Proposed mechanism for sperm chromatin condensation/decondensation in the male rat

Condensation of sperm chromatin occurs after spermatozoa have left the caput epididymis and are in transit to the cauda epididymis, during which time large numbers of disulfide bonds are formed. The formation of these disulfide bonds requires the repeated oxidation of the cofactor, NAD(P)H. To date, the means by which this oxidation is achieved has yet to be elucidated. Spermatozoa lose the bulk of their cytoplasm prior to leaving the testis; and, as a result, any shuttle systems for removing and transferring reducing equivalents into the mitochondria are unlikely to be operational. In an apparent preparation for the loss of cytoplasm, however, the following events occur during spermatogenesis. First, androgen-binding protein (ABP) is produced by the Sertoli cells of the testis; second, high affinity binding sites for ABP are inserted into the membrane surrounding the nucleus; and third, a nuclear location is acquired for the enzyme, 3α-hydroxysteroid dehydrogenase (3α-HSD).     

Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.     

Modeling of DNA condensation and decondensation caused by ligand binding

A theoretical method for computer modeling of DNA condensation caused by ligand binding is developed. In the method, starting (s) and condensed (c) states are characterized by different free energies for ligand free DNA (F(s) and F(c) respectively), ligand binding constants (K(s) and K(c)) and stoichiometry dependent parameters (c(sm) and c(cm) - maximum relative concentration of bound ligands (per base pair) for starting and condensed state respectively). The method allows computation of the dependence of the degree of condensation (the fraction of condensed DNA molecules) on ligand concentration. Calculations demonstrate that condensation transition occurs under an increase in ligand concentration if F(s) < F(c) (i.e. S(sc) = exp [- (F(c) - F(s)) / (RT)], the equilibrium constant of the s-c transition, is low (S(sc) < 1)) and K(s) < K(c). It was also found that condensation is followed by decondensation at high ligand concentration if the condensed DNA state provides the number of sites for ligand binding less than the starting state (c(sm) > c(cm)). A similar condensation-decondensation effect was found in recent experimental studies. We propose its simple explanation.     

A physical model for the condensation and decondensation of eukaryotic chromosomes

During the eukaryotic cell cycle, chromatin undergoes several conformational changes, which are believed to play key roles in gene expression regulation during interphase, and in genome replication and division during mitosis. In this paper, we propose a scenario for chromatin structural reorganization during mitosis, which bridges all the different scales involved in chromatin architecture, from nucleosomes to chromatin loops. We build a model for chromatin, based on available data, taking into account both physical and topological constraints DNA has to deal with. Our results suggest that the mitotic chromosome condensation/decondensation process is induced by a structural change at the level of the nucleosome itself.     

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 10¹⁵ or 0.5 × 10¹⁵ particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.     

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.     

Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.     

Proteolytic degradation of protamine during thiol-induced nuclear decondensation in rabbit spermatozoa

Rabbit sperm nuclei decondensed when incubated with dithiothreitol and Triton X-100. Protamine isolated from these nuclei during the course of treatment exhibited marked and progressive degradation. Timed interval experiments revealed that the degradation of protamine significantly preceded sperm nuclear decondensation and reached its maximum extent before decondensation was completed. Studies with protease inhibitors demonstrated that proteolysis was involved in both nuclear decondensation and protamine degradation.     

Sperm nuclear decondensation in mammals: role of sperm-associated proteinase in vivo

Previous studies from this (Zirkin et al., '80) and other (Marushige and Marushige, '78) laboratories have shown that proteinase associated with mammalian sperm nuclei is involved in thiol-induced sperm nuclear decondensation and protamine degradation in vitro. The results of these in vitro studies suggested the exciting possibility that the sperm nucleus itself might contribute proteinase involved in its subsequent in vivo decondensation during fertilization. In the present study, microinjection methods were used to test this possibility directly. Control hamster sperm nuclei, which exhibited proteinase activity, decondensed when incubated in vitro with disulfide reducing agent. As expected, these nuclei also decondensed when microinjected into ovulated hamster oocytes and formed morphologically normal pronuclei. When the proteinase associated with isolated sperm nuclei was removed with 0.5 M salt or inhibited with nitrophenyl-p-guanidinobenzoate, the nuclei were rendered incapable of decondensing in response to disulfide reducing agent in vitro. However, when these nuclei were microinjected into eggs, they decondensed and transformed into pronuclei. These results provide direct evidence that sperm-associated proteinase is not required for sperm nuclear decondensation and formation of the male pronucleus during fertilization.     

Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.     

Quantitative assessment of DNA condensation

A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.     

The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo

These studies were designed to test the hypothesis that sperm nuclear decondensation and male pronuclear formation during hamster fertilization depend upon the ability of the fertilized oocyte to reduce sperm nuclear disulfide bonds. In a first series of experiments, treatment of mature oocytes with the sulfhydryl blocking agent iodoacetamide or the glutathione oxidant diamide caused a dose-dependent inhibition of decondensation in microinjected sperm nuclei. Inhibition of decondensation was not observed, however, when sperm nuclei were treated in vitro with dithiothreitol (DTT) to reduce disulfide bonds prior to their microinjection. In a second series of experiments, germinal vesicle (GV)-intact oocytes and pronuclear eggs, in which mature, disulfide-rich sperm nuclei do not decondense, were found to support the decondensation of disulfide-poor DTT-treated sperm nuclei or testicular spermatid nuclei. The decondensed sperm nuclei were not, however, transformed into male pronuclei. The results of these studies suggest: (1) that sperm nuclear decondensation in the hamster requires disulfide bond reduction, (2) that GV-intact oocytes and pronuclear eggs lack sufficient reducing power to effect sperm nuclear decondensation, and (3) that disulfide bond reduction is required but not sufficient for pronuclear formation.     

Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Quantitative analysis of the changes in the nuclear apparatus of thyrocytes in experimental thyroid hyperfunction

Quantitative ultrastructural characteristics (volume, surface area, volume fraction and surface density) of the nucleus, nucleolus and cell were studied in albino rats after long-term (5, 10, 15, 20 days) melanotropin (MSH) administration. The experiments showed that enhanced functional activity of thyroid cells induced by MSH administration was accompanied by an increase in the volume, surface area of the nucleus, nucleolus and the cell proper, as well as by an increased nuclear pore diameter. Consequently, the nucleus-cytoplasm ratio in thyroid cells rises.