In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animalXenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceu) sperm. The demembranatedOrychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

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.     

Reconstitution of Sperm Nuclei of Zebrafish (Danio rerio) in Xenopus Egg Extracts

Cell-free extracts of Xenopus eggs cause cyclic change in permeabilized sperm nucleus, nuclear envelope breakdown, chromosome condensation, and reformation of nuclei. In this study, the ability of cell-free extracts to cause similar changes in zebrafish sperm was examined. When lysolecithin-treated sperm from zebrafish were incubated in Xenopus egg extracts, a series of changes in sperm nuclear morphology were observed periodically. These changes correlated with maturation-promoting factor (MPF) activity. Furthermore, sperm nuclei of zebrafish replicated DNA during reconstitution in Xenopus egg extracts. These results showed that cell-free extracts of Xenopus egg possess the ability to cause cell-cycle-dependent changes in zebrafish sperm, implying the possibility of generating transgenic zebrafish in a similar way to transgenic Xenopus. Received October 21, 1999; accepted July 18, 2000.     

ICRF-193, an Inhibitor of Topoisomerase II, Demonstrates That DNA Replication in Sperm Nuclei Reconstituted in Xenopus Egg Extracts Does Not Require Chromatin Decondensation

A bis(2,6-dioxopiperazine) derivative, ICRF-193, is a specific inhibitor of topoisomerase II without clearable complex-stabilizing activity. In Xenopus egg extract containing ICRF-193, demembranated sperm head chromatins were inhibited from decondensation. However, nuclear envelope-lamina assembled on the inhibited chromatins. The nuclear envelope-lamina continued to expand even after loss of contact with the chromatin surface. On the other hand, semiconservative DNA replication was initiated as soon as the lamina was assembled onto the surface of condensed chromatin, though the initiation was retarded and its extent was reduced, compared with that in noninhibited chromatins. Thus, it is concluded that topoisomerase II activity is not required for the formation of active DNA replication clusters and the extension of nuclear envelope-lamina on the chromatin, while the nuclear envelope-mediated decondensation of sperm chromatins is dependent on topoisomerase II activity.     

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.     

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.     

Xenopus Egg Extracts Increase Dynamics of Histone H1 on Sperm Chromatin

Background

Linker histone H1 has been studied in vivo and using reconstituted chromatin, but there have been few systematic studies of the effects of the cellular environment on its function. Due to the presence of many other chromatin factors and specific chaperones such as RanBP7/importin beta that regulate histone H1, linker histones likely function differently in vivo than in purified systems.

Methodology/Principal Findings

We have directly compared H1 binding to sperm nuclei in buffer versus Xenopus egg extract cytoplasm, and monitored the effects of adding nuclear import chaperones. In buffer, RanBP7 decondenses sperm nuclei, while H1 binds tightly to the chromatin and rescues RanBP7-mediated decondensation. H1 binding is reduced in cytoplasm, and H1 exhibits rapid FRAP dynamics in cytoplasm but not in buffer. RanBP7 decreases H1 binding to chromatin in both buffer and extract but does not significantly affect H1 dynamics in either condition. Importin beta has a lesser effect than RanBP7 on sperm chromatin decondensation and H1 binding, while a combination of RanBP7/importin beta is no more effective than RanBP7 alone. In extracts supplemented with RanBP7, H1 localizes to chromosomal foci, which increase after DNA damage. Unlike somatic H1, the embryonic linker histone H1M binds equally well to chromatin in cytoplasm compared to buffer. Amino-globular and carboxyl terminal domains of H1M bind chromatin comparably to the full-length protein in buffer, but are inhibited ∼10-fold in cytoplasm. High levels of H1 or its truncations distort mitotic chromosomes and block their segregation during anaphase.

Conclusion/Significance

RanBP7 can decondense sperm nuclei and decrease H1 binding, but the rapid dynamics of H1 on chromatin depend on other cytoplasmic factors. Cytoplasm greatly impairs the activity of individual H1 domains, and only the full-length protein can condense chromatin properly. Our findings begin to bridge the gap between purified and in vivo chromatin systems.     

Nuclear swelling and chromatin decondensation can occur without cortical granule explosion in eggs of Xenopus laevis

After injection of nuclei into Xenopus eggs which have been preimmersed in De Boer's solution for 2 h the injected nuclei and the egg pronucleus undergo normal swelling and chromatin decondensation. In these eggs explosion of the cortical granules is inhibited, and hence this feature of the activation reaction is not required for nuclear swelling. Nuclei do not swell when immersed in perivitelline fluid, so confirming that contents of the cortical granules are not involved in nuclear swelling.     

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.     

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.     

Quantitative changes in histones during the formation of structures characteristic of the male pronucleus in mammalian spermatozoa in vitro

Treatment of bull spermatozoa with DDC--Na/dithiothreitol results in the swelling and decondensation of nuclear chromatin. The structures formed at the final stages of decondensation are morphologically similar to the male pronucleus. Cytophotometric analysis has shown that decondensation of chromatin in the gametes in followed by quantitative changes of basic nuclear proteins. In partly--decondensed sperm nuclei the intensity of histone staining increases as a result of the appearance of extra reactive groups. In fully decondensed nuclei there remain only 54% of histones of the original haploid level. Nucleoproteins revealed in the sperm with fully dispersed chromatin must be histones of the somatic type.     

Nuclear reconstitution of plant (Orychophragmus violaceus) demembranated sperm in cell-free extracts from animal (Xenopus laevis) eggs

Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix--lamina showed the reconstituted nucleus to be filled with a dense network.     

The disruption in actin-perinuclear theca interactions are related with changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen

The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen–thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen–thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P < 0.001), and significant changes in sperm chromatin nuclear decondensation (P < 0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P < 0.05). Evaluation with rodamine-labeled actin (0.2 μM) showed two different patterns with differences in percentages before and after cryopreservation (P < 0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing–thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.     

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.     

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.     

THE INTRINSIC MECHANISM OF CHROMATIN DECONDENSATION AND ITS ACTIVATION IN HUMAN SPERMATOZOA*

In vitro decondensation of human sperm chromatin induced by the activation of an intrinsic mechanism was studied by light microscopy, scanning and transmission electron microscopy. Morphological evidence was provided to support the concept that this mechanism is essential for the chromatin decondensation occurring in vivo. Prostatic zinc is hypothesized to preserve this potential decondensation ability from oxidative destruction, by reversibly binding to free thiol-groups. The unique occurrence of disulphide-stabilized structures in eutherian spermatozoa may serve to protect the spermatozoon from structural degradation by its own proteolytic activity during the relatively slow passage through the eutherian egg investments.     

Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm-oocyte interaction in mice

During maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate ³H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with ³H NEM. At the same time, sperm have established pronase-resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S-S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S-S bonds may be a prerequisite for sperm chromatin decondensation.     

Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.