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Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms. [ABSTRACT FROM AUTHOR]

In spermiogenesis of many diverse organisms, it has long been well known that the nuclear chromatin achieves a high degree of condensation by passing through a series of patterns, which may be granular, fibrillar, or lamellar, but with the sequence always the same within any one species. This raises the question of what mechanism or mechanisms may determine the patterns as they form sequentially. We (LGH) were asked whether a reaction-diffusion (R-D) mechanism might be operating at any stage, with particular reference to some sequences of lamellar patterning, e.g., in the homopteran Philaenus spumarius and the whelk Murex brandaris. The patterns were, at some stages, strikingly similar to ones known to be R-D generated in inanimate chemical systems, except that the interlamellar spacings are 100,000 times greater in the latter! Here, we describe analysis of these patternings suggesting, albeit very tentatively, that the earlier stages seem to demand explanation by action-at-a-distance dynamic patterning, such as R-D, but that there is then a switch to a more crystallization-like phenomenon, which is structural and not dynamically determined. From this analysis, we have predicted where in the patterning sequence the dephosphorylation of the sperm nuclear basic proteins should occur.

Despite their relatively arginine-rich composition, protamines exhibit a high degree of structural variation. Indeed, the primary structure of these histone H1-related sperm nuclear basic proteins (SNBPs) is not random and is the depository of important phylogenetic information. This appears to be the result of their fast rate of evolution driven by positive selection. The way by which the protein variability participates in the transitions that lead to the final highly condensed chromatin organization of spermatozoa at the end of spermiogenesis is not clearly understood. In this paper we focus on the transient chromatin/nucleoplasm patterning that occurs in either a lamellar step or an inversion step during early and mid-spermiogenesis. This takes place in a small subset of protamines in internally fertilizing species of vertebrates, invertebrates and plants. It involves "complex" protamines that are processed, replaced, or undergo side chain modification (such as phosphorylation or disulfide bond formation) during the histone-to-protamine transition. Characteristic features of such patterning, as observed in TEM photomicrographs, include: constancy of the dominant pattern repeat distance λ(m) despite dynamic changes in developmental morphology, bicontinuity of chromatin and nucleoplasm, and chromatin orientation either perpendicular or parallel to the nuclear envelope. This supports the hypothesis that liquid - liquid phase separation by the mechanism of spinodal decomposition may be occurring during spermiogenesis in these species. Spinodal decomposition involves long wave fluctuations of the local concentration with a low energy barrier and thus differs from the mechanism of nucleation and growth that is known to occur during spermiogenesis in internally fertilizing mammals.

The internally fertilizing primitive frog Ascaphus truei (family Ascaphidae) from the Pacific Northwest is the only frog with an intromittent organ. The more advanced neobatrachian frog Eleutherodactylus coqui (family Leptodactylidae) from Puerto Rico has secondarily acquired internal fertilization but mates by cloacal apposition. Nonetheless, both frogs have introsperm with an elongated head containing highly condensed chromatin. Characterization of sperm nuclear basic proteins (SNBPs) in E. coqui by acid-urea polyacrylamide gel electrophoresis indicates that, as in A. truei, testes from a single animal contain several protamines. Amino acid analysis indicates a composition for the most rapidly moving protamine of each species as follows: in E. coqui, ARG (35.6 mol %) + LYS (3.8 mol %) + HIS (7.6 mol %) = 47 mol % total basic residues and in A. truei, ARG (42.1 mol %) + LYS (11.1 mol %) = 53.2 mol % total basic residues. Transmission electron microscopy shows that E. coqui introsperm, like those in A. truei, are elongate with highly condensed chromatin. However, E. coqui introsperm lacks an axial perforatorium that extends into an endonuclear canal. These morphological features are plesiomorphic (primitive) and shared by A. truei with urodeles and basal amniotes (Jamieson et al. (1993) Herpetologica 49:52-65). In E. coqui introsperm, the nucleoprotein complex has a cross-sectional axis of 420 + 20 angstroms and shows a knobby chromatin structural organization in TEM. The presence of arginine-enriched protamines in both a basal anuran like the ascaphid A. truei and a more advanced neobatrachian like the leptodactylid E. coqui supports the hypothesis that internal fertilization acts as a constraint on the range of SNBP diversity in animals., (Copyright 2005 Wiley-Liss, Inc.)

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris., (Copyright 2004 Wiley-Liss, Inc.)