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Previous studies have established that chromatin fibers which contain histones have variable diameters in different biological materials. In this paper we show that chromatin fibers are also present in spermatozoa and spermatids of bivalve molluses in which some or all histones have been replaced by protamines. In all cases thick chromatin fibers are formed, with a diameter in the range 25-50 nm, depending on the species. We conclude that the formation of chromatin fibers is determined by the partial neutralization of the DNA charges with any histone or protamine, rather than due to a precise association of nucleosomes. This conclusion is in agreement with current theoretical work which shows that the diameter of complexes of DNA with counterions is determined by an overall balance of the interaction forces present.

Nuclei of spermatozoa of the sea cucumber Holothuria tubulosa contain the five somatic-type histones plus a sperm-specific histone H1 and a unique basic protein ϑ0, which is related to H1 in amino acid composition. No proteins of the High Mobility Group (HMG) type have been detected. The structure of this chromatin has been probed by nuclease digestion. Its behaviour is anomalous, since two distinct fractions of chromatin are recovered from these spermatozoa, which differ either in the presence or absence of the sperm-specific proteins H1 and ϑ0. This heterogeneous distribution is not found in conventional materials, such as calf thymus or chicken erythrocytes. Proteins H1 and ϑ0 are not uniformly distributed and may be localized in special regions of chromatin. Fragments containing long stretches of nucleosomes lacking both proteins can be recovered. At the same time, the chromatin fractions which contain these two proteins are shown to be less soluble. When an extensive digestion of chromatin is carried out yielding only nucleosomes and small oligomers, the H1 and ϑ0 proteins redistribute themselves on chromatin, the two proteins acting in a cooperative fashion in this process. Cross-linking experiments carried out in whole cells indicate a proximity of ϑ0 and H1, whereas no crosslinks have been detected between ϑ0 and any of the four nucleosomal histones. The ϑ0 protein may thus play a role similar to histone H1 and be only loosely associated with nucleosomal histones, but contribute to the structuration of chromatin during spermiogenesis.

It is shown that spermatozoa from the marine snails Gibbula divaricata and Haliotis tuberculata contain a protamine plus a histone component. The composition of the latter is similar to H2 b histone from calf thymus.

The nuclei of sperm of the mussel Mytilus edulis contain three specific proteins: (1) a major protamine-like component (φ1); (2) a lysine-rich protein (φ3); and (3) a histone-like component (φ2b). The latter is characterized here for the first time. It is also shown that the φ3 protein does not contain modified lysines, but has some phosphorylated serines. Nuclease digestion of whole nuclei gives a continuous distribution of sizes for DNA, without any evidence of a nucleosome structure. X-ray diffraction indicates that DNA is organized as bundles of parallel molecules without any signs of either crystallinity or higher order structure.

The acid-soluble proteins from the sperm of several species of echinoderms are described. All of them are analogous to calf thymus histones, as judged by their amino acid composition, although some minor differences appear. The electrophoretic pattern is very similar for organisms belonging to the same class, but it differs among the four classes of echinoderms studied. Three to five bands appear on polyacrylamide gel electrophoresis. Two of them (γ and δ) are present and have similar mobilities for all the species studied and for calf thymus, but their relative intensity varies. On the other hand, the additional bands differ in mobility among the four classes studied.

Histone fractions have been obtained from the haemal system and male gonads of the echinoderm Holothuria tubulosa . Fractionation was achieved by the methods of Johns [5], although some anomalies were found. The fractions were purified by precipitation in adequate solvent systems. The histones obtained are similar to those found in calf thymus, although f 1, and to a lesser extent f 2 b , show significant differences. In the gonads an additional minor component with high electrophoretic mobility is found. The other histone fractions appear to be identical with those obtained from somatic tissues. The relevance of this result towards an understanding of the role played by nuclear proteins in the sperm is discussed. Some of the acidic proteins present in the nuclei of these tissues are also preliminarily characterized.

Histones in chromatin from germ cells of the echinoderm Holothuria tubulosa are retained throughout spermatogenesis. However, some alterations occur in the histone complement of the mature sperm, including the presence of a germ-line-specific H1 subcomponent unusually rich in arginine, and the appearance of a basic component termed φ 0 . Histones from ripe sperm have been extracted in a preparative scale to allow for isolation and purification of protein φ 0 . Polyclonal antibodies against φ 0 have been produced and purified by affinity chromatography. The specificity of the antibodies to φ 0 has been assessed by enzyme-linked immunosorbent assays, competition experiments, and Western immunoblotting analysis. No cross-reactivity of the antibodies with the remainder histone fractions has been observed. Immunocytolocalization of protein φ 0 by immunogold labeling has revealed that this protein is essentially confined to chromatin from ripe sperm, whereas it is wholly absent from less advanced germ cell types. From these observations, together with biochemical studies previously reported, it is inferred that protein φ 0 may well be instrumental in the known chromatin transitions occurrring in this organism during germ cell development.

The structural organization of the histone-containing spermatozoa of the goldfish C. auratus is studied. Chemical fractionation shows that the five standard histones are present. Histone H1 subfractions are also similar to those found in other organisms. This is the first organism studied in which no peculiar basic protein is found in spermatozoa, since in all the organisms described up to now, unique protein components are always present, either alone or accompanied by somatic-like histones. Ultrastructural studies show that this chromatin is organized as a bundle of fibers of about 25 nm diameter, which upon spreading give the typical ‘beads-on-a-string’ appearance. Nuclease digestion demonstrates a repeat length of 205 base pairs (bp), slightly longer than in somatic tissues, but shorter than in echinoderm spermatozoa.