Ultrastructural analysis of mouse sperm chromatin

Chromatin organization was studied during the maturation processes in the epididymis and vas deferens; these processes lead to a potentially reversible inactivation of the genome. There are progressive increases in resistance to detergent action and -S-S- bridge reduction in both head membranes and chromatin. In all the sites studied, there was a basic "knobby" chromatin fiber of 110 A diameter. In the caput epididymidis only, in addition to the knobby fibers, there were some smooth fibers, which can be considered as markers of a transient situation in which the stabilization of DNA/protamine interactions has not completely been achieved. In the vas deferens, the knobby fibers, the diameters of which are multiples of that of the basic one, can be converted to single units by increasing the ionic strength.     

Trout sperm chromatin. I. Biochemical and immunological study of the protein composition

Compact sperm chromatin was obtained from mature trout sperm nuclei resistant to sonication and detergent treatments. 0.5 to 2 M NaCl caused a gradual decondensation of this chromatin and the dependence of the percentage of dissociated proteins on the salt concentration indicated cooperativity of the dissociation process. Urea alone was insufficient to decondense the nuclei. The only proteins dissociated from the sperm nuclei by NaCl alone or combined with urea were protamines. Besides protamines, tightly bound nonprotamine proteins resisting high salt-urea extraction were detected in the sperm nucleus. Part of them could be solubilized by 1% sodium dodecyl sulphate (SDS) and displayed the characteristics of the core histones: they were soluble in 0.25 N H2SO4, their electrophoretic mobilities were similar to those of trout liver core histones, and they shared common antigenic determinants with the latter. The rest of the tightly bound proteins resisted 1% SDS treatment and could be obtained after an extensive digestion of DNA with DNase I. These were nonhistone proteins similar in mobility to the protein triplet characteristic of the lamina-pore complex and an additional high molecular weight protein.     

Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions.

A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin. Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration. The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins.     

Degradation of chromosomal proteins during dissociation and reconstitution of chromatin

Histones and nonhistone chromosomal proteins are degraded when chromatin is exposed to 2 M NaCl-5 M urea (pH 6–8) which has been most often used for disscciation and reconstitution of chromatin. Histones and nonhistone proteins are also degraded in 5 M urea (pH 6–8).     

Non-histone proteins in fractionated chromatin before and after DNAse II treatment

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.     

Comparative aspects of basic chromatin proteins in dinoflagellates

Previous work on histone-like proteins in dinoflagellates is summarized, together with some new data to give an overview of basic proteins in these algae. The first two dinoflagellates studied were both found to contain one major acid-soluble protein that migrated to the same position in acidic-urea gels. When several other genera were studied however, it became apparent that the histone-like proteins from different dinoflagellates were similar but not identical. In view of the great diversity of living dinoflagellates it is speculated that further differences in dinoflagellate basic chromatin proteins will be revealed. Electrophoretic data from the eukaryotic (endosymbiont) nucleus of Peridinium balticum showed the presence of five major components. It is speculated that two of these proteins represent an H1-like doublet and two others correspond to the highly conserved histones H3 and H4. The fifth component is a new histone that may substitute for H2A and H2B in the nucleosome. Because histones and nucleosomes are present in all higher organisms but completely lacking in procaryotes, studies on basic proteins in dinoflagellates will provides insights into the evolution of histones and eucaryotic chromatin organization.     

Ultrastructural aspects of infertility.

Organ culture studies have demonstrated that 17beta oestradiol can cause the rapid deterioration and dissolution of pre-existing nuclear channel systems in the human endometrial glandular cell. The importance of the nuclear channel system in implantation is emphasized and the implications of secretory activity in the endometrium of an infertile woman are discussed.     

Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin

Zona-free hamster eggs were fertilized in vitro with human spermatozoa in a culture medium enriched with either 3H-arginine or 3H-tryptophan. Autoradiography was used to investigate decondensing sperm heads and all pronuclei for the presence of newly synthesized, 3H-labelled proteins. In the case of 3H-arginine-labelled proteins, an intense accumulation of radioactivity was detected in all autoradiograms of chromatin structures. On the other hand, no comparable accumulation was seen for 3H-tryptophan-labelled proteins up to the progressed-pronucleus stage. It is concluded that, as a part of changes of the nucleoproteins in decondensing sperm chromatin, there is an accumulation in the male (as well as in the female) pronucleus of basic nuclear proteins synthesized by the egg during fertilization. Since non-histone, 3H-tryptophan-labelled proteins were not incorporated in the same way, these 3H-arginine-labelled proteins accumulating in pronuclear chromatin during the earliest phase of pronucleus formation are probably histones.     

Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin

Summary Zona-free hamster eggs were fertilized in vitro with human spermatozoa in a culture medium enriched with either ³H-arginine or ³H-tryptophan. Autoradiography was used to investigate decondensing sperm heads and all pronuclei for the presence of newly synthesized, ³H-labelled proteins.In the case of ³H-arginine-labelled proteins, an intensen accumulation of radioactivity was detected in all autoradiograms of chromatin structures. On the other hand, no comparable accumulation was seen for ³H-tryptophan-labelled proteins up to the progressed-pronucleus stage. It is concluded that, as a part of changes of the nucleoproteins in decondensing sperm chromatin, there is an accumulation in the male (as well as in the female) pronucleus of basic nuclear proteins synthesized by the egg during fertilization. Since non-histone, ³H-tryptophan-labelled proteins were not incorporated in the same way, these ³H-arginine-labelled proteins accumulating in pronuclear chromatin during the earliest phase of pronucleus formation are probably histones.     

Enzymatic unpacking of bull sperm chromatin.

When isolated bull sperm chromatin is incubated with 0.1 M 2-mercaptoethanol at pH 8, an extensive proteolytic degradation of sperm histone occurs, being accompanied by a marked swelling of the chromatin masses. The degradation of sperm histone is strongly inhibited by monovalent or divalent metal ions. The protease found in isolated bull sperm chromatin possesses properties indistinguishable from those of an acrosomal protease of trypsin-type, acrosin (EC 3.4.21.10), and requires a combination of NaCl, urea and 2-mercaptoethanol for its extraction. Evidence suggests that the protease travels along chromatin strands and hydrolyzes essentially all the sperm histone molecules within the chromatin masses.     

Acid-extracted nuclear proteins and ultrastructure of human sperm chromatin as revealed by differential extraction with urea,mercaptoethanol, and salt

Basic proteins were differentially extracted from the purified heads of ejaculated human sperm by successive treatment with (1) 1% Triton X-100, 1% mercaptoethanol (ME); (2) 8 M urea, 1% ME; (3) 8 M urea, 1% ME, 0.2 M NaCl; and (4) 8 M urea, 1% ME, 0.6 M NaCl. Nonhistones were extracted in the first, second, and third treatments; histones, in the second and third; and most of the protamines, in the fourth, together with DNA. Corresponding electro microscopic studies of the sperm pellets after each treatment suggest that the nucleoprotamines are discrete portions of chromatin which are organized in the form of long thick cords and oval bodies of about 400–550 Å in diameter interlinked with very thin strands of fibers about 20–50 Å thick, which could represent the naked DNA depleted of its constituent histones.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

Ultrastructural aspects of human nonunion.

A histological study on the tissue of nonunion of tibias of two young patients was performed to evaluate the ability of cells to start the mineralization of the matrix. The observations can be summarized as follows: 1) Tissue vessels often appear occluded by thrombotic material; 2) Fibroblasts and chondrocytes found in the nonunion tissue seemed normal, with a good secretion apparatus; 3) The cell membranes were able to produce matrix vesicles; 4) Matrix vesicles and cell membrane looked positive to ALPase reaction, 5) Hydroxyapatite crystals could be observed in the cell matrix or inside matrix vesicles. It may be concluded that cells populating nonunion tissue are well equipped to induct the mineralization of the matrix, but the absence of a blood supply, enough to bring them a normal calcium amount, is the real reason for the nonunion.