Sperm Histone Research Articles

SpH‐protease exists in Caco‐2 cells

We have described a 60 kDa nuclear cysteine protease that degrades sperm histones during male chromatin remodeling after fertilization in sea urchins (SpH protease). This enzyme was shown to be localized in the mitotic furrow and to be involved in cell cycle control in sea urchin embryos. The N terminal sequence and the gene encoding this protease have homology with human cathepsin L family; therefore we investigated with antibodies raised against the sea urchins protease if a similar protease is present in Caco‐2 cells. Among Caco 2 chromosomal proteins obtained from confluent cell cultures, we found by Western blots, two bands migrating at 60 and 100 kDa in SDS/PAGE. We have also used these anti SpH protease antibodies to immunolocalize this protease in the intracellular context. This protease in mitotic cells localize on the mitotic furrow and remains in the nucleus in binuclear cells. On the other hand, when inhibitors of cysteine‐proteasas were used in different phases of the cell cycle we observed a disorganization of the mitotic spindle and an alteration in the localization of the protease during the S phase affecting DNA replication. Our results demonstrate that in Caco 2 cells exist a variant of cathepsin L protease that exhibits a similar localization to that was described for embryos of sea urchin, indicating a putative rol in cell proliferation. FONDECYT 11070067.

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.

Self-association of Chaetopterus variopedatus sperm histone H1-like. Relevance of arginine content and possible physiological role.

Self-association of histones H1 from calf thymus and from sperm of the marine worm Chaetopterus variopedatus was studied on native and glutaraldehyde cross-linked molecules by PAGE and by salt-induced turbidity measurements. Multiple polymers were generated by native sperm histone H1-like after glutaraldehyde cross-linking while the same treatment on its lysine- or arginine-modified derivatives and on somatic histone H1 failed to induce polymerization. This result suggests the relevance of arginine content in the formation of histone H1-like polymers particularly because Chaetopterus variopedatus and calf thymus histones H1 have similar content of lysine but different K/R ratio (2 and 15, respectively). Salt-induced turbidity experiments confirmed the high tendency of sperm histone H1-like to form oligomers, particularly in the presence of phosphate ions. Native PAGE analysis in the presence of phosphate supported this hypothesis. The reported results suggest that phosphate ions connecting lysine and arginine side chain groups contribute to the interaction of sperm histone H1-like with DNA in chromatin and play a key role in organization and stabilization of the chromatin higher order structures.

Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus.

To simultaneously determine the localization of histones and protamines within human sperm nuclei. Immunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei. Immunofluorescent localization of histones, protamine 1 (PRM1) and protamine 2 (PRM2) along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region (i.e. the sperm nuclear annulus), whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus. The co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.

Labelling of the rat spermatid nucleus with 3H-arginine.

The incorporation of 3H-arginine by the nucleus of the rat spermatid was studied with electron microscopic autoradiography. The nucleus is slightly labelled with 3H-arginine until step 9 of spermiogenesis, where the labelling ceases when the nucleus condenses. Thereafter there is a high level of 3H-arginine incorporation into the spermatid nuclei at steps 15–16, reflecting the formation of the arginine rich sperm histone. This histone is synthesiszed in the cytoplasm close to the nuclear membrane. Based on this it is suggested that the arginine rich sperm histone does not play any role in the condensation of the sperm nuclei or in the cessation of RNA synthesis. It appears to be the package form of the DNA during the period of its transit from one generation to the next.

Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones

To compare the aniline blue assay with and without eosin, and to correlate the results with pregnancy outcome after intracytoplasmic sperm injection (ICSI) procedure. A retrospective study. University-based fertility center. One hundred thirty infertile patients. Left-over washed sperm after each ICSI procedure were fixed on glass slides and stained with aniline blue with and without eosin. Chromatin condensation, pregnancy, and age. Percentage chromatin condensation assessed by aniline blue-eosin was higher compared with standard aniline blue (72.4 +/- 2.4% vs. 64.0 +/- 2.4% [mean +/- SEM]). Chromatin condensation was higher in pregnant (86.6 +/- 0.9%) versus nonpregnant (80.9 +/- 2.1%) women age 35 years or more. In younger women, chromatin condensation was not correlated with pregnancy outcome. There was no correlation between chromatin condensation and ICSI fertilization or male age. Adding eosin counterstain to aniline blue improved assessment of chromatin condensation, suggesting that the standard assay underestimated chromatin condensation. The association between chromatin condensation and pregnancy in older but not younger women suggests that oocytes of younger women had the capacity to compensate for the immature sperm shortcomings.

Presence and Release of Bovine Sperm Histone H1 During Chromatin Decondensation by Heparin-Glutathione

During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 μM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37°C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 μM heparin and 5.0 mM GSH for 4.0 h at 37°C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the bovine sperm nuclei. H1 depletion may participate in chromatin decondensation and nuclear swelling induced by heparin-GSH.

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.

Testis/Sperm Specific Histone H2B-127: Expression in Human Sperm and Correlations With Semen Quality and Fertility Status

A significant proportion of infertile men have sperm with chromatin defects including abnormal DNA packaging in the nuclei. Such abnormalities have been suggested as a possible reason for infertility. In human sperm, DNA is packaged by protamines (85%) and histones (15%). A significant portion of the histones in sperm is represented by testis /sperm specific variants. Interestingly, immunolocalization procedures have revealed that one of the members of the histone complement, testis / sperm specific histone H2B which is made up of 127 amino acids (TSH2B-127), is present in only 20% of mature human sperm cells.

During male pronuclei formation chromatin remodeling is uncoupled from nucleus decondensation

Male pronucleus formation involves sperm nucleus decondensation and sperm chromatin remodeling. In sea urchins, male pronucleus decondensation was shown to be modulated by protein kinase C and a cdc2-like kinase sensitive to olomoucine in vitro assays. It was further demonstrated that olomoucine blocks SpH2B and SpH1 phosphorylation. These phosphorylations were postulated to participate in the initial steps of male chromatin remodeling during male pronucleus formation. At final steps of male chromatin remodeling, all sperm histones (SpH) disappear from male chromatin and are subsequently degraded by a cysteine protease. As a result of this remodeling, the SpH are replaced by maternal histone variants (CS). To define if sperm nucleus decondensation is coupled with sperm chromatin remodeling, we have followed the loss of SpH in zygotes treated with olomoucine. SpH degradation was followed with anti-SpH antibodies that had no cross-reactivity with CS histone variants. We found that olomoucine blocks SpH1 and SpH2B phosphorylation and inhibits male pronucleus decondensation in vivo. Interestingly, the normal schedule of SpH degradation remains unaltered in the presence of olomoucine. Taken together these results, it was concluded that male nucleus decondensation is uncoupled from the degradation of SpH associated to male chromatin remodeling. From these results, it also emerges that the phosphorylation of SpH2B and SpH1 is not required for the degradation of the SpH that is concurrent to male chromatin remodeling.

Persistent histones in immature sperm are associated with DNA fragmentation and affect paternal contribution of sperm: A study of aniline blue staining, fluorescence in situ hybridization (FISH) and DNA nick translation

We have tested whether aniline blue staining of sperm is related to DNA chain breakage. During sperm development there are concurrent nuclear and cytoplasmic processes, including meiosis, DNA repair, histone-protamine replacement, and cytoplasmic extrusion. Aniline blue, a marker of persistent histones in immature sperm, may stain sperm light (L, mature sperm), intermediate (IN) and dark (D, immature sperm, high level of persistent histones). It was reported that the immature D sperm do not show FISH chromosome labeling, whereas in L and IN there are well characterized FISH signals. We have examined, with a combination of aniline blue staining and FISH or aniline blue staining and DNA nick translation, whether the lack of FISH signals in D sperm is caused by an interference of aniline blue staining with FISH, or if in immature D sperm there is also DNA fragmentation, thus the FISH probes fail to recognize the chromosomes. Sperm were stained with aniline blue, and the microscopic fields of aniline blue stained sperm were digitized and captured by the computer assisted Metamorph program (Universal Imaging Co., PA). Subsequently, the aniline blue stain was bleached with 30% methanol-DDW, and the slides were subjected to either multicolor FISH using X, Y, and 17 chromosome probes, or to DNA nick translation. Nick translation generates color via enzyme linked nucleotides inserted at DNA chain breaks. The color intensity is proportional with the extent of DNA fragmentation, and graded as light nick (LN), intermediate nick (IMN) and dark nick (DN) sperm. Further, the saved aniline blue stained sperm images were compared with those of the same sperm after FISH or DNA nick translation treatment. Statistical evaluation by Student t, Mann-Whitney, and the Spearman’s correlation tests was carried out by the SigmaStat program (Jandel, CA). We confirmed that D sperm indeed fail to show FISH signal, whereas in L and IN sperm the fluorescence chromosomes are well detectable. The sperm stained for aniline blue and nick translation provided evidence for extensive DNA degradation in D sperm. In 5 men we studied 600 spermatozoa. There was an agreement between the staining patterns with aniline blue and subsequent nick translation in 82.8% of sperm (500 of 600): 46.5% of sperm stained as L and LN, 27.7% of sperm stained IM and IMN, and 7.6% of sperm stained D and DN, respectively. Thus, sperm with high level of persistent histones have also shown extensive DNA degradation. Only 10 sperm of 600 had an L-DN or D-LN discordant pattern. The correlation between the aniline blue and nick translation staining patterns was r=0.82, p<0.001. Using probes for persistent histones and fragmented DNA in the same sperm indicates that diminished maturity sperm show defects in both histone-protamine replacement and DNA fragmentation. Both persistent histones and DNA fragmentation affect the paternal contributions of sperm to the zygote. Thus, aniline blue staining may facilitate the quick and inexpensive prospective evaluation of DNA integrity in husbands treated with assisted reproduction.

Cysteine-protease involved in male chromatin remodeling after fertilization co-localizes with alpha-tubulin at mitosis.

We postulated an essential role for a cysteine-protease in sea urchins sperm histones degradation which follows fertilization. We now report the purification of this enzyme, the determination of its N-terminal amino acid sequence and the localization of the protein with antibodies generated against this amino-terminal peptide. The immunofluorescence data confirmed the presence of this enzyme in the nucleus of unfertilized eggs. After fertilization labeling is observed both in female and male pronuclei suggesting a rapid recruitment of the enzyme to the male pronuclei. Interestingly, we have found that this cysteine-protease persists in the nucleus of the zygotes during S phase of the cell cycle and co-localizes with alpha-tubulin that organizes the mitotic spindle during the initial embryonic cell division.

Remodeling of sperm chromatin after fertilization involves nucleosomes formed by sperm histones H2A and H2B and two CS histone variants.

The composition of nucleosomes at an intermediate stage of male pronucleus formation was determined in sea urchins. Nucleosomes were isolated from zygotes harvested 10 min post-insemination, whole nucleoprotein particles were obtained from nucleus by nuclease digestion, and nucleosomes were subsequently purified by a sucrose gradient fractionation. The nucleosomes derived from male pronucleus were separated from those derived from female pronucleus by immunoadsorption to antibodies against sperm specific histones (anti-SpH) covalently bound to Sepharose 4B (anti-SpH-Sepharose). The immunoadsorbed nucleosomes were eluted, and the histones were analyzed by Western blots. Sperm histones (SpH) or alternatively, the histones from unfertilized eggs (CS histone variants), were identified with antibodies directed against each set of histones. It was found that these nucleosomes are organized by a core formed by sperm histones H2A and H2B combined with two major CS histone variants. Such a hybrid histone core interacts with DNA fragments of approximately 100 bp. It was also found that these atypical nucleosome cores are subsequently organized in a chromatin fiber that exhibits periodic nuclease hypersensitive sites determined by DNA fragments of 500 bp of DNA. It was found that these nucleoprotein particles were organized primarily by the hybrid nucleosomes described above. We postulate that this unique chromatin organization defines an intermediate stage of male chromatin remodeling after fertilization.

Characterization of human lactoferricin as a potent protein kinase CK2 activator regulated by A-kinase in vitro.

Lactoferricin (LFcin) hydrolyzed from lactoferrin (LF), a major 80 kDa iron-binding protein in milk and other exocrine secretions, was characterized as a potent activator of protein kinase CK2 (CK2) in vitro. Human LFcin (hLFcin) at 0.5 microg stimulated approx. 5-fold CK2 activity [phosphorylation of 60S acidic ribosomal proteins (P0, P1, P2) and Hsp90 (p98)] in a manner similar to other functional proteins with oligo-Arg clusters, such as salmine A1, sperm histone H2B and HIV-1 Rev. Interestingly, this stimulatory effect of hLFcin was significantly reduced when it was phosphorylated by A-kinase in vitro. These results suggest that (i) hLFcin acts as a potent CK2 activator in vitro; and (ii) the stimulatory effect of hLFcin on CK2 activity is regulated by its phosphorylation by A-kinase in vitro.

Variability of sperm specific histones in sea urchins

The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0–8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.

Cytoplasm of sea urchin unfertilized eggs contains a nucleosome remodeling activity.

After fertilization the sea urchin sperm nucleus transforms into the male pronucleus which later fuses with the female pronucleus re-establishing the diploid genome of the embryo. This process requires remodeling of the sperm chromatin structure including the replacement of the sperm histones by maternally derived cleavage stage histone variants. In recent years, a group of protein complexes that promote chromatin-remodeling in an ATP-dependent manner have been described. To gain understanding into the molecular mechanisms operating during sea urchin male pronuclei formation, we analyzed whether chromatin-remodeling activity was present in unfertilized eggs as well as during early embryogenesis. We report that in the sea urchin Tetrapygus niger, protein extracts from the cytoplasm but not from the nucleus, of unfertilized eggs exhibit ATP-dependent nucleosome remodeling activity. This cytosolic activity was not found at early stages of sea urchin embryogenesis. In addition, by using polyclonal antibodies in Western blot analyses, we found that an ISWI-related protein is primarily localized in the cytoplasm of the sea urchin eggs. Interestingly, SWI2/SNF2-related proteins were not detected neither in the nucleus nor in the cytoplasm of unfertilized eggs. During embryogenesis, as transcriptional activity is increased an ISWI-related protein is found principally in the nuclear fraction. Together, our results indicate that the cytoplasm in sea urchin eggs contains an ATP-dependent chromatin-remodeling activity, which may include ISWI as a catalytic subunit.

Phosphorylation protects sperm-specific histones H1 and H2B from proteolysis after fertilization.

At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.

Histone- and protamine-DNA association: conservation of different patterns within the beta-globin domain in human sperm.

Most DNA in human sperm is bound to highly basic proteins called protamines, but a small proportion is complexed with histones similar to those found in active chromatin. This raises the intriguing possibility that histones in sperm are marking sets of genes that will be preferentially activated during early development. We have examined the chromatin structure of members of the beta-globin gene family, which are expressed at different times in development, and the protamine 2 gene, which is expressed in spermatids prior to the widespread displacement of histones by transition proteins. The genes coding for epsilon and gamma globin, which are active in the embryonic yolk sac, contain regions which are histone associated in the sperm. No histone-associated regions are present at the sites tested within the beta- and delta-globin genes which are silent in the embryonic yolk sac. The trends of histone or protamine association are consistent for samples from the same person, and no significant between-subject variations in these trends are found for 13 of the 15 fragments analyzed in the two donors. The results suggest that sperm chromatin structures are generally similar in different men but that the length of the histone-associated regions can vary. The association of sperm DNA with histones or protamines sometimes changes within as little as 400 bp of DNA, suggesting that there is fine control over the retention of histones.

Cytoplasmic control of nuclear assembly.

The reconstitution of a replication-competent, transcriptionally active nucleus following mitosis, fertilization or nuclear transplantation involves a stepwise series of reactions, most (if not all) of which are controlled by the cytoplasmic environment. This review discusses the nature of cytoplasmic contributions to the development of the male pronucleus at fertilization, and the effect of altering the cytoplasmic environment on nuclear assembly. The system used to investigate these regulations consists of permeabilized sea urchin sperm nuclei incubated under controlled conditions in a cell-free extract of fertilized sea urchin eggs. (1) In egg cytoplasmic extract, male pronuclear formation is initiated by the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation by a cytosolic protein kinase C. (2) Sperm histones are phosphorylated by an as yet unidentified soluble kinase. (3) The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-and cytosolic pH-dependent manner. (4) Chromatin decondensation is associated with the replacement of sperm histones by maternal histones. (5) Nuclear membranes form by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of these vesicles to one another. (6) Three cytoplasmic vesicle populations with distinct biochemical, chromatin-binding and fusion properties are required for nuclear envelope assembly. (7) Targeting of the bulk of nuclear membrane vesicles to chromatin is mediated by an integral membrane protein similar to human lamin B receptor. (8) The last step of male pronuclear formation, nuclear swelling, is promoted by the assembly of nuclear pores, nuclear import of soluble lamins and growth of the nuclear membranes. (9) Once inside the nucleus, lamin B associates with lamin B receptors, presumably to tether the inner nuclear membrane with the lamina. Overall, these processes are similar to those characterizing nuclear reconstitution after mitosis in somatic cells or nuclear remodeling following transplantation into oocytes or eggs. The influence of the egg cytoplasmic environment on some aspects of nuclear remodeling after nuclear transplantation is also discussed.