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In amphibians, sperm histone transition post-fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin-like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin-like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post-fertilization and this phosphorylation is dependent on CDK-cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin-like activity in sea urchins participating during male pronucleus formation post-fecundation., (Copyright © 2013 Wiley Periodicals, Inc.)

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell-cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH-protease) participates in male chromatin remodeling and in cell-cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38-42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco-2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non-proliferative cells., (Copyright © 2010 Wiley-Liss, Inc.)

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.

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., ((c) 2007 Wiley-Liss, Inc.)

Previously we have identified a cysteine-protease involved in male chromatin remodeling which segregates into the nuclei of the two blastomeres at the first cleavage division. Here we have investigated the fate of this protease during early embryogenesis by immunodetecting this protein with antibodies elicited against its N-terminal sequence. As shown in this report, the major 60 kDa active form of this protease was found to be present in the extracts of chromosomal proteins obtained from all developmental stages analyzed. In morula and gastrula the 70 kDa inactive precursor, which corresponds to the major form of the zymogen found in unfertilized eggs, was detected. In plutei larvas, the major 60 kDa form of this enzyme was found together with a higher molecular weight precursor (90 kDa) which is consistent with the less abundant zymogen primarily detected in unfertilized eggs. As reported here, either the active protease or its zymogens were visualized in most of the embryonic territories indicating that this enzyme lacks a specific pattern of spatial-temporal developmental segregation. Taken together our results indicate that this protease persists in the embryo and is ubiquitously distributed up to larval stages of development, either as an active enzyme and/or as an inactive precursor. These results suggest that this enzyme may display yet unknown functions during embryonic development that complement its role in male chromatin remodeling after fertilization., ((c) 2007 Wiley-Liss, Inc.)

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development., (Copyright 2006 Wiley-Liss, Inc.)

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., (Copyright (c) 2005 Wiley-Liss, Inc.)

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs., ((c) 2005 Wiley-Liss, Inc.)

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., (2004 Wiley-Liss, Inc.)

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2002 Wiley-Liss, Inc.)

Chromatin remodeling of the bone-specific rat osteocalcin (OC) gene accompanies the onset and increase in OC expression during osteoblast differentiation. In osseous cells expressing OC, the promoter region contains two nuclease hypersensitive sites that encompass the elements that regulate basal tissue-specific and vitamin D-enhanced OC transcription. Multiple lines of evidence indicate that DNA methylation is involved in maintaining a stable and condensed chromatin organization that represses eukaryotic transcription. Here we report that DNA methylation at the OC gene locus is associated with the condensed chromatin structure found in cells not expressing OC. In addition, we find that reduced CpG methylation of the OC gene accompanies active transcription in ROS 17/2.8 rat osteosarcoma cells. Interestingly, during differentiation of primary diploid rat osteoblasts in culture, as the OC gene becomes increasingly expressed, CpG methylation of the OC promoter is significantly reduced. Inhibition of OC transcription does not occur by a direct mechanism because in vitro methylated OC promoter DNA is still recognized by the key regulators Runx/Cbfa and the vitamin D receptor complex. Furthermore, CpG methylation affects neither basal nor vitamin D-enhanced OC promoter activity in transient expression experiments. Together, our results indicate that DNA methylation may contribute indirectly to OC transcriptional control in osteoblasts by maintaining a highly condensed and repressed chromatin structure.

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., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 1999 Wiley-Liss, Inc.)

The role of proteolysis during fertilization has been investigated only to a very limited extent as compared with its role on the control of cell cycle progression. In this report, we discuss briefly the proteases involved in fertilization, their relevance in the egg-sperm interaction and in the chromatin remodeling that occurs before the reestablishment of the diploid condition of the zygote. We further emphasize how the post-translational modifications of target proteins modulate these proteolytic events. J. Cell. Biochem. Suppls. 32/33:149-157, 1999., (Copyright 1999 Wiley-Liss, Inc.)

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.

To determine the changes in chromatin organization during male pronucleus remodeling, we have compared the composition of nucleoprotein particles (NP-ps) resulting from digestion with endogenous nuclease (ENase) and with micrococcal nuclease (MNase). Whole nuclei were isolated from sea urchin gametes and zygotes containing partially decondensed (15 min postinsemination, p.i.) or a fully decondensed (40 min p.i.) male pronucleus and digested with nucleases. The NP-ps generated were analyzed in agarose gels, and their histone composition was determined. Sperm core histones (SpH) and cleavage stage (CS) variants were identified by Western immunoblots revealed with specific antibodies. A single NP-ps was generated after digestion of sperm nucleus with MNase, which migrated in agarose gels between DNA fragments of 1.78-1.26 Kb. Sperm chromatin remained undigested after incubation in ENases activating buffer, indicating that these nuclei do not contain ENases. One type of NP-ps was obtained by digestion of unfertilized egg nuclei, either with ENase or MNase; the NP-ps was located in the region of the agarose gel corresponding to DNA fragments of 3.4-1.95 Kb [Imschenetzky et al. (1989): Exp Cell Res 182:436-444]. When whole nuclei from zygotes containing the female pronucleus and a partially remodeled male pronucleus were digested with ENase, a single NP-ps was generated, which migrated between DNA fragments of 2.5-1.9 Kb. This particle contained only CS histone variants. Alternatively, when these nuclei were digested with MNase, two NP-ps were generated; the slower migrating NP-ps (s) was located in the same position of the agarose gel as those resulting from ENase digestion and the faster migrating NP-ps (f) migrated between DNA fragments of 1.95-1.26 Kb. It was found that NP-ps (s) contained only CS histone variants, whereas NP-ps (f) were formed by a subset of SpH and by CS histone variants. When nuclei from zygotes containing a fully decondensed male pronucleus were digested either with ENase or MNase, a single type of NP-ps was observed, which migrated in the same position as NP-ps (s) in agarose gels. This particle contained only CS histone variants. On the basis of the histone compositions and on electrophoretic similarities, it was concluded that NP-ps (s) originated from the female pronucleus and that NP-ps (f) were generated from the partially remodeled male pronucleus. Consequently, our results indicate that at an intermediate stage of male pronucleus remodeling the chromatin is formed by NP-ps containing a subset of both SpH and of CS histone variants, whereas at final stages of male pronucleus decondensation chromatin organization is similar to that of the female pronucleus.

Sea urchin CS histone variants are electrophoretically heterogeneous when analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previous results suggested that this heterogeneity is due to the poly (ADP-ribosylation) of these proteins. Consequently, native CS histone variants were subjected to different treatments to remove the ADP-ribose moiety. The incubation in 1 M hydroxylamine was not effective in eliminating the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the ADP-ribose moiety was successfully removed from the CS variants by incubation with phosphodiesterase (PDE). To eliminate contamination of CS histone variants with PDE extract, the enzyme was covalently bound to Sepharose 4B prior to its utilization. Treatment of native CS histone variants with this immobilized phosphodiesterase removed around 85% of the total ADP-ribose moiety from these proteins. After S-PDE treatment the complex electrophoretic pattern of CS histone variants in 2-D PAGE decreases to five major fractions. From these results we conclude that the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are poly(ADP)-ribosylated).

To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP-ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benzamide (3 ABA) during the interval between 3 and 30 min post-insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP-ribosylation) of CS histone variants.

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of 3H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with 3H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased 3H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation.(ABSTRACT TRUNCATED AT 250 WORDS)

To obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western immunoblot analysis with polyclonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5-15 min post insemination (p.i.), SpH2B and SpH2A disappear 20-40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.

The patterns of poly(ADP-ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post-translational modification was detected by Western immunoblots with rabbit sera anti-poly(ADP-ribose) that was principally reactive against ADP-ribose polymers and slightly against ADP-ribose oligomers. The effect of 3 aminobenzamide (3-ABA), an inhibitor of the poly(ADP-ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti-poly(ADP-ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3-ABA inhibited the poly(ADP-ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP-ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.