Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.     

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.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

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.     

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.     

Determinants of sperm nuclear shaping in the genus Xenopus

The morphogenesis of sperm nuclei was investigated in six different species or subspecies of the genus Xenopus (Pipidae, Anura). The sequence of nuclear morphogenesis was similar in each species used in this study. Electrophoretic comparison of the basic chromatin proteins from late spermatids and sperm of each species demonstrated that the complements of histones and spermatid-sperm-specific basic proteins were extremely diverse suggesting that shape was not determined by specific basic proteins or mechanisms of histone removal. This conclusion was reinforced by the observation that Xenopus sperm DNA decondensed by 2.0 M NaCl remained contrained in residual structures which resembled intact sperm nuclei. These observations suggested that morphogenesis of sperm nuclei is directed by proteins or RNA molecules which are not directly responsible for chromatin condensation.     

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.     

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.     

Turnover of basic chromosomal proteins in fertilized eggs: a cytoimmunochemical study of events in vivo

The chromosomal complements of mouse oocytes, ova, and fertilizing sperm have been studied by immunofluorescence with specific antisera to the basic protein fraction of sperm nuclei and to histones H2b and H4, and by staining with ethidium bromide. These studies support the hypothesis, previously proposed (Rodman and Barth, 1979, Dev. Biol. 68:82-95), that the chromosomes of the oocyte in maturation incorporate unique basic protein(s) similar to those incorporated during spermiogenesis. That similarity is characterized, here, by immunologic cross-reactivity. The basic proteins of the fertilizing sperm nucleus and the cross-reactive moiety of the two haploid complements of the ovum are displaced simultaneously, shortly after sperm entry. However, because the unique basic proteins incorporated into the oocyte chromosomes do not, as in the spermatogenic sequence, entirely replace the histones, the maternal chromosomes display histones H2b and H4 at all postfertilization stages examined, whereas the decondensing paternal complement, for an interval before maturation of the pronuclei, contains neither sperm basic chromosomal proteins nor histones. Sequential staining of the same specimens with ethidium bromide revealed well-organized nuclear morphology of the residual DNA complex. Those observations suggest that, for an as yet undefined period in the transformation from spermatozoal to embryonic genome, the chromatin is devoid of a complement of basic proteins.     

Complete displacement of somatic histones during transformation of spermatid chromatin: a model experiment.

Displacement of histones from calf thymus chromatin has been studied in an attempt to postulate the mechanisms involved in the total removal of somatic-type histones during transformation of spermatid chromatin. When chromatin is saturated with protamine (protamine/DNA, 0.5), histone I becomes displaceable at 0.15-0.3 M NaCl, suggesting that direct replacement by highly basic sperm histone could be a mechanism for its removal. While histone I is the only histone which is extensively degraded upon incubation of chromatin and, therefore, proteolysis might provide an additional mechanism for the removal of this histone, acetylation of chromatin by acetic anhydride greatly increases suscpetibility of histones IIb1, IIb2, and III to the chromosomally associated protease. These histones are extensively degraded and displaced from the DNA upon incubation of the acetylated chromatin. Although histone IV is not appreciably degraded, the proteolytic removal of acetylated histone III from chromatin weakens the interaction of acetylated histone IV to the DNA, and this histone becomes dissociable at 0.3 M NaCl. A comparison of the extent of chemical acetylation of individual histones observed in this investigation with that of enzymatic acetylation which can be achieved in vivo suggests that acetylation and proteolysis could be a mechanism for the removal of histone IIb2 and III. The displacement of histones IIb1 and IV could be explained on the basis of decreased binding to DNA as a result of their acetylation together with the proteolytic removal of their respective partner histones, IIb2 and III.     

Chromatin and chromosomal fine structure in spermatogenesis of some species of amphibians

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.     

Cell specific nuclear antigens of boar spermatozoa

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins, hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.     

In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation.

In this study, we describe an efficient protocol for the formation of in vitro developed pronuclei for micromanipulation techniques. Our approach involved incubation of demembranated or permeabilized mammalian sperm in a phosphate buffer supplemented with heparin and beta-mercaptoethanol. Under the prevailing conditions, we achieved a uniform and reliable synchronous decondensation of sperm nuclear DNA. This initial decondensation facilitated the removal of mammalian protamines upon subsequent incubation in an amphibian egg extract. The interchange of protamines for histones to stabilize the DNA structure is recognized as a prerequisite for pronuclear formation. Furthermore, immunocytochemical studies have revealed that pronuclear development is accompanied by the formation of a nuclear lamina with corresponding DNA synthesis. The method described gave a high yield of nuclei during pronuclear formation. Ultimately, our aim is to transfer the in vitro-developed pronuclei into mammalian oocytes by micromanipulation. This novel procedure may prove useful in alleviating severe male factor problems especially in oligozoospermic cases in our in vitro fertilization center.     

Replication of Xenopus erythrocyte nuclei in a homologous egg extract requires prior proteolytic treatment

Reactivation and reinitiation of DNA replication in quiescent frog erythrocyte nuclei has been analyzed following incubation in extracts prepared from activated Xenopus eggs. Nuclear decondensation and DNA synthesis only occurred if nuclei were pretreated with low doses of trypsin. This protease treatment did not digest histones, but did degrade several nonhistone proteins. Activated erythrocyte nuclei swell and begin DNA synthesis by 30 min after being mixed with the egg extract. In some extracts virtually complete genome replication was achieved in all nuclei after 2-3 hr. Addition of several protease inhibitors during sperm nuclear isolation significantly reduced the template efficiency of these preparations. We concluded that proteolytic alteration of nonhistone nuclear structural proteins may be a general mechanism which permits quiescent nuclei to reenter the replication cycle. Erythrocyte nuclei and egg extracts provide an excellent experimental system in which to investigate the processes of nuclear reactivation.     

Mitotic chromosome condensation in the sperm nucleus during post fertilization maturation division in urechis eggs

Changes in the morphology of the sperm nucleus in the egg cytoplasm are mong the immediate events in nucleocytoplasmic interactions during early embryogenesis. Soon after its entrance into the egg cytoplasm, the sperm nucleus of various organisms increases in size with the transformation of condensed chromatin to a diffuse state, resembling the chromatin of an interphase nucleus (2, 13, 15, 16). This is followed by a close association or fusion of male and female pronuclei (2, 13, 15, 16). Cytoplasmic influences on nuclear morphology have also been demonstrated clearly in nuclear transplantation and cell fusion studies (10, 11). Reactivation of the nucleus, such as the transplanted brain nucleus in Xenopus egg cytoplasm or the hen erythrocyte nucleus in interphase cytoplasm of HeLa cells, is accompanied by nuclear enlargement and chromatin dispersion (10, 11). However, premature mitotic-like chromosome condensation takes place in the nuclei of sperm or interphase cells fused with mitotic cells (9, 12). Thus, chromosome dispersion and condensation seem to depend on the state of the cytoplasm in which the nucleus is present. These observations imply that the initial morphological changes in the sperm nucleus after fertilization may very well be dependent on the state of maturation of eggs at the time of sperm entry. Unfertilized eggs of Urechis caupo, a marine echiuroid worm, are stored at the diakinesis stage. These eggs complete maturation division after insemination and this is followed by fusion of male and female pronuclei (5, 8). Therefore, Urechis caupo is a suitable organism in which to study the response of the sperm nucleus to the changing state of the egg cytoplasm during and after postfertilization maturation division.     

Dispersion of mammalian sperm chromatin during fertilization: an in vitro study

When "denuded spermatozoa" (spermatozoa stripped of the greater part of their acrosomes and resembling in may respects spermatozoa after acrosomal reaction) of the bull are incubated with 0.1 M 2-mercaptoethanol (pH 8), sperm chromatin is degraded extensively by a protease in the sperm head. The morphological pattern of sperm nuclear dispersion upon in vitro incubation is similar to that observed in the newly fertilized egg. Following disintegration of the outer layers of the sperm nucleus, chromatin dispersion commences from the periphery of the posterior half and proceeds to the anterior end and to the core of the head. Less basic N- and C-terminal portions of bull sperm histone molecules are digested quickly. The central, very arginine-rich portions of the molecules degrade gradually, yielding an heterogeneous series of arginine-rich peptides (molecular weight, 400-1500). Evidence suggests that the protease which is responsible for the degradation of sperm chromatin is a small fraction of acrosin. This fraction of acrosin appears to be arranged along the nuclear surface and to become associated with sperm chromatin during structural changes of the nuclear surface. A similar proteolysis of rabbit, hamster and guinea pig sperm chromatin has also been observed. The resulting pattern of dissolution of the sperm nucleus is proposed as a model of some of the steps involved in male pronucleus formation from the sperm head after fertilization. Histones H2a, H2b, H3, and H4 associated with DNA are relatively resistant to acrosin.     

Histone-DNA interactions and their modulation by phosphorylation of -Ser-Pro-X-Lys/Arg- motifs.

The sea urchin sperm-specific histones H1 and H2B are multiply phosphorylated in spermatids, dephosphorylated in the final stages of spermatogenesis to give mature sperm, and rephosphorylated upon fertilization. Phosphorylation in spermatids, and probably at fertilization, occurs at repeated -Ser-Pro-X-Basic-motifs in the distinctive N-terminal basic domains of both histones and at the end of the much longer C-terminal domain of H1. Here we identify the consequences of multiple phosphorylation through comparison of some physical and biochemical properties of spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. Study of the DNA binding properties of the intact histones and isolated basic domains suggests that phosphorylation at three dispersed sites in the C-terminal tail of H1 has little effect on its overall DNA binding affinity, whereas, strikingly, binding of the N-terminal domains of H2B and H1 is abolished by phosphorylation at four or six tandemly repeated sites respectively. Together with the relative timing of events in vivo, this suggests that phosphorylation/dephosphorylation of the N-terminal (and distal end of the C-terminal) tail of H1, and/or the N-terminal tail of H2B, effectively controls intermolecular interactions between adjacent chromatin filaments, and hence chromatin packing in the sperm nucleus.