Two kinase activities are sufficient for sea urchin sperm chromatin decondensation in vitro

Stephens, Sahar M.; Beyer, Birgit; Balthazar‐Stablein, U.; Duncan, Richard P.; Kostacos, M.; Lukoma, M.; Green, G.R.; Poccia, Dominic

doi:10.1002/mrd.90005

Cited by 22 publications

(25 citation statements)

References 42 publications

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“…In sea urchins, previous attempts to detect a NMP2-like activity in vitro were unsuccessful and it was suggested that this cells lack nucleoplasmin [Stephens et al, 2002]. In contrast, NMP-like molecules were found to be encoded in the genomes of the sea urchins Lyctechinus pictus and Strogylocentrutus purpuratus.…”

Section: Discussionmentioning

confidence: 99%

“…We think that the apparent contradiction of the detection of the SNDA in this report with the lack of detection of a NMP-like activity by Stephens et al may be explained by the fact that the previous detection system was based on the information concerning the activity of NMP2 from Xenopus. Stephens et al [2002] tested the ability of sea urchin thermo-heated nuclear extract to promote sperm nuclear swelling in vitro. In contrast our assay was performed measuring the ability of the nuclear extracts obtained at low temperatures to disorganize isolated poly-nucleosomes.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleoplasmin participates as a chaperone in the assembly of nucleosome cores, by facilitating the addition of H2A-H2B dimers to the preformed H3-H4 tetramers organized by the chaperones N1/N2 (reviewed by Philpott et al [2000]). Unfortunately, previous attempts to detect nucleoplasmin in sea urchins were unsuccessful [Stephens et al, 2002]. In this context we have further investigated if a sperm nucleosome disassembly activity (SNDA) was detectable in sea urchins after fertilization.…”

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confidence: 99%

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

Iribarren

Morín

Puchi

et al. 2007

J of Cellular Biochemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Iribarren

Morín

Puchi

et al. 2007

J of Cellular Biochemistry

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“…Step 1, condensed chromatin (gray) deconsenses in the presence of soluble cytosolic proteins (25). Simultaneously MVs (black) bind to the surface of the chromatin, initially around regions of the lipophilic structures (white circles), detergent-resistant membranes in the centriolar and acrosomal fossae (8).…”

Section: Pretreatment Of Mv1 With Pi-plc Renders It Fusogenicmentioning

confidence: 99%

Diacylglycerol Induces Fusion of Nuclear Envelope Membrane Precursor Vesicles

Barona

Byrne

Pettitt

et al. 2005

Journal of Biological Chemistry

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Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.At the end of each mitosis in eukaryotes, the nuclear envelope is typically reconstituted by membrane fusion, forming the nuclear compartment and segregating the chromosomes from the cytoplasm. A similar process encloses sperm chromatin in egg cytoplasm following fertilization. A number of studies emphasizing the role of proteins have addressed the mechanism of nuclear envelope assembly, many utilizing cell-free systems derived from eggs or somatic cells (1-4). However, relatively little attention has been paid to the essential role(s) played by membrane lipids in this process.Male pronuclear or somatic nuclear envelope formation involves binding of nuclear membrane precursors to the chromatin surface followed by fusion to create a double membrane enclosing the chromatin (1, 5-7). We have previously reported that envelope formation in a cell-free system derived from sea urchin eggs requires the fusion of three egg membrane vesicle populations and remnants of the sperm nuclear envelope at the tip and base of the conical nucleus (8 -10).One of the egg vesicle populations (MV1) 3 is particularly unusual. It is a low density fraction highly enriched in the membrane lipid phosphatidylinositol (PtdIns) (9, 11). MV1 binds at the tip and base of the sperm nucleus and is required for nuclear envelope formation, which can be induced by addition of GTP or a bacterial PtdIns-specific phospholipase C (PI-PLC) (9, 12). The endogenous sea urchin PI-PLC activity probably resembles a typical eukaryotic enzyme whose substrate is PtdIns(4,5)P 2 . GTP-initiated envelope formation is inhibited by GTP␥S and by the PI 3-kinase inhibitors, wortmannin and LY294002 (12, 13). Initi...

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“…It was further postulated that the phosphorylation of these sperm histones (SpH) is catalyzed by a cdc-2 like protein kinase [Stephens et al, 2002]. More recently, these phosphorylation events associated to male pronucleus formation, visualized as changes in male pronucleus shape, has been elucidated by in vitro assays.…”

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During male pronuclei formation chromatin remodeling is uncoupled from nucleus decondensation

Monardes

Iribarren

Morín

et al. 2005

J of Cellular Biochemistry

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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.

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Two kinase activities are sufficient for sea urchin sperm chromatin decondensation in vitro

Cited by 22 publications

References 42 publications

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

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

Diacylglycerol Induces Fusion of Nuclear Envelope Membrane Precursor Vesicles

During male pronuclei formation chromatin remodeling is uncoupled from nucleus decondensation

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