Enhancement of sperm acrosin activity by glycerol-pretreatment - quantitative estimations

Acetic acid treatment of spermatozoa, a method suitable for optimal extraction of the acrosomal proteinase acrosin, was used to show the influence of glycerol on sperm acrosin activity. Short-time pretreatment of semen samples with glycerol in concentrations up to 35% (v/v) caused a 1.5-2.5 fold increase in sperm acrosin activity. Higher glycerol concentrations caused a decrease in sperm acrosin activity due to leakage of the enzyme into the suspension medium. A further increase in sperm acrosin activity was observed during aging of semen in the presence of glycerol. In this case, the height of the increase depends on the glycerol concentration applied as well as incubation temperature and time. The acrosin activation pattern induced by glycerol was not influenced by proteinase (acrosin) inhibitors. On the other hand, addition of glycerol to spermatozoa from which the seminal plasma had been removed and substituted by physiological saline caused only a small increase in sperm acrosin activity. This indicates the occurrence of a seminal plasma factor which is either stimulated (activated) by glycerol or which can penetrate the membranes and subsequently activate acrosin only in the presence of glycerol. This seminal plasma factor was not consumed during activation and could be transfered to another sperm sample. However, a protecting influence of such a factor on the sperm head membranes and thus an indirect activation effect, i.e. better extractibility of acrosin, has also to be considered. The glycerol-induced rise of the acrosin activity is not caused by reversible conformational changes of the enzyme molecules: acrosin activity was not diminished if glycerol was removed from semen samples or extracts later on. The possibility that the observed increase in BAEE-splitting activity is due to a so far unknown proteinase may be excluded: the activity completely disappeared by neutralization due to formation of the acrosin-inhibitor complex and appeared again by acidification, a well known characteristic of acrosin and its inhibitors. Factors which are probably responsible for the glycerol-induced activation of acrosin-membrane effects and the activation of a precursor from of acrosin-are discussed.