Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope . The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (201uM A23187) suspended in sea water containing 50 mM excess CaC1 2.Before activation of the sperm, the acrosomal region appears as a 1 .1-,um diameter sphere, slightly less dense than the rest of the sperm head . Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After^"1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material .In this paper, we also consider the meaning of the enhanced DIC image seen in the highresolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope .

In an attempt to investigate the role of water influx in the extension of the acrosomal process of Thyone sperm, we induced the acrosomal reaction in sea water whose osmolarity varied from 50 to 150% of that of sea water. (a) Video sequences of the elongation of the acrosomal processes were made; plots of the length of the acrosomal process as a function of (time) t/2 produced a straight line except at the beginning of elongation and at the end in both hypotonic and hypertonic sea water (up to 1.33 times the osmolarity of sea water), although the rate of elongation was fastest in hypotonic sea water and was progressively slower as the tonicity was raised. (b) Close examination of the video sequences revealed that regardless of the tonicity of the sea water, the morphology of the acrosomal processes were similar. (c) From thin sections of fixed sperm, the amount of actin polymerization that takes place is roughly coupled to the length of the acrosomal process formed so that sperm with short processes only polymerize a portion of the actin that must be present in those sperm. From these facts we conclude that the influx of water and the release of actin monomers from their storage form in the profilactin (so that these monomers can polymerize) are coupled. The exact role of water influx, why it occurs, and whether it could contribute to the extension of the acrosomal process by a hydrostatic pressure mechanism is discussed.When a Thyone sperm comes in contact with the outer surfaces of an egg, a series of reactions are elicited which culminate in the growth of a process which can form in less than 10 s, yet exceed 90 um in length. In fact, the rate of elongation of this process, the acrosomal process, is comparable to the rate of contraction of some of the fastest skeletal muscles. Of the many questions that one could ask about this extraordinary motile event perhaps the most basic is, What generates the force required to drive the extension process? In earlier publications, we demonstrated that at all stages during the growth of the acrosomal process there is an explosive, yet controlled assembly of actin and concluded that the assembly per se provides the force for the elongation of the process. The assembly of actin is initiated by a rise in internal pH (20); this is followed by the nucleation of actin monomers on a cytoplasmic organelle, the actomere, which simultaneously nucleates and directs the assembly of filaments (19) to form a directed spear used to penetrate the jelly which surrounds the egg. Thus, it is clear that actin polymerization is involved

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