Decondensed Sperm Chromatin Research Articles

Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin.

Cysteine oxidation in protamines leads to their oligomerization and contributes to sperm chromatin compaction. Here we identify the Drosophila thioredoxin Deadhead (DHD) as the factor responsible for the reduction of intermolecular disulfide bonds in protamines and their eviction from sperm during fertilization. Protamine chaperone TAP/p32 dissociates DNA-protamine complexes in vitro only when protamine oligomers are first converted to monomers by DHD. dhd-null embryos cannot decondense sperm chromatin and terminate development after the first pronuclear division. Therefore, the thioredoxin DHD plays a critical role in early development to facilitate the switch from protamine-based sperm chromatin structures to the somatic nucleosomal chromatin.

Deciphering the structure of DNA toroids

Toroids are small donut shaped organizational units within sperm chromatin and viruses containing DNA and protein. Investigators first characterized the dimensions of toroids created in vitro, in viruses and in decondensed sperm chromatin using transmission electron and atomic force microscopy. More recent measurements, performed using cryo-electron microscopy, have allowed experimenters to observe the hexagonal organization of DNA within viruses, and toroids created from DNA and cobalt hexammine. However, it has been difficult to obtain information about the assembly of DNA into a toroid, its structure and the biomechanical forces involved because of the limitations of these techniques. Similarly, biophysical studies of toroids utilizing techniques such as circular dichroism or light scattering are difficult to perform and interpret because toroids created using bulk DNA can aggregate and precipitate out of solution even at very low concentrations. The development of optical and magnetic traps has allowed experimenters to manipulate single DNA molecules within microfluidic, multichannel flow cells and measure the structural changes they undergo as they are transformed into toroids. During the past few years investigators have demonstrated that toroids consist of loops of DNA. They have observed the stepwise incorporation of these loops into a toroid that is not in contact with charged surfaces, which might affect its formation. The condensation of a constrained DNA molecule into a toroid was observed to significantly increase its tension, which reduced the size of the DNA loops that form the toroid. This structural information is important for understanding how genomic DNA is assembled and organized within the sperm cell and viruses. In this perspective we discuss what is known about the structure and formation of toroids, what has been learned recently using single molecule techniques and what remaining questions have the potential to be answered using these emerging technologies.

Expression of human oocyte-specific linker histone protein and its incorporation into sperm chromatin during fertilization

To investigate the expression of oocyte-specific linker histone protein (hH1FOO) in human ovaries and its incorporation into sperm chromatin after intracytoplasmic sperm injection (ICSI). Laboratory study. University hospital. Human ovarian tissues were obtained from patients at oophorectomy. Human oocytes and embryos were obtained from infertile patients undergoing IVF and ICSI. A polyclonal rabbit antibody targeting the predicted hH1FOO protein was used for immunohistochemical analysis. Western blot analysis and the reverse transcriptase-nested polymerase chain reaction were done to detect hH1FOO in chromatin of germinal vesicle-stage oocytes through to two-cell embryos. The hH1FOO antibody reactivity of oocytes, ovarian tissues, and sperm chromatin after ICSI. hH1FOO protein was localized in all oocytes from primordial to Graafian follicles. In unfertilized oocytes after ICSI, the chromatin of injected sperm was condensed without hH1FOO incorporation in 45.5% of oocytes, condensed with hH1FOO incorporation in 9%, and decondensed with hH1FOO incorporation in 45.5%. None of the oocytes contained decondensed sperm chromatin without hHFOO incorporation. hH1FOO protein was expressed by human oocytes from primordial follicles to early embryogenesis. Sperm nuclei that were still condensed after ICSI could be separated into two categories by hH1FOO incorporation, which might provide valuable information regarding failed fertilization.

Nuclear competence for maturation and pronuclear formation in mouse oocytes

In response to gonadotrophins, a fully grown mouse oocyte matures to the metaphase of the second meiotic division and becomes competent for the development of female and male pronuclei after fertilization. The present study was carried out to clarify when during the growth period an oocyte nucleus acquires the ability to promote pronuclei formation after fertilization. Fully grown germinal vesicle (GV) oocytes were enucleated and fused with nuclei from growing oocytes from 1-20 day old mice by standard nuclear transfer technique. The reconstructed oocytes were matured and fertilized in vitro, and pronuclear formation was assessed. The oocytes whose nuclei were exchanged for those of the non-growing-stage oocytes matured to the metaphase of the second meiotic stage, but no normal female pronuclei were formed. Female pronuclei first formed in 27% of the oocytes reconstituted with the nuclei of oocytes from 8 day old pups after fertilization. Recondensed sperm chromatin was detected in 27% of the oocytes reconstructed with oocyte nuclei from 8 day old mice, and a male pronucleus was first formed in 6% of the oocytes that had been reconstructed with the nuclei of oocytes from 15 day old mice. The sizes of the female and male pronuclei increased with oocyte donor age, and reached normal size when the oocytes from 15 and 20 day old mice respectively were used. An electron microscopic study using oocytes that had received the oocyte nuclei of 8 day old mice confirmed these results. The factors required for pronuclear formation are derived from fully grown GV oocytes, and the transformation from decondensed sperm chromatin to a recondensed male pronucleus is governed by GV-derived factors.

Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF-I, and NAP-I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.

Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins

BackgroundIn order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle.ResultsHere we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occuring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-γ-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-γ-S.ConclusionsNucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

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.

Transformation of sperm nuclei into metaphase chromosomes in maturing pig oocytes penetrated in vitro.

Cumulus-free pig oocytes at the germinal vesicle (GV) stage were incubated in modified Brackett & Oliphant's medium with 5% fetal calf serum and 5mM caffeine with or without cryopreserved, ejaculated spermatozoa. When oocytes were transferred into modified tissue culture medium (TCM-199B at pH 7.4) supplemented with 10 IU/ml eCG, 10 IU/ml hCG and 1 microg/ml oestradiol-17beta after 8 h of incubation with spermatozoa and cultured for 0-48 h, 86-99% of oocytes were penetrated. Most (95-100%) oocytes penetrated 0-16 h after transfer had decondensed sperm chromatin. However, 24 h after transfer 47% and 33% of penetrated oocytes contained recondensed sperm chromatin and sperm metaphase chromosomes, respectively. The proportion of penetrated oocytes containing sperm metaphase chromosomes increased after 36-48 h of transfer (51-65%). The transformation of sperm nuclei to metaphase chromosomes was obtained in 75% and 79% of anaphase I (AI) to telophase I (TI) and metaphase II (MII) oocytes, respectively, but only in 38% of metaphase I (MI) oocytes. Moreover, such transformation was observed only in 1 of 30 oocytes at the stages of GV breakdown to prometaphase I and none of 69 oocytes at the GV stage. The transformation of sperm nuclei into metaphase chromosomes was completely inhibited in oocytes penetrated by eight or more spermatozoa. Well-developed male and female pronuclei were observed in only 3 (4%) of 77 oocytes penetrated 48 h after transfer. The proportion of oocytes reaching MII was greatly inhibited by sperm penetration; only 18% of penetrated oocytes, but 87% of non-inseminated oocytes, reached MII by 48 h after transfer. None of the oocytes penetrated by seven or more spermatozoa reached MII. Most (75%) oocytes were inhibited from the transition from MI to MII even though they were cultured for 48 h. The present results indicate that: (1) the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes, (2) immature pig oocytes penetrated by spermatozoa can undergo meiotic maturation to MI, and (3) the transition of such oocytes from MI to MII is inhibited, suggesting that an activity of mitogen-activated protein kinase may be retarded.

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs.

During fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entry into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been a point of dispute. To resolve this issue, the fate of the SINEs of hamster sperm nuclei was traced after incorporation into immature oocytes at the germinal vesicle stage (GV oocytes) or fertilised eggs at the pronuclear stage (PN eggs). The SINEs disappeared quickly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelopes around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3 h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle, i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase promoting factor (MPF) is absent from the ooplasm.

Hyperphosphorylation of Nucleoplasmin Facilitates Xenopus Sperm Decondensation at Fertilization

Previous studies showed that the nuclear phosphoprotein nucleoplasmin performs the first stage of chromatin decondensation of Xenopus sperm at fertilization. It binds and removes sperm basic proteins replacing them with histones. We now show that this activity depends upon the massive hyperphosphorylation of nucleoplasmin that occurs when oocytes mature into eggs. Egg extracts or purified hyperphosphorylated egg nucleoplasmin decondense sperm chromatin and remove sperm basic proteins much faster than oocyte extracts or hypophosphorylated oocyte nucleoplasmin. Furthermore, dephosphorylation of egg nucleoplasmin slows sperm decondensation and prevents basic protein removal from sperm chromatin. We conclude that hyperphosphorylation of nucleoplasmin is used to modulate the rapid changes in chromatin structure that accompany early development in Xenopus.

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.

Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts

Nucleoplasmin is necessary and sufficient for the initial stage of Xenopus sperm decondensation in egg extracts. In this article we show that sperm decondensation is accompanied by loss of two sperm-specific basic proteins (X and Y) and gain of histones H2A and H2B, resulting in nucleosome formation. Purified nucleoplasmin alone removes X and Y and assembles purified H2A and H2B on decondensing sperm chromatin, forming nucleosome cores. Immunodepletion of nucleoplasmin from extract prevents removal of X and Y and addition of H2A and H2B, while adding back nucleoplasmin restores decondensation and X and Y removal. Thus, nucleoplasmin acts as both an assembly and a disassembly factor for remodeling sperm chromatin at fertilization.

Extracts from eggs and oocytes of Xenopus Laevis differ in their capacities for nuclear assembly and dna replication

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

The silver-stained NOR and argentophilic nuclear proteins in early mouse embryogenesis: a cytological study

Silver staining (Howell and Black, 1980) was used in light and electron microscopic studies for detecting the localization of argentophilic nuclear proteins in fertilized ova and cleaving mouse embryos. No silver-stained nucleolus organizing regions (NORs) (Ag-NORs) were visualized in the metaphase chromosomes of the first cleavage mitosis. From the 2-cell stage on, metaphase chromosomes contained Ag-NORs. Argentophilic proteins were detected in the pronuclei of the 1-cell embryos, i.e. before transcription of the ribosomal genes started. After fertilization these proteins accumulated on the decondensing sperm chromatin and telophase maternal chromosomes, then migrated into the pronuclei to be stored in pronucleoli, and, during mitosis, were transferred into the cytoplasm. In the metaphase chromosomes of the cleaving embryos Ag-NORs adequately reflected the transcription activity of the ribosomal genes, whereas in pronuclei of the 1-cell stage embryos argentophilic proteins were not involved in this process, but are likely to play a part in the formation and maturation of pronucleoli, and in the cell cycle regulation.

Fine structure of sheep fertilization in vitro.

The fertilization events of the sheep oocyte have been investigated by electron microscopy. Thirty-two oocytes recovered from the oviducts of superovulated ewes were fertilized in vitro by a procedure that has given rise to full embryonic development (Crozet et al.: Gamete Res 16:159-170, 1987) and were analyzed at different stages of fertilization. Sperm incorporation in the ooplasm occurred as early as 2 h postinsemination (PI). One hour later, a fertilization cone containing very few cytoplasmic organelles developed above the decondensing sperm chromatin. It was a transitory structure which disappeared when the male pronucleus (PN) was formed. The second meiotic division of the oocyte resumed soon after sperm incorporation; abstriction of the second polar body occurred by 3-4 h PI; the polar body constriction was associated with microfilaments. Both the male and female PN were formed at 5 h PI and were surrounded by nuclear envolopes containing pore complexes. The female PN was characterized by patches of condensed chromatin; the male PN was closely associated with the sperm neck and proximal midpiece. Thereafter the PN migrated toward the center of the egg, where the spindle of the first cleavage division was observed at 21 h PI. Dispermic ova analyzed at 2-4 h PI contained 2 decondensing sperm heads and 2 fertilization cones which have reached the same stage, thus suggesting that a few sperm may penetrate quite simultaneously. Multipronucleate eggs were characterized by disturbance of the PN migration.

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.

Effects of Ca 2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 m M EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca 2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca 2+ ion levels are kept low, but when Ca 2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca 2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.

Association of newly synthesized 3H‐arginine‐labeled proteins with chromatin structures in fertilization

AbstractZona‐free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine‐3H and the sites of localization of newly synthesized arginine‐3H–labeled proteins have been investigated using fine‐structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.

The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm.

The chromatin of Xenopus laevis sperm nuclei was induced to decondense, swell and form mitotic chromosomes following its injection into mature Rana pipiens oocytes. In contrast, the sperm chromatin did not decondense or form mitotic chromosomes when injected into oocytes from which the germinal vesicle (GV) was removed prior to the initiation of maturation. Injection into enucleated oocytes of the material extracted from manually-isolated GVs restored their ability to decondense sperm nuclei. This soluble GV material was stable at 18 degrees C for 16 h but was inactivated by heating to 80 degrees C for 10 min. We examined the distribution of this GV material in a cytoplasmic preparation from activated eggs which can induce sperm pronuclear formation in vitro. The cytoplasmic preparation was separated into soluble and particulate fractions by centrifugation and then each fraction was injected into enucleated eggs to determine whether or not it restored the ability to decondense sperm nuclei. We found that the soluble, but not the particulate fraction could restore the ability to decondense sperm nuclei to enucleated oocytes. This result clearly indicates that the soluble fraction contains most of the GV material required for chromatin decondensation. However, since the soluble fraction fails to decondense sperm chromatin in vitro in the absence of material from the particulate fraction, sperm pronuclear formation appears to require both the soluble material derived from the GV and particulate material which can develop in the oocyte cytoplasm in the absence of the GV.

The effect of the T/t locus on sperm penetration in vivo in the house mouse.

The timing and morphology of sperm penetration in vivo after insemination by C57BL/6�tW32/+ male mice were compared to sperm penetration after insemination by C57BL/6-+/+ males. Morphologically abnormal decondensing sperm chromatin was occasionally seen, indicating that abnormal spermatozoa may sometimes be successful in egg penetration. Spermatozoa from BL/6�tw32/+ males appeared to penetrate eggs sooner than spermatozoa from BLI6-+I+ males, suggesting that the T/t locus may have an effect on sperm function in fertilization, perhaps because of the other known abnormalities of these spermatozoa, such as an increased energy metabolism and unique surface antigens.