Chromosomes of mouse oocytes in maturation: differential trypsin sensitivity and amino acid incorporation.

In the period of maturation in vivo, the chromosomes of mouse oocytes display a spectrum of unique configurations that is postulated to be related to a sequence of turnover of chromosomal proteins. Evidence on behalf of that hypothesis is provided by the following cytologic observations: The chromosomes of the diakinesis-metaphase I complement are resistant to disruption by mild treatment with trypsin. Following metaphase I, the chromosomes become exceedingly compact and display correlated increased resistance to trypsin. At telophase I, when the complements of the secondary oocyte and the first polar body have each coalesced into a “chromatin mass,” the chromosomes are greatly sensitive to trypsin. Following separation from the mass, the definitive oocyte chromosomes decompact into a “relaxed coil” conformation and display moderate trypsin sensitivity comparable to that of mitotic metaphase chromosomes. Autoradiography of [³H]-arginine and [³H]tryptophan incorporation show that while both amino acids are incorporated into the ooplasm, arginine, but not tryptophan, is incorporated into the chromosomal material. Analysis of the data indicates that incorporation takes place as two separate events, one in late dictyotene and the other post-telophase I and that the arginine-containing proteins incorporated into the dictyate chromosomes are transient and are not retained on the metaphase II chromosomes.     

Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes

The mouse oocyte expresses a polypeptide of Mr 120,000 that cross-reacts with an antibody to the brain membrane skeletal protein adducin. Immunofluorescence localization showed a bright chromosomal staining reaction in metaphase I and metaphase II oocytes. Following in vitro fertilization the maternal chromosomes lost their immunoreactivity during pronuclear development. The fertilizing sperm chromatin and male pronucleus did not show any detectable staining reaction. Bright chromosomal fluorescence was again observed in the first mitotic metaphase when both maternal and paternal chromosomes gave a positive staining reaction. In contrast to the immunoreactivity of the maternal meiotic chromosomes, the meiotic chromosomes of male germ line cells failed to exhibit any detectable staining reaction and this difference was confirmed by immunolabeling of oocyte and spermatocyte karyotypes. Mitotic chromosomes in preimplantation embryos, fetal liver, adult intestinal epithelium, and MDCK cells also failed to show any detectable labeling reaction. The results suggest that expression of the immunoreactive chromosomal adducin may be a unique feature of oogenesis.     

Analyse de la structure chromatinienne du sperme par la cytométrie en flux à l’acridine orange. — intérêt clinique

During spermatozogenesis, sperm nucleosomal DNA type, linked to histones, is transformed into a special form bound to small basic proteins, the protamines. This process allows sperm nucleus to condense and mature. This maturation process is achieved in the epididymis. When a spermatozoon enters an oocyte, protamines are released and replaced by histones, enabling the sperm nucleus to expand into a male pronucleus. Flow cytometry using acridine orange staining is an objective and quantitative technique well-adapted for the investigation of the chromatin structure at the level of each spermatozoon, as well as the whole ejaculate. This technique allows tracing the young sperm cell nuclei from the diploid stage, through tetraploid until the final haploid stage in mature spermatozoa. It allows also following the sperm maturation process during the epididymal transit. It detects various sperm chromatin condensation defects, hypocondensation, hypercondensation or other aberrations, as well as decondensation defects by using an in vitro assay. These defects may impair sperm fertilizing ability, even after sperm microinjection into the oocyte. Better understanding of sperm chromatin integrity and stability prerequisites might help us improving the quality of various technologies used in assisted medical procreation.     

Histone- and chromatin-binding activity of template activating factor-I

Template activating factor-I (TAF-I) is a histone-binding chromatin remodeling factor. We recently found that TAF-I is capable of mediating decondensation of Xenopus sperm chromatin by releasing sperm-specific basic proteins. Here we present evidence that TAF-I preferentially binds to histone H3 among four core histones. Immunofluorescent staining revealed that TAF-I binds to the decondensed sperm chromatin, of which protein components predominantly consist of histones H3 and H4.     

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.     

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.     

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.     

Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein

In order to assess the selectivity of the distribution patterns of individual nonhistone chromosomal proteins (NHC proteins), immunofluorescent staining experiments were performed on Drosophila polytene chromosomes. Antisera have been prepared against three individual NHC proteins which were isolated by sequential preparative slab gel isoelectric focusing and SDS polyacrylamide gel electrophoresis. In two cases, immunofluorescent staining of the chromosomes indicated a specific limited distribution pattern; apparently the antigen in each case is present at a reproducible and distinct subset of chromomeres. This type of pattern has also been obtained with antisera prepared against molecular weight subfractions of NHC proteins (Silver and Elgin, 1977). Each selective fluorescence distribution pattern obtained so far is reproducible and unique to the antiserum under study. In a third case, an antiserum caused prominant staining at dense chromomeres and the chromocenter in a pattern mimicking DNA (and presumably histone) distribution. Indirect radioimmunostaining of SDS and isoelectric focusing gels on which total NHC proteins had been separated confirmed that this antiserum reacted specifically with a protein(s) of molecular weight 21,000 D and pI 5.2. The data in conjunction with absorption experiments indicates that the chromosomal staining is due to an interaction of antibodies with NHC protein(s) and not with histones. This finding suggests that at least one major acidic NHC protein plays a very general role (comparable to that of the histones) in maintaining chromatin structure.     

Detection of Sperm Histone Diversity Among Vertebrates by Alkaline Fast Green Staining

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.     

Detection of sperm histone diversity among vertebrates by alkaline fast green staining

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.     

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.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Dependence of removal of sperm-specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin

In Xenopus laevis , nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.     

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.     

Cytoplasmic factors that control nuclear behavior in mammalian oocytes: a re-evaluation of studies performed as a student of Yoshio Masui

Studies performed by the author in the laboratory of Dr Yoshio Masui are reviewed and interpreted in the light of subsequent findings. The first series of studies indicated that that chromosome condensation during meiotic maturation of mouse oocytes is controlled initially by a stable protein that decays during maturation and subsequently by an unstable protein synthesized after germinal vesicle breakdown. Cyclin B is present in immature oocytes, becomes partially degraded near metaphase I and then re-accumulates, suggesting that this may be protein whose activity was inferred from the original results. The second series of experiments indicated that factors which appear in the oocyte cytoplasm during maturation are able to remodel the sperm into metaphase-like chromosomes, and that the supply of these factors is limited. Recent work indicates that these factors are required for the assembly of histones onto the sperm DNA, and has identified two molecular species, mNAP-1 and NPM-3, known to promote replication-independent chromatin assembly in somatic cells, that are expressed in oocytes.     

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.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Role of nonhistone proteins in metaphase chromosome structure

In this paper, we show that HeLa metaphase chromosomes still possess a highly organized structure retaining the familiar metaphase morphology following removal of virtually all the histones and most of the nonhistone proteins. The structure is stabilized by a relatively small number of nonhistones, which we call scaffolding proteins.These results are based on a method which allows the removal of the histones, and most of the nonhistone proteins, by competition with polyanions such as dextran sulfate and heparin.The histone-depleted chromosomes sediment in sucrose gradients as a broad peak between 4000 to 7000S. These structures are dissociated by mild trypsin or chymotrypsin treatment, or by 4 M urea, but are stable in 2 M NaCl and insensitive to treatment with RNAase A. The histone-depleted chromosomes have a DNA to protein ratio of about 6:1; gel electrophoresis reveals the presence of about 30 nonhistone proteins and the virtual absence of histones. These experiments suggest that nonhistone proteins exist in metaphase chromosomes which maintain the DNA chain in a highly folded conformation.Structural studies support this conclusion. Analysis by fluorescence microscopy of histone-depleted chromosomes stained with ethidium bromide shows that each chromatid is still paired with its sister chromatid, and consists of a central structure surrounded by a halo of DNA. The length of the central structure in each chromatid is about 2–3 times longer than the chromatid length in the original chromosome.     

Calreticulin as a new histone binding protein in mitotic chromosomes

Calreticulin (CRT) is a multifunctional Ca(2+)-binding protein that mainly functions in the endoplasmic reticulum as a molecular chaperone for newly synthesized proteins. Recently we reported the protein composition of human metaphase chromosomes (Uchiyama et al., 2004), which included CRT. Here we describe new characteristics of CRT in vitro as well as its localization on the surface of metaphase chromosomes in vivo. CRT was detected in the chromosomal fraction by Western blotting and its binding partners were identified as core and linker histones by ligand overlay assay. Surface plasmon resonance sensor analyses revealed that CRT is bound to chromatin fibers. Moreover, we found that CRT has both supercoiling activity, which assists core histone assembly into chromatin fibers, and binding ability to histone H2A/H2B dimers and histone H3/H4 tetramers. Unlike the chromosome scaffold proteins, indirect immunofluorescent staining revealed that CRT is located on the surface of metaphase chromosomes. These results suggest that CRT plays a role which involves chromatin dynamics on the surface of mitotic chromosomes.