which the percentage of eggs fertilized was 70%, equal to that observed in medium CM. This percentage applies to cumulus-free, zona-intact eggs. With zona-free eggs, the percentage fertilization increased to 90% in media CM and HMB, but remained zero in medium HM. This result shows that HEPES does not inhibit fertilization while omission of CO2/HCO-3 blocks it completely. Capacitation of mouse sperm, as determined by chlortetracycline fluorescence assay, proceeded at the same rate in media CM and HMB, but was retarded in medium HM. Sufficient sperm became capacitated, however, that this could not be the block of fertilization. The acrosome reaction in sperm bound to isolated zonae pellucidae, as also determined by chlortetracycline fluorescence assay, occurred with the same time course in media CM and HMB, but did not occur in medium HM.(ABSTRACT TRUNCATED AT 250 WORDS)