Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Identification and isolation of soluble histones from bovine milk and serum.

An immunoassay for soluble histones as trace components of biological fluids was developed on the basis of the dual capacity of histones to bind solid-phase DNA and monoclonal anti-histone antibody. Application of this histone-capture assay to bovine milk resulted in a positive signal, and DNA-cellulose chromatography was used to isolate histone-like material in microgram quantities. Western-blot analysis using a panel of anti-histone antibodies demonstrated the presence of histones H2A, H2B and H4 in apparently intact form. DNAase digestion experiments indicated that at least a portion of milk histone was complexed to DNA. Bovine serum was analysed in the same manner on serial DNA-cellulose columns, and H4 and partially degraded H2A were detected by Western-blot analysis. The finding of soluble histones in bovine milk and serum may account for unexpected results when these biological fluids are used as blocking reagents in Western blots and other immunoassays and may have ramifications in the origin and significance of anti-histone antibodies in human disease.     

Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

Anti-histone antibodies in idiopathic and drug-induced lupus recognize distinct intrahistone regions

We have identified regions within core histones that are antigenic for autoantibodies in systemic lupus erythematosus (SLE) and drug-induced lupus. An immunoblotting technique was used to determine the reactivity of lupus antibodies for intact histones and for trypsin-resistant histone fragments that lack the amino- and carboxyl-terminal amino acids that are normally exposed in native nucleosomes. In SLE, the predominant anti-histone response was restricted to epitopes in the trypsin-sensitive regions. Of 20 SLE sera that had strong antibody activity for multiple intact histones, 17 showed minimal activity with any of the corresponding trypsin-resistant fragments. A markedly different pattern of reactivity was present in sera of patients with procainamide (Pr)-induced lupus in which antibodies to H2A, H2B, and the H2A-H2B complex had strong fragment activity. Interestingly, recognition of trypsin-resistant fragments was also noted in a small number of SLE sera that contained antibodies to the H2A-H2B complex. In contrast to both SLE and Pr-induced lupus, antibodies induced by hydralazine (Hy) reacted primarily with H3 and H4. Furthermore, these antibodies bound equally well to the corresponding trypsin-resistant regions that are thought to be relatively unexposed in native nucleosomes. Thus, the specificities of anti-histone antibodies in SLE, Pr-induced lupus, and Hy-induced lupus are markedly different, but in each disease reactivity appears to be restricted to a limited number of histone determinants. The data raise the possibility that autoantigen in the form of native nucleosomes may be recognized in SLE and possibly in Pr-induced lupus. In contrast, the propensity of Hy to induce autoantibodies to determinants usually not recognized in SLE or Pr-induced lupus may suggest a different immunogenic stimulus in this disease.     

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.     

Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones

Using high performance liquid chromatography we have successfully purified four core histones from mature human sperm chromatin. The H2A variants present in sperm (H2A.X and limited H2A.Z) have been shown previously to be minor variants in somatic chromatin. The histones are highly modified as evidenced by extensive acetylation and an as yet uncharacterized multicharge modification of H2B. Based on our data, we conclude that histone proteins are a minor component of each mature spermatozoa. Given the unique nature of the histone variants present in sperm, we propose that this chromatin component has a specific function and may possibly facilitate the programming of genes which will be active in early development.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

Characterization of boar sperm cytoskeletal cylicin II as an actin-binding protein

The presence of actin-binding proteins in the perinuclear theca of boar spermatozoa has been investigated, using stepwise extractions of proteins from sperm heads. Proteins extracted with the alkaline buffer 1M Na(2)CO(3), pH 11, were found to contain a 66kDa protein that binds F-actin in actin pelleting assays. Sequence studies and immunological characterization with antibodies specific for human cylicin II identified the 66kDa protein as the homologue of bovine and human cylicin II. Immunocytochemical studies showed the presence of porcine cylicin II in the acrosomal region of round spermatids and in the postacrosomal region of late spermatids and spermatozoa, in agreement with the previously described localization of cylicins. Taken together, the results suggest that cylicin II, a protein of the sperm perinuclear cytoskeleton, is a novel actin-binding protein, which probably plays a role in the actin-related events that occur during spermiogenesis and the early events of fertilization.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Replacement of nucleosomal histones by histone H1-like proteins during spermiogenesis in Cnidaria: Evolutionary implications

We have analyzed the chromosomal protein composition of the sperm from several species belonging to three different classes (Hydrozoa, Scyphozoa, Anthozoa) of the phylum Cnidaria. In every instance, the sperm nuclear basic proteins (SNBPs) were found to consist of one to two major protein fractions that belong to the histone H1 family, as can be deduced from their amino acid composition and solubility in dilute perchloric acid, and the presence of a trypsin-resistant core. In those species where mature spawned sperm could be obtained, we were able to show that these proteins completely replace the somatic histones from the stem cells that are present at the onset of spermatogenesis. The presence of a highly specialized histone H1 molecule in the sperm of this phylum provides support for the idea that the protamine-like proteins (PL) from higher groups in the phylogenetic tree (and possibly protamines as well) may all have evolved from a primitive histone H1 ancestor.     

Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis.

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking.     

Nucleosomal structure as probed by H3 histone thiol reactivity. Conformation of H3 histone variants is differently affected by thiol group reagents

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available to N-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking. Copyright 2001 Academic Press.     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.     

Nucleosomal structure as probed by H3 histone thiol reactivity

Two H3 histone variants are found in equal amount in HeLa cells, and they have been characterized by two-dimensional gel electrophoresis followed by reaction with specific antibodies. These molecules are the only cysteine-containing histones, and they have been used as the target for thiol-specific reagents, in intact nuclei, isolated nucleosomes, histone complexes, and purified histones. Cysteine residues are available toN-ethylmaleimide only when histones are disassembled from the core particles. Upon reaction with these reagents, one of the H3 variants undergoes profound conformational changes, as revealed by an altered electrophoretic mobility.