CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.     

Hormonal control of meiosis reinitation in starfish oocytes. New evidence for the absence of efficient intracellular receptors for l-methyladenine recognition.

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

Fractionation of cricket testis nuclei on gradients of colloidal silica for study of basic protein changes during spermiogenesis

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

Protamines in plant sperm

Sperm protamines have been isolated from representatives of three major plant groups: algae (Chara corallina ), bryophytes ( Marchantia polymorpha), and ferns ( Marsilea vestitia ). We previously reported the complete displacement of histones by protamines in Marchantia (Reynolds W F & Wolfe, S L, Exp cell res 116 (1978) 269 [8] ). Marchantia protamines appear as four components on acid-urea gels, whereas Chara and Marsilea protamines comigrate as a single band with a mobility comparable to salmon protamine. The amino acid compositions of the plant protamines show these to be arginine-rich, highly basic (35-42%) proteins which display overall similarity in amino acid composition (84-91%). The molecular weights of Chara and Marsilea protamines are approx. 4700-5300 D.     

Identification and characterization of the packaging proteins of core 40S hnRNP particles

The proteins involved in protein-RNA and protein-protein interactions to form the core structure of nuclear 40S hnRNP particles in HeLa cells have been identified and characterized. Through complete analysis of nuclear extracts on sucrose density gradients and controlled salt dissociation of particle proteins, six lower molecular weight polypeptides are identified as the protein constituents of the 40S ribonucleoprotein complex which appears in the electron microscope as 210 A spherical particles. 40S hnRNP particles isolated from Chinese hamster lung fibroblasts show a strikingly similar protein composition to the human cells. The proteins are specifically associated with rapidly labeled nonribosomal nuclear RNA. Particle proteins from HeLa cells migrate in polyacrylamide gels as three groups of closely spaced doublets (groups A, B and C) and are present in a simple fixed stoichiometry. The group C proteins (C₁ and C₂ of 42,000 and 44,000 daltons) interact directly with RNA to form a smaller high salt-resistant RNP complex. The group A proteins (A₁ and A₂ of 32,000 and 34,000 daltons) are major nuclear proteins and constitute 60% total particle protein mass. These two proteins are basic with isoelectric points near 9.2 and 8.4, respectively, and are characterized by an unusual amino acid composition, including high glycine (25%) and the unusual modified basic residue identified as N^G,N^G-dimethylarginine. The major particle proteins (A₁ and A₂) interact electrostatically with nucleic acids and apparently function structurally in the packaging and stabilization of hnRNA in a manner analogous to the histones in chromatin υ bodies. The similarity in protein composition of core RNP particles from different cell types (especially in the basic proteins, A₁, A₂ and B₁) is consistent with a conserved particle structure and function in eucaryotes.     

Arginine-rich nucleoprotein transition occurs in the two size classes of spermatozoa of Drosophila subobscura males

The normal male of Drosophila subobscura displays polymegaly, which is the presence of two sizes of spermatozoa in the same testis. It is still unknown whether both kinds of sperm are able to fertilize the egg. An indicator of normal functioning of Drosophila spermatozoa is the replacement of the somatic histones by sperm-specific arginine-rich nucleoproteins during spermiogenesis. The appearance of these arginine-rich nucleoproteins in the two kinds of sperm was investigated using the fluorescent dye sulfoflavine, which stains basic proteins at pH 8. In the spherical nuclei of early spermatids of Drosophila subobscura the somatic histones fluoresced strongly, but fluorescence could not be detected in later stages when the spermatid nuclei were elongating. After elongation, however, the nuclei of both kinds of sperm, long and short, fluoresced brightly again, due to the presence of sperm-specific arginine-rich nucleoproteins. Half of the cysts of both types contained spermatid nuclei with aberrant fluorescent pattern including 5–9% of both cyst types which do not undergo histone transition at all. These results indicate that both sperm types may be functional.     

Dynamic aspects of spermiogenic chromatin condensation patterning by phase separation during the histone-to-protamine transition in charalean algae and relation to bryophytes

During early-to-middle spermiogenesis in multicellular, internally fertilizing charalean green algae (Chara fibrosa, Chara vulgaris, Chara tomentosa, Nitella missouriensis), patterning of chromatin/nucleoplasm in developing spermatid nuclei changes from granules → fibers → contorted lamellae → condensed chromatin. Cytochemical, immunocytochemical, electrophoretic studies on C. vulgaris and C. tomentosa spermatids (Kwiatkowska, Poplonska) and amino acid analysis of protamines in Chara corallina sperm (Reynolds, Wolfe), indicate that more positively charged protamines replace histones directly during spermiogenesis, not indirectly through other intermediate transitional proteins as in internally fertilizing neogastropods and sharks with more ordered spermatid lamellae. We hypothesize that such lamellar-mediated patterning is due to liquid–liquid phase separation by spinodal decomposition. This is a spontaneous thermodynamic process that involves diffusive instability of a lamellar chromatin network, a dominant pattern repeat distance and bicontinuity of chromatin/nucleoplasm phases. C. vulgaris sperm show contorted lamellae in the posterior region, whereas C. corallina sperm display contorted peripheral lamellae and interior fibrils. Among internally fertilizing liverworts, which may have evolved from Zygnematales, mid-spermatid nuclei lack lamellae. Instead they display self-coiled chromatin rods in Blasia pusilla, contain short chromatin tubules in Haplomitrium hookeri resembling those in internally fertilizing mosses and a hornwort and indirectly replace histones with protamines in Marchantia polymorpha.     

Purification and characterization of two basic spermatid-specific proteins isolated from the dog-fish Scylliorhinus caniculus

In dog-fish spermatid nuclei two intermediate proteins S1 and S2 replace histones before the setting down of protamines. These spermatid-specific proteins were isolated by carboxymethyl-cellulose chromatography and purified by high pressure liquid chromatography. S1 and S2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N- and C-terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.     

Protamines and related proteins from spermatozoa of molluscs. Characterization and molecular weight determination by gel electrophoresis

The spermatozoa of most species of molluscs contain a mixture of proteins with compositions related to those of histones and protamines. The various components present in the spermatozoa of Cryptochiton stellerii, Donax trunculus, Mactra corallina and Gibbula divaricata have been isolated and characterized. The results obtained, taken together with those previously found (Subirana, J.A., Cozcolluela, C., Palau, J. and Unzeta, M. (1973) biochim. Biophys. Acta 317, 364--379), show that in all the molluscs studied one or more arginine-rich components are present. The molecular weight of these proteins varies vary much in different species and is usually much greater than in conventional fish protamines. Conventional histones, as well as lysine-rich proteins of low molecular weight, have also been found in ripe spermatozoa of several species. The molecular weights have been estimated by gel electrophoresis, using polymerized iridine as a standard.     

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, <Emphasis Type="Italic">Bandicota indica</Emphasis>

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.     

An analysis of the influence of age and school-attendance status on the spread of variola minor.

Definitions are proposed for the independent and joint contributions that the chemical groups A and B make to the free energy of association of the ligand A?B with a receptor. The definitions are independent of the choice of the standard state and are consistent with the basic thermodynamic cycle relating the association of the ligands A?B, A?Y and X?B to the receptor Rappaport 1976. The basic idea is the use of the excess free energy of association of the ligand A?Y over the free energy of association of the reference ligand X?Y as the measure of the “independent” contribution of the group A to the binding. This definition allows the free energy of association of the ligand A?B to be written as the sum of the independent contributions of the groups A and B, their joint contribution, and an invariant free energy of association of the reference ligand with any receptor. With the appropriate definition of the receptor-reference ligand complex, water can be chosen as the reference ligand. Using ΔG(A?OH)?AG(HOH), ΔG(H?B?H)?ΔG(HOH) and ΔG(HO?C)?ΔG(HOH) as the definitions of the “independent” contributions of the chemical groups A, B and C to the binding of the ligand A?B?C, the joint contribution of the groups A and C to the binding is ΔG(A?B?C) ? ΔG(A?B?H) ? ΔG(H-B-C) + ΔG(H?B?H).     

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus . Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.     

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab,Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.     

A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization

Protamine-like proteins constitute a group of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1 family). Like protamines, they usually replace the chromatin somatic histone complement during spermiogenesis; hence their name. Several of these proteins have been characterized to date in invertebrate organisms, but information about their occurrence and characterization in vertebrates is still lacking. In this sense, the genus Mullus is unique, as it is the only known vertebrate that has its sperm chromatin organized by virtually only protamine-like proteins. We show that the sperm chromatin of this organism is organized by two type I protamine-like proteins (PL-I), and we characterize the major protamine-like component of the fish Mullus surmuletus (striped red mullet). The native chromatin structure resulting from the association of these proteins with DNA was studied by micrococcal nuclease digestion as well as electron microscopy and X-ray diffraction. It is shown that the PL-I proteins organize chromatin in parallel DNA bundles of different thickness in a quite distinct arrangement that is reminiscent of the chromatin organization of those organisms that contain protamines (but not histones) in their sperm.     

Proteolysis of somatic type histones in transforming rat spermatid chromatin

Elongated rat spermatid nuclei have been isolated on the basis of their resistance to sonication in 0.32 M sucrose containing 1.5 mM CaCl₂. Chemical analyses indicate that approx. 35% of the DNA in these nuclei is associated with somatic type histones, while the remainder represents sperm histone-DNA complex. In contrast to nuclei of somatic cells, when elongated spermatid nuclei are incubated under appropriate conditions, somatic type histones but not sperm histone are rapidly degraded. Differential extraction of elongated spermatid nuclei with 5 mM HCl and then with various concentrations of NaCl followed by 0.2 M HCl has revealed that they contain two kinds of proteases. The protease in the 5 mM HCl extract is acrosin (EC 4.3.21.10). Rapid degradation of somatic type histones is, however, observable upon incubation of elongated spermatid nuclei which have been treated with 5 mM HCl and are therefore free of acrosin or upon incubation of elongated spermatid chromatin where the majority of acrosin is removed, suggesting that the observed proteolysis of somatic type histone is not due to acrosin. Proteases which may represent the enzymes responsible for the histone degradation are extractable from acrosin-free spermatid nuclei with NaCl (0.9 M) and by subsequent treatment of the salt-extracted nuclei with 0.2 M HCl. The proteases in the NaCl and the 0.2 M HCl extract possess identical properties and appear to be the same enyzyme which may exist in spermatid chromatin in two different forms.     

Persistence of protamine precursors in mature sperm nuclei of the mouse

During mouse spermiogenesis, two protamines, mP1 and mP2, are synthesized in replacement of histones. One of them (protamine mP2, 63 residues) appears at first in elongating spermatid nuclei as a pro-protamine of 106 residues (pmP2) with an amino-terminal extension that is progressively excised. The two protamines were previously described as the only proteins associated with DNA in sperm chromatin. This paper shows that the nuclear proteins of mouse spermatozoa are indeed heterogeneous: at least six minor polypeptides in addition to protamines can be identified. The primary structure of four of them has been established. They are intermediate in the maturation of the precursor of protamine mP2 and correspond to polypeptides pmP2/11, pmP2/16, pmP2/20, and pmP2/32, characterized previously in mouse testis. Therefore, these intermediates of proteolysis generated from pmP2 inside spermatid nuclei persist in mature sperm, whereas the largest precursors, pmP2 and pmP2/5, disappear. These findings clearly indicate that limited proteolysis events still occur outside of the testis. © 1995 Wiley-Liss, Inc.     

Nature et évolution des protéines basiques au cours de la spermiogenèse chez Pleurodeles waltlii Michah., Amphibien urodèle

Résumé La spermatogenèse du Triton Pleurodeles waltlii a été étudiée au moyen de techniques cytochimiques et autoradiographiques. Les phénomènes spermiogénétiques ont été divisés en six stades caractéristiques en fonction de la forme du noyau et de l'évolution des structures extranucléaires: cou et filament de soutien de la queue.Les protéines basiques du noyau du spermatozoïde sont du type protamine, tandis que certaines structures extranucléaires se montrent riches en protéines basiques libres. Les transitions depuis les histones de type somatique présentes dans les jeunes spermatides I et II jusqu'aux protamines du spermatozoïde mûr par l'intermédiaire d'histones enrichies en arginine, montrent des phénomènes intéressants.La première transition depuis les histones de type somatique riches en lysine vers les histones enrichies en arginine est progressive. Elle se produit au cours des stades III et IV. Un gradient apparaît dans le noyau depuis la base vers le sommet. Au même moment, les protéines basiques libres apparaissent dans le cou.La perte des histones enrichies en arginine puis leur substitution par des protamines au niveau des noyaux des spermatides V est beaucoup plus brutale, elle intéresse tout le noyau. Ce dernier montre de même une disparition complète des acides aminés soufrés. Au même moment, les protéines basiques apparaissent dans la queue, de même que des acides aminés soufrés.Toutes ces observations plaident en faveur d'une origine nucléaire possible de certains composants de ces structures extranucléaires originales et riches en protéines basiques libres.

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Nature and development of the basic proteins during the spermiogenesis in Pleurodeles waltlii michah., urodele amphibian

Summary The spermiogenesis of the newt Pleurodeles waltlii has been studied with cytochemical and autoradiographical methods. The process of spermiogenesis has been divided into six characteristic steps in relation with the nuclear shape and the evolution of the extranuclear structures: the neck and the supporting filament of the tail.The nuclear basic proteins of the spermatozoon are of protamine type while the well developed extra-nuclear structures show a free basic proteins rich content. The transition from early spermatids somatic type histone to the mature sperm protamine via arginine-rich histone and the appearance of the free basic proteins in the tail show different interesting features. The first transition from the somatic rich lysine histone to the arginine rich histone which occurs at the spermatid III and IV steps, is gradual. A gradient appears in the nuclei from the base to the apex. Concurrently the free basic proteins appear in the neck. The loss of the arginine rich histone and its substitution by protamine in the spermatid V is more sudden and affects all the nuclei without a gradient, the sulfhydryl group content of the nuclei also disappear. At the same time, the basic protein of the tail appear as well as the sulfhydryl group.All these results are discussed in view of the hypothetical nuclear origin of these very unusual extranuclear basic proteins.

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Equipe de Recherche associée au C.N.R.S. Cytologie Ultrastructurale No. 129.

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Avec la collaboration technique de Melle F. de Sallier Dupin et de M. B. Morille.     

Nuclear proteins in spermatogenesis

Mammalian somatic type histone variants are replaced or supplemented in early primary spermatocytes and possibly spermatogonia by testis specific and testis enriched histone variants. The testis complement of histones is replaced entirely by transition basic proteins in mid-spermatids. This transition is accompanied by a dramatic reduction of thermal stability of mid-spermatid chromatin which may be due in part to hyperacetylation of histone H4. The transition basic proteins are replaced by protamines which are arginine-rich and contain cysteine.