Formyl-MET-Leu-Phe induces chemotaxis and acrosomal enzyme release in bull sperm.

The chemical nature of the factors that induce the selective fusion and vesiculation of the outer acrosomal and plasma membranes in sperm cells is yet to be established [ 1,2]. This process, known as the acrosome reaction, precedes fertilisation of the egg and is accompanied by the release of the degradative enzymes sequestered in the acrosomal vesicle of sperm [l]. The synthetic tripeptide, formyl-MetLeu-Phe has been shown to induce chemotaxis in rabbit neutrophils [3]. It also causes the release of lysosomal enzymes, a process which must necessarily involve a fusion of the lysosomal and plasma membranes [4]. Functional analogies between lysosomes and the acrosome in sperm have been drawn [5,6]. Here we describe the selective release of bull sperm acrosomal enzymes, induced by formyl-Met-leuPhe. The specificity of this process is established by the inability of related diand tripeptide analogs to cause enzyme release. Further, formyl-~et-leuPhe is also shown to induce chemotaxis in bull sperm. This observation demonstrates for the first time that low molecular weight peptides may indeed be capable of stimulating release of acrosomal contents, a process closely identified with the acrosome reaction. NMR. The extent of acrosomal enzyme release was monitored by incubating the sperm cells with the peptide followed by centrifugation and assay of the enzymatic activities in the supernatant. Typically, 4 ml fresh bull semen was diluted with 12 ml KrebsRinger bicarbonate buffer (pH 7.4) containing fructose (1 mg/ml) [7]. About 5 ml of the diluted semen was layered over 5 ml 1.3 M sucrose/0.9% NaCl and centrifuged at 6000 rev./min for 10 min. The pellet was resuspended in 20 ml buffer and spun down at 6000 rev./rnin-'/lS min-'. The resultant sperm pellets were resuspended in a minimum volume of buffer. Peptides (100 pM) and BSA' (2 mglml) were added to 1 ml sperm suspension (-5 X lo6 cells). The controls lacked the peptide. The mixtures were incubated at 37°C for 1 h and the cells, pelleted. The supernatants were collected and assayed for hyaluronidase [8], acrosin [9], lactate dehydrogenase (LDH) [lo], aryl sulfatase [ 111, N-acetyl-0-D-glucosaminidase [ 121 and acid phosphatase [ 131 by standard procedures. The chemotactic response of bull sperm to the peptides was measured by the inverted capillary assay [14]. Freshly collected semen was suspended into yolk-citrate buffer containing 4 mM C a Q . Experiments were carried out at 37°C.