Effects of Protease Inhibitors and Substrates on Motility of Mammalian Spermatozoa

A series of protease inhibitors were tested on the motility of human, rat, bull, and rabbit demembranated reactivated spermatozoa. Some inhibitors, including aprotinin, boc-gln-leu-lys-H, and Dphe-pro-arg-H, could inhibit motility as well as prevent initiation of motility. In general, with the exception of aprotinin, protease inhibitors were more potenl in preventing the initiation of movement than in blocking motility of demembranated spermatozoa. Protease substrates could also block sperm motility. Of the substrates tested only those with arg or lys ester bonds were active. The inhibition of motility by protease substrates was reversible, as once spermatozoa hydrolyzed the added exogenous protease substrates, motility reappeared. The importance of ester bond in the inhibitory action of protease substrates was confirmed by experiments that showed the lack of effect of pre-hydrolyzed protease substrates. The results suggest that a serine prmease with lys and arg ester bond specificity is involved in the control of sperm motility. The fact that protease substrates also block motility of intact spermatozoa further emphasizes the physiological relevance of this new regulatory system. p ROTEASES fulfill two main functions: protein degradation as m protein turnover and protein limited degradation as in the activation or modulation of protein action. The importance of this second function is becoming more and more recognized. Several peptide hormones exist in a precursor form, and the action of specific proteases, often localized in the same storage organelles, yields active hormones (6). Proteases have also been shown to be involved in receptor-mediated events such as in the modulation of the ~adrenergic-receptor adenylate cyclase system (I 8) and in insulin action (1). Acrosin, the most extensively studied protease associated with spermatozoa, is a trypsin-like protease involved in fertilization (21 ). Acrosin has been reported both in the acrosomal sac and on the inner acrosomal membrane where it exists in the form of proacrosin ( 14, 21). A different trypsin-like protease has also been isolated from bull spermatozoa. This enzyme can activate adenylate cyclase from rat brain (10) and human plalelels (11). Kallikreins have been reported to affect sperm motility by generating kinins, which presumably act on motility (17). However, no protease has been directly implicated in sperm motility. Recently, we have reported that aprotinin, a protease inhibitor, blocked the motility of demembranated reactivated mammalian spermatozoa (2, 3) without significantly affecting the force-generating dynein ATPase (8). In the present study, we investigated in more detail the possible involvement of a protease in sperm motility. We now report that a series of protease inhibitors as well as a series of protease substrates with lys or arg ester bonds inhibit the motility of both demembranated reactivated and intact spermatozoa. These results suggest that a serine protease modulates sperm motility. Materials and Methods