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

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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.

supporting

confidence: 71%

Microinjection of an antibody against the cysteine‐protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle

Puchi

Quiñones

Concha

et al. 2006

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“…The maintenance of the polynucleosomes organization after the incubation with the protease was analyzed by following its electrophoretic migration on 1% (w/v) agarose gels. The integrity of the SpH that were forming these polynucleosomes were further analyzed by SDS-PAGE followed by Western blots revealed with polyclonal antibodies against SpH that were performed as described previously [Oliver et al, 2002]. Western blots final detection was performed using a quimioluminiscence ECL Kit (Amersham Pharmacia Biotech, UK).…”

Section: Proteolytic Assaymentioning

confidence: 99%

“…Nucleosomes were obtained by incubating the isolated nuclei with 72 units/ml of micrococcal nuclease (MNase) (Worthington, New Jersey) in a buffer 0.01 M Tris-HCl at pH 7.6, 0.01 M NaCl, 2.5 mM MgCl 2 , 1 mM CaCl 2 at 378C for 10 min. The nucleoprotein particles derived from MNase digestion were further purified on a sucrose density gradient 5-20% (w/v) in a buffer 10 mM Tris-HCl pH 7.2 containing 0.7 mM Na 2 EDTA and analyzed by electrophoresis on horizontal 1% (w/v) agarose gels in 1 mM EDTA and 0.04 M Tris-acetate buffer pH 8.0, as described by Oliver et al [2002]. The initial fractions (1-2) of the sucrose gradient containing polynucleosomes free of unbound DNA were used to determine the effect of the protease on the polynucleosomes in vitro or alternatively for the screening of a SNDA after fertilization.…”

Section: Polynucleosomes Isolationmentioning

confidence: 99%

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

Iribarren

Morín

Puchi

et al. 2007

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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.

“…Subsequently, the histone variants isolated either from unfertilized eggs or from the plutei larvas, were separated by electrophoresis in one-dimensional 18% (w/v) polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). After electrophoresis, these histones were transferred to nitrocellulose membranes, and immuno detected with antibodies anti-CS histone variants that were obtained as described previously [Oliver et al, 2002]. Since native CS histone variants were previously found to be poly(ADP-ribosylated) [Imschenetzky et al, 1996], the anti-CS variants antibodies were incubated with (ADP-ribose) polymers covalently bound to Sepharose 4B to remove the fraction of antibodies that may recognize the polymers of ADP-ribose.…”

Section: Isolation Of Histone Variants and Western Immunoblotsmentioning

confidence: 99%

Conservative segregation of maternally inherited CS histone variants in larval stages of sea urchin development

Oliver¹,

Rodríguez²,

Bustos³

et al. 2002

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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.