Isolation and Characterization of Chromatin and Dna from the Sperm of the Spider Crab, Libinia Emarginata

Abstract

An isolation technique is described which yields highly purified sperm from various regions of the reproductive tract of the crab Libinia emarginata. Sperm chromatin was isolated and analysed with respect to ultrastructure, ultraviolet absorption spectrum, thermal melting profile, behaviour in CsCl gradients, and chemical composition. Sperm-isolated chromatin not stabilized with uranyl acetate prior to ethanol dehydration, like prokaryotic chromatin, has aggregated fibres with a diameter in the range 30-95 nm or more. With uranyl acetate stabilization, only very fine fibres are seen. Pronase treatment of the thick fibres leads to formation of very fine fibres, showing that protein is associated with this isolated chromatin. Polyacrylamide electrophoretic analysis of the chromatin protein shows that it is not basic. Standard basic protein staining of sperm taken from testes, vas deferens, and seminal receptacles after various periods of storage there gives a negative nuclear reaction in all cases. This suggests the absence of sperm nuclear histones and protamines. Sperm chromatin protein is salt-dissociable and has a ratio of basic to acidic amino acids of 0.43-0.48 The protein/DNA ratio is 2.3-3.2 and the ratio E280/E260 is 0.59-0.67 Thermal denaturation curves are identical for sperm chromatin and purified sperm DNA, supporting the other data showing lack of nuclear basic proteins. An (A + T)-rich satellite DNA component comprises about 7-9 % of the total sperm genome. It is suggested that the genes which specify gamete-type nuclear basic proteins are either permanently repressed or deleted in decapod crustacea.