Decondensation Of Nuclei Research Articles

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.

179 Decondensation of bovine spermatozoa after dithiothreitol treatment

Dithiothreitol (DTT) has been shown to reduce protamine disulfide bonds present in the outer acrosomal membrane of sperm. These disulfide bonds provide bull sperm with greater membrane stability and an increased resistance to decondensation compared with other species. (Hutchinson et al. 2017 Biophysical J. 113, 1925-1933). This can result in asynchronous fertilization due to the sperm nucleus failing to properly decondense and form male pronuclei after assisted reproductive techniques, such as IVF or intracytoplasmic sperm injection (ICSI) (Águila et al. 2017 Reproduction 154, 307-318). By reducing these bonds and disrupting the acrosomal membrane, DTT can minimize this complication, allowing the sperm nucleus to decondense more readily. This study aimed to assess bovine sperm, treated or not with DTT, for changes in morphology and to track the progress of decondensing sperm nuclei after treatment. Frozen-thawed bovine semen, utilising only egg yolk extender, was prepared by the swim-up method in the presence or absence of DTT at 5mM. After a 1-h incubation time, permitting sperm to swim up, sperm in the top 0.5mL in each culture tube were washed by centrifugation and observed hourly. Changes in morphology of DTT-treated and nontreated sperm were observed through a combination of phase-contrast and Hoffman modulation contrast microscopy. To assess DNA dispersal, DTT-treated sperm were stained with Hoechst and viewed under fluorescence microscopy. The DTT-treated sperm exhibited time-dependent changes in morphology, including altered movement, head folding at the equatorial segment, bending and swelling of the mid-piece, and expansion of the sperm head accompanied by a cratered appearance, signalling a partially decondensed nucleus, which progresses to full decondensation, and last, complete dispersal of DNA. These changes were seen over a 7-h incubation period in HEPES-TALP. No such changes were observed in nontreated sperm. The proportion of sperm in each stage of decondensation was estimated by a single observer at hourly time points in at least 8 repetitions. Mean estimated proportions of each stage of decondensation over time was analysed through factorial ANOVA and found significant (P<0.001). An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the third hour and increased to 62% by the fifth hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at 5h to 50% at 7h. A remaining 10% of sperm remained unaffected and showed no changes to morphology. These results were found to be in agreement with other studies and indicate that 5mM DTT treatment induces decondensation of bovine sperm nuclei, which may assist in male pronuclei formation for other assisted reproductive technologies.

H3K9ac involved in the decondensation of spermatozoal nuclei during spermatogenesis in Chinese mitten crab Eriocheir sinensis.

As a well-known crustacean model species, the Chinese mitten crab Eriocheir sinensis presents spermatozoa with decondensed DNA. Our aim was to analyze structural distribution of the histone H3 and its acetylated lysine 9 (H3K9ac) during spermatogenesis for the mechanistic understanding of the nuclear decondensation of the spermatozoa in E. sinensis. Using specific antibodies, we followed the structural distribution and acetylated lysine 9 of the histone H3 during spermatogenesis, especially spermiogenesis, of E. sinensis. Various spermary samples at different developmental stages were used for histological immunofluorescence and ultrastructural immunocytochemistry. Our results demonstrate a wide distribution of the histone H3 and H3K9ac during spermatogenesis, including spermatogonia, spermatocytes, spermatids, and immature and mature spermatozoa except for absence of H3K9ac in the secondary spermatocytes. Especially during the initial stage of nuclear decondensation, histone H3 lysine 9 was acetylated and then an amount of H3K9ac was removed from within to outside of the nuclei of late spermatids. The portion of remaining H3K9ac was gradually transferred from the nuclei during the stages of spermatozoa maturation. Our findings suggest both the acetylation of histone H3 lysine 9 and the remain of H3K9ac to contribute to the nuclear decondensation in spermatozoa of E. sinensis.

Possible participation of calmodulin in the decondensation of nuclei isolated from guinea pig spermatozoa

The guinea pig spermatozoid nucleus contains actin, myosin, spectrin and cytokeratin. Also, it has been reported that phalloidin and/or 2,3-butanedione monoxime retard the sperm nuclear decondensation caused by heparin, suggesting a role for F-actin and myosin in nuclear stability. The presence of an F-actin/myosin dynamic system in these nuclei led us to search for proteins usually related to this system. In guinea pig sperm nuclei we detected calmodulin, F-actin, the myosin light chain and an actin-myosin complex. To define whether calmodulin participates in nuclear-dynamics, the effect of the calmodulin antagonists W5, W7 and calmidazolium was tested on the decondensation of nuclei promoted by either heparin or by a Xenopus laevis egg extract. All antagonists inhibited both the heparin- and the X. laevis egg extract-mediated nuclear decondensation. Heparin-mediated decondensation was faster and led to loss of nuclei. The X. laevis egg extract-promoted decondensation was slower and did not result in loss of the decondensed nuclei. It is suggested that in guinea pig sperm calmodulin participates in the nuclear decondensation process.

Y-chromosome-specific fluorescence (f-body) of poorly decondensed bovine spermatozoa.

An account is given in this paper of a method of identifying the fluorescence body (f-body) as a marker of the Y-chromosome. Also covered by this method is poor decondensation of spermatozoal nuclei when exposed to action of 1.25% of papain, 0.155% of DTE, and 0.025% of DMSO. Quinacrine mustard was used as fluorescent stain, its final concentration being 0.0025%. For staining, spermatozoa were suspended for 1-5 h. Average f-body frequency accounted for 41.0 +/- 5.1% in 35 ejaculates from 22 bulls. The overall variation coefficient amounted to 12.4% and thus was higher than each of the single values individually recorded from six bulls which were involved with three or four ejaculates (3.6-7.0%). F-bodies could not be detected by the method generally used on human spermatozoa. The applicability of the f-body test to quantification of Y spermatozoa in experimental separation of androspermatozoa and gynospermatozoa is discussed and is demonstrated by an example.

Desintegration von Spermatozoen bei der impulscytophotometrischen Analyse des menschlichen Spermas

In some cases, the pretreatment of human spermatozoa produced desintegration of various degrees, while in others a reduced staining was observed. For the evaluation of these phenomena the following techniques were applied: papain/DTE solution at pH 5.5 and 6.4, pepsin solution and a staining procedure without any preceeding decondensation of the spermatozoa nuclei. Pathological spermatozoa from patients with severe disorders of spermatogenesis showed an extended resistance to the pretreatment procedures, the spermatozoa being stained after papain/DTE pretreatment at pH 5.5 only. Contrary to this finding, normal spermatozoa and spermatozoa with slight morphological aberrations desintegrated when papain/DTE solutions were used. In this group the abortion rate was high.

Chromatin remodeling in somatic cells injected into mature pig oocytes

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

Acrocentric centromere organization within the chromocenter of the human sperm nucleus.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.

STUDY ON THE MODE OF PRIMARY CONTROL IN THE EGG OF GYNOGENETIC CRUCIAN CARP FOR INHIBITING THE DEVELOPMENT OF TWO TYPES OF SPERM NUCLEI

By comparing the different developmental characteristics of two types of sperm nuclei which were from gynogenetic fish (crucian carp) and amphimictic fishes (red carp, red goldfish and sex-reversal red carp) respectively in the eggs of gynogenetic crucian carp, it was preliminarily revealed that there existed selective inhibiting actions of the primary control in the eggs of crucian carp for inhibiting the development of the two types of sperm nuclei. To homologous sperms, the primary control showed weak effect, thus leading to the decondensation of homologous sperm nuclei at different degrees in the eggs of crucian carp. But to heterologous sperms, the primary control showed strong effects, resulting in the total inhibition of the development of heterologous sperm nuclei. Moreover, our experimental results also showed that the different developmental behavior of the two types of sperm nuclei might have a great relationship to the changes of the sex ratio in the population of gynogenetic crucian carp. The infiltration of qthe genetic materials in sperm nucleiq into the female nucleus at random might play an important role in male emergence in the naturally gynogenetic population of crucian carp.

Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin

At fertilization, sperm chromatin decondenses in two stages, which can be mimicked in extracts of Xenopus eggs. Rapid, limited decondensation is followed by slower, membrane-dependent decondensation and swelling. Nucleoplasmin, an acidic nuclear protein, occurs at high concentration in Xenopus eggs and has a histone-binding role in nucleosome assembly. Immunodepleting nucleoplasmin from egg extracts inhibits the initial rapid stage of sperm decondensation, and also the decondensation of myeloma nuclei, relative to controls of mock depletion and TFIIIA depletion. Readdition of purified nucleoplasmin recues depleted extracts. A physiological concentration of purified nucleoplasmin alone decondenses both sperm and myeloma nuclei. We conclude that nucleoplasmin is both necessary and sufficient for the first stage of sperm decondensation in Xenopus eggs.

Studies on the decondensation of human, mouse, and bull sperm nuclei by heparin and other polyanions

We report heparin-induced decondensation of human, mouse, and bull sperm nuclei. Decondensation did not occur if the spermatozoa were intact but only if the membranes were severely damaged by freezing and thawing or by treatment with a detergent. If a disulphide bond reducing agent (thiol) was absent, decondensation of human sperm nuclei was usually a relatively slow process, with large interindividual variation. Mouse and bull sperm nuclei did not decondense in the absence of a thiol. With a thiol relatively low concentrations of heparin induced a rapid decondensation of the sperm nuclei of all three species. The decondensation activity was not specific for heparin; other polyanions were also active, with heparin being the most effective compound. It is supposed that heparin and other polyanions induce sperm nuclear decondensation because they deplete protamines from the chromatin. Thus the negatively charged phosphate groups of the DNA are no longer opposed by positively charged protamines. Consequently the mutual repulsion of unopposed phosphate groups causes the DNA molecules to stretch, which results in an increase of the sperm nuclear volume. Since heparin and other polyanions induce decondensation under physiological pH and temperature, polyanions might also be active in the oocyte.

Cation-dependent solubilization of rat thymocyte chromatin is closely related to decondensation of the nuclei

The cation-dependent solubilization of rat thymocyte chromatin has been compared with decondensation of the nuclei as a function of sodium phosphate-mediated changes in the concentration of Mg 2+ and Na +. After digestion of the nuclei with DNase I or Micrococcus nuclease for a time just sufficient to permit extraction of a maximal amount of chromatin (minimum digestion), solubilization of most of the chromatin was found to occur with the same cation dependency as decondensation of untreated nuclei, while further digestion changed the ionic requirements for solubilization. The cation-dependency of the chromatin solubility and of the nuclear decondensation also exhibited the same variations with temperature. The chromatin in the nuclei became up to 4-times more sensitive to DNase I by decondensation, which also induced a shift in the DNase I cleavage mode from a 200 bp to a 100 bp repeat pattern. In contrast, the sensitivity to Micrococcus nuclease appeared to be nearly unchanged. These results suggest that solubilization of chromatin prepared by a mild endonuclease treatment occurs as a direct consequence of structural changes in the chromatin which take place during decondensation of the nuclei.

Effect of zinc on decondensation of human spermatozoa nuclei by heparin.

Swelling sperm nuclei were assayed, exposing them to the combine action in increasing amounts of seminal plasma 0.1-1.0 ml (124 +/- 21 micrograms of Zn++/ml). An inhibition of almost 30% in the swollen spermatozoa nuclei was observed with 0.1 ml of seminal plasma (12 micrograms of Zn++/ml) reaching to 71% with 74-124 micrograms of Zn++/ml. Inactivated seminal plasma (boiling) induced the same percentage of inhibition (73%) than normal seminal plasma. Dialyzed seminal plasma (31 micrograms of Zn++/ml) produced an inhibition of 88% of swollen spermatozoa nuclei, the same percentage produced with 30-35 micrograms of zinc ions (ZnCl2). Previous release of sperm zinc by preincubation with EDTA 6 mM changed the decondensation kinetics, making sperm nuclei more susceptible to the action of the glycosaminoglycan. No effect was observed in the presence of calcium ions. Therefore, zinc, among its several physiological roles, may act as a nuclear chromatin stabilizer.

Heparin-induced nuclei decondensation of mammalian epididymal spermatozoa.

Decondensation of mammalian epididymal spermatozoa nuclei has been induced by exposure of intact spermatozoa to heparin, including those species in which ejaculated sperm were not susceptible to this treatment. This process occurred in the absence of any disulfide bond cleaving reactant. Swelling of caput epididymal spermatozoa nuclei commenced about 30 min after the addition of heparin, reaching 88% in rat, 33% in rabbit, 26% in pig, and 62% in bull of swelled nuclei after 6 hr of incubation at 37 degrees C with 5000 USP of heparin per ml. Corpus epididymal spermatozoa nuclei of rat and rabbit underwent decondensation at 50 degrees C reaching 24% and 22% of swelled nuclei, respectively, after 6 hr of incubation. The nuclei of the sperm cells of pig and bull from this epididymal region remained highly condensed as well as the nuclei of the cauda epididymal spermatozoa of all the species assayed. Electron microscope observations of the caput epididymal spermatozoa nuclei treated with heparin revealed that the chromatin is organized into nuclear bodies joined by a network of cross-linked and branched chromatin fibers in the species studied.

Decondensation of human sperm nuclei by glycosamineglycan‐sulfate from sea urchin egg

Decondensation of human and sea urchin spermatozoan nuclei has been induced by exposure of intact spermatozoa to glycosamineglycan-sulfate (GAGs) extracted from eggs of the sea urchin. Toxopneustes roseus. Swelling of human spermatozoan nuclei commenced about 60 min after the addition of GAGs and, depending on the extract concentration, the proportion of swollen nuclei reach 90% after 6 h of incubation when 20 mg of GAGs per ml was used. Swelling of sea urchin spermatozoan nuclei could be induced also with GAGs and/or with heparin. Swelling commenced about 10 min after the addition of GAGs/heparin reaching 90-92% after 60 min of incubation with 5 mg GAGs/500 USP heparin per ml. Preincubation of spermatozoa with 10 mg/ml of trypsin soybean inhibitor did not interfere with the swelling action of the GAGs.

Species specific decondensation of human spermatozoa nuclei by heparin.

Decondensation of human spermatozoa nuclei has been induced by exposure of intact spermatozoa to heparin, while the spermatozoa of rabbit, ram, and bull remained highly condensed under similar experimental conditions. This process occurred in the complete absence of any disulfide bond cleaving reactant. Swelling of human spermatozoa nuclei commenced about 30 min after the addition of heparin and depended on heparin concentration reaching 83% of swelled nuclei after 6 hr. of incubation with 5000 USP of heparin per ml. Addition of 10 mg/ml of trypsin soybean inhibitor did not interfere with the swelling action of heparin. Electron microscope observations of human spermatozoa nuclei treated with heparin revealed that the chromatin is organized into "hublike" nuclear bodies joined by a network of cross-linked and branched chromatin fibers ranging in thickness from 25 to 1.5 nm.

Subunit structure of Eutherian sperm chromatin.

The beta-mercaptoethanol induced decondensation of spermatozoon cell nuclei from several Eutherian species has been followed from the intact spermatazoon cell to the solubilized linear unit sperm chromosomal fiber using fluorescence and electron microscopy. Data from nuclease digestion studies in conjunction with electron microscopic evidence indicate that the gross structure of the unit Eutherian sperm chromosomal fiber consists of DNA folded around sperm specific histone multimers spaced regularly along the fiber generating a linear array of sperm nucleosomes connected by short stretches of uncomplexed DNA. The sperm nucleosomes, 80 A in diameter are separated by 20 A filaments of DNA. This structure is remarkably similar to the structure of somatic chromatin although the protein components of the two chromosomes are markedly different. It seems likely that chromosomal fibers, similar to those described herein, may be present in the male pronucleus following fertilization.

Behavior of nuclei of testicular, caput and cauda epididymal spermatozoa injected into hamster eggs.

The behavior of isolated nuclei of testicular and cauda and caput epididymal spermatozoa of the hamster injected into mature unfertilized hamster eggs was studied. Decondensation of nuclei within the egg cytoplasm was observed regardless of the type of spermatozoa used and whether the eggs were activated or not. In activated eggs the decondensed nuclei of testicular and cauda but not caput epididymal spermatozoa transformed into pronuclei. This failure of caput epididymal spermatozoa nuclei to transform into pronuclei may be due to a factor in the nuclei which prevents such a transformation. It is possible that this factor may be inactive or absent in the nuclei of testicular spermatozoa or may be inactivated by another factor while spermatozoa are present in the cauda epididymis.