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The spermatozoon of the Japanese abalone, Haliotis discus, and its structural changes during the acrosome reaction were observed by electron microscopy. The spermatozoon has a huge acrosome in the shape of a hanging bell or a forefinger with a deep fossa at the posterior end being filled with a bundle of microfilaments. The membranes of the acrosomal apex, the so-called trigger region, are structurally discernible from those of other acrosomal regions. Following the trigger region, a unique structure under the acrosomal membrane covers the surface of the acrosomal content in the form of a truncated cone.First, the membranes at the apex of the acrosome are vesiculated, followed by the formation of a narrow gap between the outer acrosomal membrane and the ac;osomal content. Next, the bundle of microfilaments elongates, running through the center of the acrosome, reaching the trigger region and protruding out of the acrosomal top. Then release of the acrosomal content occurs in two steps, disclosing the "membrane undercoating structure" that comprises globular particles with a fuzzy material connecting them.The acrosome reaction occurs in the jelly layer very close to the egg envelope.This resembles the undercoat network found in erythrocytes.

An electron-dense structure termed the "truncated cone" covers the apical surface of the acrosomal contents except for the trigger region in Haliotis discus spermatozoa. The truncated cone, having a slant height of 0.3 pm and diameters of circular top and base of 0.3 and 0.6 pm, shows striations with a periodicity of 6.6 to 8.0 nm. During the acrosome reaction, the truncated cone elongates simultaneously with the protrusion of the acrosomal process through the truncated cone. As the growth of the acrosomal process further proceeds, the truncated cone transforms into a cylindrical shape and eventually reaches 1 pm in length and 0.2 pm in diameter. The elongated truncated cone is characterized by regularlly helical striations with a periodicity of 19 to 21 nm with an inclination of 40" to 46". These results may suggest that the truncated cone is composed of coiled filaments, which coil up further during the acrosome reaction causing the truncated cone to slenderize and elongate. The elongation is also achieved by stretching of the coil. In H . discus hannai Ino, structural changes in the truncated cone show close homology to those in H . discus. No such morphologically unique organelle has been found in other species thus far.

Since the teleostean egg is enclosed in the chorion which spermatozoa cannot penetrate except the region of the micropyle, polyspermic fertilization never occurs even if the egg is inseminated by dense sperm suspension. As a matter of fact, once the chorion is removed, both herring and salmon eggs receive more than one spermatozoon (YANAGIMACHI, 1957;YAMAMOTO, T. S., 1958;KANOH and T. S. YAMAMOTO, 1957). In the Medaka, Oryzias latipes, the present author found a way to remove the chorion and also succeeded in monospermic fertilization of the naked eggs. The naked embryos can be kept alive for various lengths of time in sterilized medium. These results will be presented in the following pages. (1957) succeeded in removing the chorion from the unfertilized herring egg by mechanical means. KANOH and T . S. YAMAMOTO (1957) and T. S. YAMAMOTO (1958) removed the chorions of the unfertilized eggs of both salmon and herring by a dual treatment with acidulated and pancreatin-containing Ringer. On the other hand, in Oryzias latipes, ISHIDA (1944ISHIDA ( a, b, 1948 reported that the use of pancreatin or trypsin singly dissolves the chorions if left in the solution for more than 20 hours, although he casually mentioned the ultimate dissolution of the chorion by the natural hatching enzyme too. METHODS YANAGIMACHIAs far as the author's experience goes, the methods reported in the first three papers are not effective for oryzias eggs and ISHIDA'S method requires too long a treatment to obtain viable eggs. However, it was noticed that a mixture of the natural hatching enzyme and pancreatin could attack the chorion without affecting the development 357