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.

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. © 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.

Three sets of hi stone 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.