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Sperm chromatin of Murex brandaris (a neogastropod mollusc) undergoes a series of structural transitions during spermiogenesis. The DNA-interacting proteins responsible for these changes as well as the mature protamines present in the ripe sperm nucleus have been characterized. The results reveal that spermiogenic nuclear proteins are protamine precursors that are subjected to a substantial number of small N-terminal deletions that gradually modify their overall charge. The composition of mature protamines is remarkably simple in turn, promoting an efficient and extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that corresponding to vertebrate chromatin.The current model for the nuclear changes occurring in spermiogenesis has been essentially drawn from studies on bony fishes and other vertebrates (1-5). The model considers that in early spermatids, histones become acetylated preceding their replacement by a highly basic protein (protamine). During histone displacement, the protamine is found polyphosphorylated, although it undergoes progressive dephosphorylation in the subsequent spermiogenic development. The processes of protamine phosphorylation and dephosphorylation imply a mechanism regulating the interaction with DNA. This mechanism allows for the orderly substitution of protamine for the nucleohistones and for the ensuing binding to DNA. The protamine is a small molecule displaying a very high positive charge density (6, 7). Consequently, the nuclear replacement of histones by protamine induces profound transitions in chromatin structure directly leading to the compactness of the sperm nucleus.Although these events are well established for some bony fishes and related vertebrates, there is not a universal pattern, or even a "more frequent" model, to account for spermiogenic processes. Evolution has generated an enormous diversity of both DNA-condensing proteins (8 -10) and structural conformations for spermiogenic and/or sperm chromatin (11, 12).Traditionally, electron microscopy studies have focused on cenogastropod molluscs due to the complexity of their chromatin condensation patterns (13-16). In this regard, Murex brandaris is a paradigmatic species (17). Chromatin in the early haploid spermatid displays a somatic-like appearance, which immediately undergoes a peripheral migration, giving rise gradually to granular and fibrilar structures and finally to 18-nm lamellae (18). The nascent lamellar structures are first seen in a disordered arrangement but later become progressively ordered in a regular concentric pattern surrounding the nuclear axis.The progression of structural changes in chromatin should be paralleled by simultaneous modifications of DNA-protein interactions, concurrently with thermodynamic restrictions on the size of the DNA-protein complexes (19,20). It was found in a previous study (18) that the histone complement of the immature gonads of M. brandaris becomes replaced by a large set of intermediate proteins that...

The amino acid sequences of two cuttlefish protamine variants Spl and Sp2 have been established from automated sequence analysis and mass spectrometry data. Spl (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Spl; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.In invertebrates, the nuclear basic proteins associated with DNA in mature sperm cells show a great variability, according to the degree of evolution of the concerned species. In echinoderms, for example, histones persist in spermatozoa [l -31. In other species, such as annelids [4] and molluscs [5 -81, histones together with protamine-like proteins are present in spermatozoa. A third situation can be found in more evoluted species as cephalopods : histones are completely replaced by arginine-rich proteins similar to protamines observed in teleost fishes [9].In a previous work [lo], we reported the characterization of a typical protamine from mature cuttlefish spermatozoa. This protamine, called protein Sp, already appears in elongated spermatids in a phosphorylated form [I 11, whereas it is found dephosphorylated in mature sperm cells.Here, we present the amino acid sequence of protein Sp, which is constituted of two different structural variants Spl and Sp2. MATERIALS AND METHODS MaterialsMature male cuttlefishes Sepia officinalis were collected in May at the Station Marine de Wimereux (France). Spermatophores were excised and spermatozoa were immediatly frozen in liquid nitrogen and stored at -80 "C until use.Correspondence to P. Sautiere, URA 409 CNRS, Universitk de Lille 11, Institut de Recherches sur le Cancer, Place de Verdun, F-59045 LILLE CCdex, FranceAbbreviations. Sp, cuttlefish sperm protamine; FAB-MS, fastatom-bombardment mass spectrometry; '"Cf PD-MS, 252Cf plasma-desorption mass spectrometry; ES-MS, electrospray mass spectrometry.Enzymes. Chymotrypsin (EC 3.4.21 .l); pancreatic elastase (EC 3.4.21.36); Astacusfluviatilis protease (EC 3.4.99.6).Note. The novel amino acid sequence data published here have been deposited with the EMBL sequence data bank. Purijication and fractionation of protein SpNuclear basic proteins were extracted from sperm nuclei and the protamine (protein Sp) subsequently purified as described previously [lo].The separation of protamine variants was performed in some experiments by reverse-phase HPLC on a CI8 pBondapak column (Waters Associates) eluted with a gradient of 0 -50% acetonitrile in 0.05% heptafluorobutyric acid. In other experiments, the fractionation was assayed by ion-exchange HPLC on a Spherogel TSK...