Lysis of photographic emulsions by mammalian and chicken spermatozoa.

Abstract

Gaddum & Blandau (1970) studied the proteolytic activity of individual mammalian spermatozoa by directly observing the dissolution of pure gelatin membranes which had been blackened by the inclusion of India ink and fixed with glutaraldehyde. Their observations showed that lytic enzymes were released from the acrosomal region of the sperm head, but not from other parts of the cell. Gaddum & Blandau's technique has two important practical disadvantages. Firstly, the `home-made' gelatin membranes used are tedious to prepare and variable in thickness, and, secondly, the incorporation of India ink obscures the images of the spermatozoa. To counter the second of these disadvantages, Ben\l=i'\tez-Bribiesca& Ve\l=a'\zques-Meza(1972) used Ponceau stain instead of India ink, and Gaddum-Rosse & Blandau (1972) used clear unstained membranes. To eliminate both kinds of disadvantage, we have explored the use of commercial photographic emulsions as a source ofgelatin substrate, a technique which has been used for many years to study the enzymatic activity of tissue slices (Gordon, Levin & Whitehead, 1952; Adams, Fernand & Schneiden, 1958; Adams & Tuquan, 1961; Kirchner, 1972). Our first observations were made with blackened photographic plates, such as Kodak P300. After exposure to light, these plates were processed in the usual way and, after washing, were fixed in 0-05 % glutaraldehyde in veronal-acetate buffer (pH 8-2) for 2 min and then rinsed in buffer and distilled water. Washed spermatozoa, suspended in a mixture of Hanks' BSS medium (B.D.H.) and veronal acetate buffer, were spread onto these plates and incubated at 37° C in a humid atmosphere. The results obtained were essentially the same as those described by Gaddum & Blandau (1970) but with less variability. The areas of depolymerization were very clear, but details of the reacting spermatozoa remained obscured by the blackened emulsions. Very much better images were then obtained by using colour film, such as Kodachrome II, exposed and developed to produce a light red or light blue background. These films are, however, relatively expensive and celluloid film is much less convenient to use than glass plate, which can be cut to the size of a microscope slide and remains solid and flat without additional mounting. We therefore returned to the use of black and white photographic plates but fixed them with Kodafix without exposure either to light or chemical development. These plates are easy to prepare and have given excellent results. Their gelatin coats are transparent,