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

Abstract

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.

The Protease Degrading Sperm Histones Post-Fertilization in Sea Urchin Eggs Is a Nuclear Cathepsin L That Is Further Required for Embryo Development

“…Previous pharmacological experiments demonstrated that a cathepsin L inhibitor I-sensitive protease remains necessary beyond the first cell cycle post-fertilization suggesting that this protease activity can play a role in regulation of chromatin structure along embryogenesis [6]. In agreement, northern blot and semi-quantitative PCR analyses demonstrated that the transcripts encoded by the identified cathepsin L gene are constantly expressed during sea urchin development.…”

“…This substrate selectivity is also regulated by post-translational modification of the substrates: poly(ADP-ribosylation) of CS histones and phosphorylation of sperm specific histones [33], [34]. During this dramatic sperm chromatin remodelling it has been shown that previous disassembly of nucleosomes is required for complete histone proteolysis however the activity in charge of this displacement remains unidentified [6]. While in that case deposition of maternal histones on sperm DNA is replication independent, it has been suggested that H3 replacement in ESC is S-phase/replication coupled [3].…”

“…We subsequently showed that a cathepsin L protease is necessary for mitotic chromosomes decondensation during cleavage cell cycles of these embryos [6]. These suggested that proteases of the cathepsin L type should specifically proteolyze proteins essential for cell division in early embryos.…”

“…In sea urczhin, this event involves the replacement of sperm histones (SpH) by maternally inherited cleavage stage (CS) histone variants [7]. The SpH are released from male chromatin and subsequently degraded by a nuclear cysteine protease that catalyzes SpH proteolysis and leaves the CS histone variants unaffected [6], [8]. This SpH protease (SpHp) is present as an inactive precursor in the nucleus of unfertilized eggs and was found to be activated and mobilized into male pronucleus after fertilization [5].…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…A candidate for the maternal kinase is Cdk2, because it colocalizes with the male pronucleus after fertilization (Schnackenberg and Marzluff, 2002;reviewed in Imschenetzky et al, 2003). Once their attachment has been loosened, sperm-specific histones are removed from sperm chromatin by a maternal sperm-nucleosome disassembly activity (SDNA) and then degraded by a maternal protease (Imschenetzky et al, 1997;Iribarren et al, 2008).…”