Ultrastructural and biochemical analysis of the apical cap: A contractile element of the lobster sperm acrosome

Abstract

AbstractSperm from the American lobster, Homarus americanus, have a large cylindrical 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 extracellularly to eject the acrosomal filament forward toward the oocyte. The purpose 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 acrosome reaction, zone 1 appears crystalline, while after the reaction, it is swollen and contains filamentous material. Zones 2 and 3 are crystalline and electron dense before the reaction. In reacted sperm, zone 2 remains electron dense, while zone 3 becomes swollen. Zone 4, which is homogenous and moderately electron dense prior to the reaction, is largely absent from reacted sperm. A procedure was developed to isolate apical caps using Percoll density gradient centrifugation. The purity of the cap fraction, which has a density of 1.06 gm/ml, was evaluated by light and electron microscopy and found to be enriched in apical caps by as much as 81%. On SDS‐PAGE, isolated apical caps show 8 major polypeptide bands with estimated molecular weights ranging from 37 to 66 kDa and 5 additional bands at 18 to 29.5 kDa. Isolated caps appear similar structurally to caps in unreacted sperm, except that most of zone 4 is absent. When acrosome reacted sperm were incubated with RCA60 gold (Ricinus communis agglutinin), only zone 3 of the apical cap was labeled, and 0.2 M D‐galactose inhibited RCA60 binding. When apical caps were subjected to Western blot analysis with RCA60‐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) was totally released into the exudate. It is probable this band comes from zone 4 which is the most soluble component of the acrosome. An additional 10 bands (17, 21.5, 23, 26, 27.5, 37, 39, 41, 59, and 87 kDa) were partially released into the exudate. Seven of these bands (21.5, 23, 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 present in isolated apical caps and probably come from the inner and/or outer acrosomal material. Finally, lobster abdominal muscle, pleopod tegumental glands containing non‐muscle actin, whole lobster sperm, and isolated apical caps all contain a 44 to 45 kDa and that comigrates with purified rabbit muscle actin on SDS‐PAGE. However, Western blot analysis using a monoclonal antibody to chicken gizzard actin showed cross reaction with all samples except whole sperm and the apical cap fraction. The contractile apparatus in apical caps of lobster sperm is therefore unlikely to be an actin‐myosin based system and may contain novel types of proteins that function in contraction extracellularly. © 1994 Wiley‐Liss, Inc.