ULTRASTRUCTURAL AND BIOCHEMICAL-ANALYSIS OF THE APICAL CAP - A CONTRACTILE ELEMENT OF THE LOBSTER SPERM ACROSOME

Sperm from the American lobster, Homarus americanus, have a large cyli ndrical acrosome containing inner acrosomal material, outer acrosomal material, and an apical cap. During the acrosome reaction, the apical cap binds to the vitelline envelope and later contracts extracellularl y to eject the acrosomal filament forward toward the oocyte. The purpo se of this study was to characterize the apical cap ultrastructurally and biochemically. Based on its ultrastructural appearance, the apical cap in unreacted sperm can be divided into four zones. Prior to the a crosome reaction, zone 1 appears crystalline, while after the reaction , it is swollen and contains filamentous material. Zones 2 and 3 are c rystalline and electron dense before the reaction. In reacted sperm, z one 2 remains electron dense, while zone 3 becomes swollen. Zone 4, wh ich is homogenous and moderately electron dense prior to the reaction, is largely absent from reacted sperm. A procedure was developed to is olate apical caps using Percoll density gradient centrifugation. The p urity of the cap fraction, which has a density of 1.06 gm/ml, was eval uated by light and electron microscopy and found to be enriched in api cal caps by as much as 81%. On SDS-PAGE, isolated apical caps show 8 m ajor polypeptide bands with estimated molecular weights ranging from 3 7 to 66 kDa and 5 additional bands at 18 to 29.5 kDa. Isolated caps ap pear similar structurally to caps in unreacted sperm, except that most of zone 4 is absent. When acrosome reacted sperm were incubated with RCA(60) gold (Ricinus communis agglutinin), only zone 3 of the apical cap was labeled, and 0.2 M D-galactose inhibited RCA(60), binding. Whe n apical caps were subjected to Western blot analysis with RCA(60)-HRP as the probe, a single 41 kDa band, which probably represents a major component of zone 3, was labeled. To determine what proteins comprise the soluble components of the acrosome, SDS-PAGE analysis was done on acrosome intact sperm, acrosome reacted sperm, and supernatants from sperm induced to undergo reactions. One band (approximately 29 kDa) wa s totally released into the exudate. It is probable this band comes fr om zone 4 which is the most soluble component of the acrosome. An addi tional 10 bands (17, 21.5, 23, 26, 27.5, 37, 39, 41, 59, and 87 kDa) w ere partially released into the exudate. Seven of these bands (21.5, 2 3, 26, 37, 39, 41, and 59 kDa) are components of the apical cap. Three of the partially released bands (17, 27.5, and 87 kDa) are not presen t in isolated apical caps and probably come from the inner and/or oute r acrosomal material. Finally, lobster abdominal muscle, pleopod tegum ental glands containing non-muscle actin, whole lobster sperm, and iso lated apical caps all contain a 44 to 45 kDa and that comigrates with purified rabbit muscle actin on SDS-PAGE. However, Western blot analys is using a monoclonal antibody to chicken gizzard actin showed cross r eaction with all samples except whole sperm and the apical cap fractio n. The contractile apparatus in apical caps of lobster sperm is theref ore unlikely to be an actin-myosin based system and may contain novel types of proteins that function in contraction extracellularly. (C) 19 94 Wiley-Liss, Inc.