Basic nuclear proteins of transition during spermiogenesis in Scylliorhinus caniculus

During dog-fish spermiogenesis, 2 basic nuclear protein transitions occur: the first from histones to spermatid-specific proteins S1 and S2, the second leading to protamines. S1, the most abundant transition protein, is a polypeptide containing 87 residues (Mr = 11,179 Da) whereas S2, the minor transition protein, contains 80 residues (Mr = 9,726 Da). The 2 proteins are mainly characterized by an asymmetry of the molecule, a very high content of basic residues, a relatively high level of hydrophobic residues and a cluster of acidic residues in the carboxy-terminal quarter of the molecule. The 2 proteins are phosphorylated on serine residues and the degree of phosphorylation is relatively important in protein S1. The 2 transition proteins are structurally unrelated to testis histones or sperm protamines and cannot be considered either as their proteolytic degradation products or as their precursors.     

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.     

Nuclear basic protein transition during sperm differentiation. Primary structure of the spermatid-specific protein S2 from the dog-fish Scylliorhinus caniculus

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.     

Protamine precursors in human spermatozoa

Basic proteins isolated from human sperm nuclei are highly heterogeneous. Three groups of nuclear basic proteins have been characterized: somatic-type as well as testis-specific histones, protamines and basic proteins with an electrophoretic mobility which is intermediate between that of histones and that of protamines. Human protamines can be separated into 2 protein families with different amino acid composition and amino-acid sequence. Protamines HP1 differ in their degree of phosphorylation. Protamines HP2, 3 and 4 differ by their amino-terminal sequence. Intermediate basic proteins (HPI1, HPI2, HPS1, HPS2) share a common C-terminal sequence of 54 residues identical to the amino-acid sequence of protamine HP3; only their N-terminal regions are different. Taking into account these structural homologies, the intermediate basic protein HPI1 appears as a precursor of protamines HP2 and HP3.     

Basic homopolyamino acids, histones and protamines are potent antagonists of angiogenin binding to ribonuclease inhibitor

A radio-ribonuclease inhibitor assay based on the interaction of 125I-angiogenin with ribonuclease inhibitor (RI) was used to detect pancreatic-type ribonucleases and potential modulators of their action. We show that highly basic proteins including the homopolypeptides poly-arginine, poly-lysine and poly-ornithine, core histones, spermatid-specific S1 protein and the protamines HP3 and Z3 were strong inhibitors of angiogenin binding to RI. A minimum size of poly-arginine and poly-lysine was required for efficient inhibition. The inhibition likely resulted from direct association of the basic proteins with the acidic inhibitor, as RI bound to poly-lysine and protamines while 125I-angiogenin did not. Antagonists of the angiogenin-RI interaction are potential regulators of either angiogenin-triggered angiogenesis and/or intracellular RI function, depending on their preferential target.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.     

Purification and characterization of nuclear basic proteins of human sperm

High purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins as in fact the same protein with different degrees of phophorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.     

Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids

The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths.     

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.     

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.     

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.     

Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain

Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.     

Common phylogenetic origin of protamine-like (PL) proteins and histone H1: Evidence from bivalve PL genes

Sperm nuclear basic proteins (SNBPs) can be grouped into three main categories: histone (H) type, protamine (P) type, and protamine-like (PL) type. Protamine-like SNBPs represent the most structurally heterogeneous group, consisting of basic proteins which are rich in both lysine and arginine amino acids. The PL proteins replace most of the histones during spermiogenesis but to a lesser extent than the proteins of the P type. In most instances, PLs coexist in the mature sperm with a full histone complement. The replacement of histones by protamines in the mature sperm is a characteristic feature presented by those taxa located at the uppermost evolutionary branches of protostome and deuterostome evolution, while the histone type of SNBPs is predominantly found in the sperm of taxa which arose early in metazoan evolution; giving rise to the hypothesis that protamines may have evolved through a PL type intermediate from a primitive histone ancestor. The structural similarities observed between PL and H1 proteins, which were first described in bivalve molluscs, provide a unique insight into the evolutionary mechanisms underlying SNBP evolution. Although the evolution of SNBPs has been exhaustively analyzed in the last 10 years, the origin of PLs in relation to the evolution of the histone H1 family still remains obscure. In this work, we present the first complete gene sequence for two of these genes (PL-III and PL-II/PL-IV) in the mussel Mytilus and analyze the protein evolution of histone H1 and SNBPs, and we provide evidence that indicates that H1 histones and PLs are the direct descendants of an ancient group of "orphon" H1 replication-dependent histones which were excluded to solitary genomic regions as early in metazoan evolution as before the differentiation of bilaterians. While the replication-independent H1 lineage evolved following a birth-and-death process, the SNBP lineage has been subject to a purifying process that shifted toward adaptive selection at the time of the differentiation of arginine-rich Ps.     

Comparison of the high-mobility-group chromosomal proteins in rainbow-trout (Salmo gairdnerii) liver and testis.

Chromatography and characterization of the proteins extracted by 5% (w/v) HClO4 from rainbow-trout (Salmo gairdnerii) liver and testis show that the two tissues present a characteristically different spectrum of high-mobility-group (HMG) proteins. A variant subfraction of HMG C is found in liver, but is not detectable in testis, where even the main fraction of HMG C is present in only very low quantity. A protein, F, which appears to be related to protein H6 has similarly been isolated only from liver and not from testis. Quantification of the HMG proteins in total 5%-HClO4 extracts of trout liver and testis nuclei shows that, in relation to DNA, levels of HMG T1 and T2, and D are more than 2-fold, and C, 20-fold higher in liver than in testis. However, these differences do not result merely from the sequential withdrawal of HMG proteins at the same time that histones are replaced by protamines in the developing spermatid, since in testis, at some stages of maturation, levels of H6 are almost 2-fold higher than in liver. The implications of these findings for the function of HMG proteins are discussed.     

Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein

The manchette, which is the structure that appears around the nuclei of elongated spermatids, is assumed to be involved in nuclear shaping during spermiogenesis and the transport of various proteins between the nucleus and sperm tail. In this report, we describe the molecular cloning and characterization of a mouse spermatid-specific manchette-related protein 1 (Smrp1) from a spermatid-specific subtracted mouse testis cDNA library. The isolated Smrp1 cDNA clones could be divided into three variants based on sequence analysis. Computer-assisted analysis showed that these variants were splice variants from a single locus of the mouse genome. The three putative proteins consisted of 296, 260, and 175 amino acids, respectively. Although 155 amino acids of the N terminus were common to the three proteins, they were distinguished by their C-terminal regions. Western blot analyses using specific antisera showed that SMRP1 expression was specific to the testes and that only the 261-amino-acid form was translated into protein. Immunohistochemistry revealed that SMRP1 was localized to the cytoplasm of step 9-12 elongated spermatids. The protein appeared in a cap formation that covered the caudal sides of the elongated nuclei. This localization pattern coincided with that of the manchette. SMRP1 may play an important role as a functional protein that co-operates with manchette proteins.     

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.     

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.