Anne Marie Genevière

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein–protease (SpH‐protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per‐se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno‐inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti‐SpH‐protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N‐terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z–Phe‐Phe–CH2F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno‐inhibition of this enzyme obtained by microinjecting the anti‐SpH–protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis. J. Cell. Physiol. 216: 790–795, 2008,

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

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. J. Cell. Biochem. 98: 335–342, 2006.

Cysteine-protease involved in male chromatin remodeling after fertilization co-localizes with α-tubulin at mitosis

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 α‐tubulin that organizes the mitotic spindle during the initial embryonic cell division.

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

Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

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

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchins 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.

Cell Dynamics in Early Embryogenesis and Pluripotent Embryonic Cell Lines: From Sea Urchin to Mammals

From oogenesis through fertilization and gastrulation, embryos use various mechanisms to regulate cell expansion, keeping a strict balance between cell proliferation, cell differentiation and cell death. While rapid divisions are necessary at the initial stage to ensure early embryo survival, further developmental transitions are marked by changes in cell cycle and transcriptional regulation. Pluripotency and capability of self-renewal are maintained in a low percentage of cells, the embryonic stem cells (ESCs), which will be later used as a cellular source for tissue replacement. Clearly, some essential characteristics of cell cycle and transcriptional regulation of the early embryo will be conserved in ESC lines. We addressed here the peculiarities of these developmental programs in early embryos and pluripotent embryonic cell lines, considering examples from marine invertebrates to mammals. Finally, we discussed the importance of transcription regulation and chromatin remodelling and their peculiar features in embryonic cells from these species.