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1.
Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin 总被引:9,自引:0,他引:9
Immunohistochemical studies with antiserum against the protamines of the toad, Bufo japonicus, revealed that the sperm nucleus loses protamines within 5 min after entry into the egg. Likewise, lysolecithin-permeabilized sperm incubated with the egg extract lose the protamines within 1 min, accompanied by nuclear decondensation. The activities that induce both protamine removal and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos or adult tissues. SDS-PAGE analyses revealed that the egg extract removed not only protamines from the Bufo sperm, but also selectively the sperm-specific basic proteins from sperm nuclei of Xenopus laevis. The protamine-removing activity (PRA) was partially purified from egg extracts as negatively charged macromolecules by anion-exchange chromatography and gel filtration. The PRA was heat-stable (100 degrees C, 10 min) and sensitive to proteinase K, but not to RNase A and DNase I. Immunoblot analysis of the supernatant after incubation of Bufo sperm in the fraction with the PRA revealed that protamines derived from sperm nuclei were associated with a major protein of the fraction. This protein exhibited mobilities of 140 and 36 kDa on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 to 4.5 and possessed an amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine. 相似文献
2.
Stephens S Beyer B Balthazar-Stablein U Duncan R Kostacos M Lukoma M Green GR Poccia D 《Molecular reproduction and development》2002,62(4):496-503
Decondensation of compact and inactive sperm chromatin by egg cytoplasm at fertilization is necessary to convert the male germ cell chromatin to an active somatic form. We studied decondensation of sea urchin sperm nuclei in a cell-free extract of sea urchin eggs to define conditions promoting decondensation. We find that egg cytosol specifically phosphorylates two sperm-specific (Sp) histones in vitro in the same regions as in vivo. This activity is blocked by olomoucine, an inhibitor of cdc2-like kinases, but not by chelerythrine, an inhibitor of protein kinase C (PKC). PKC phosphorylates and solubilizes the sperm nuclear lamina, one requirement for decondensation. Olomoucine, which does not inhibit lamina removal, blocks sperm nuclear decondensation in the same concentration range over which it is effective in blocking Sp histone phosphorylation. In a system free of other soluble proteins, neither PKC nor cdc2 alone elicit sperm chromatin decondensation, but the two act synergistically to decondense sperm nuclei. We conclude that two kinases activities are sufficient for sea urchin male pronuclear decondensation in vitro, a lamin kinase (PKC) and a cdc2-like Sp histone kinase. 相似文献
3.
4.
Okuwaki M Sumi A Hisaoka M Saotome-Nakamura A Akashi S Nishimura Y Nagata K 《Nucleic acids research》2012,40(11):4861-4878
Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins-NPM1, NPM2 and NPM3-expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells. 相似文献
5.
Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts. 总被引:18,自引:0,他引:18
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. 相似文献
6.
Xenopus nucleoplasmin: egg vs. oocyte 总被引:14,自引:0,他引:14
Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis. We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis. Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins. The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro. Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested. Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein. The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered. 相似文献
7.
Massive phosphorylation distinguishes Xenopus laevis nucleoplasmin isolated from oocytes or unfertilized eggs 总被引:7,自引:0,他引:7
Nucleoplasmin isolated from unfertilized Xenopus laevis eggs possesses an in vitro chromatin assembly activity which is superior to nucleoplasmin isolated from oocytes. It is demonstrated here that the two forms of the protein differ in the amount of attached phosphate, with the egg protein possessing nearly 20 phosphate groups per protein monomer and the oocyte protein possessing less than 10 phosphate groups per monomer. A kinase preparation from unfertilized eggs is shown to be capable of modifying oocyte nucleoplasmin so that it displays the electrophoretic heterogeneity of egg nucleoplasmin. Furthermore, when the egg protein is treated with phosphatase and repurified, the chromatin assembly activity deteriorates to the level of the oocyte protein. 相似文献
8.
Tamada H Van Thuan N Reed P Nelson D Katoku-Kikyo N Wudel J Wakayama T Kikyo N 《Molecular and cellular biology》2006,26(4):1259-1271
Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation. 相似文献
9.
During maturation, the mouse oocyte is transformed into a highly polarized egg, characterized by an actin cap and cortical granule-free domain (CGFD) overlying the meiotic spindle that is in close proximity to the cortex. The presence of spindle/chromosomes or microinjected sperm chromatin in the cortical region initiates this cortical reorganization, but the pathway is unknown. We report that cortical reorganization induced by microinjected sperm chromatin is blocked by inhibitors of microfilament assembly or disassembly. Active mitogen-activated protein kinase (MAPK), which becomes enriched in the region of sperm chromatin, is required for cortical reorganization, because microinjected sperm chromatin fails to induce cortical reorganization in Mos-/- eggs, which lack MAPK activity. Last, myosin light chain kinase (MLCK), which can be directly phosphorylated and activated by MAPK, appears involved, because the MLCK inhibitors ML-7 and Peptide 18 prevent sperm chromatin-induced cortical reorganization. These results provide new insights into how cortical reorganization occurs independently of extracellular signals to generate egg polarity. 相似文献
10.
We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S. 相似文献
11.
Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation 总被引:17,自引:0,他引:17
Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin. 相似文献
12.
Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions. 总被引:18,自引:3,他引:15
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The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin. 相似文献
13.
A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1. 总被引:20,自引:2,他引:18
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M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B. 相似文献
14.
Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc. 相似文献
15.
At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation 总被引:10,自引:1,他引:9
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《The Journal of cell biology》1993,123(4):859-868
MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope. 相似文献
16.
17.
The cyclin B2 component of MPF is a substrate for the c-mos(xe) proto-oncogene product 总被引:15,自引:0,他引:15
Previous studies from this laboratory have shown that purified MPF from Xenopus eggs contains cyclin B2 complexed with cdc2 kinase. The activation of MPF during oocyte maturation is known to require expression of the c-mos(xe) proto-oncogene. We show here that immunoprecipitates of either v-mos from Moloney murine sarcoma virus-transformed NIH 3T3 cells or c-mos from Xenopus eggs phosphorylate cyclin B2 in vitro. Phosphopeptide analysis reveals a pattern similar to that observed with cdc2 kinase. Moreover, ablation of c-mos(xe) from oocytes by antisense oligonucleotide injection reduces the rate of cyclin B2 phosphorylation in oocyte extracts by 40%. These results suggest that the mechanism of activation of MPF by c-mos(xe) involves phosphorylation of the cyclin component. 相似文献
18.
Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor
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Expression of the mosxe protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mosxe functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mosxe or kinase-inactive mosxe(R90), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mosxe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mosxe, this indicates that the major phosphopeptides of mosxe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mosxe protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mosxe were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine----alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mosxe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine----alanine mosxe mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mosxe mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF. 相似文献
19.
Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation. 总被引:25,自引:9,他引:16
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Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase. 相似文献
20.
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. 相似文献