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STUDY QUESTION: What is the expression status and subcellular localization of the maternally expressed Bcl-2 family member, BCL2L10, in early human embryos of diverse developmental stages and quality? SUMMARY ANSWER: The anti-apoptotic protein, BCL2L10, is expressed in human preimplantation embryos at least until the blastocyst stage and appears to be differentially distributed at the subcellular level between viable embryos and fragmented or arrested embryos. WHAT IS KNOWN ALREADY: BCL2L10 is an anti-apoptotic member of the BCL-2 family that shows abundant expression in human oocytes and limited sequence conservation to its mouse homologue. STUDY DESIGN, SIZE, DURATION: Embryos donated with informed consent by couples consulting for infertility in the Department of Reproductive Medicine (Hopital Femme Mere Enfant, Bron, France) were divided into two groups: high quality embryos (n = 18) and poor quality embryos (n = 30). Semen samples (n = 4) were obtained after informed consent from men consulting for couple infertility. Experiments involving human preimplantation embryos were performed between January and December 2009. PARTICIPANTS/MATERIALS, SETTING, METHODS: We examined BCL2L10 expression and subcellular localization in early human embryos by using immunofluorescence and confocal microscopy. The subcellular distribution of BCL2L10 was also studied in ejaculated sperm cells and in isolated mouse skeletal muscle fibres. MAIN RESULTS AND THE ROLE OF CHANCE: The BCL2L10 protein was detectable in healthy human preimplantation embryos at least until the blastocyst stage. In high-quality embryos, BCL2L10 was predominantly cytoplasmic with mitochondrial localization. In contrast, BCL2L10 exhibited extra-mitochondrial localization in abnormal embryos, and was nuclear-cytoplasmic in approximately half (17/30) of the poor-quality embryos. Morphologically fragmented embryos showed coexistence of blastomeres with BCL2L10-positive expression and blastomeres or fragments negative for BCL2L10. LIMITATIONS, REASONS FOR CAUTION: Future studies are needed to evaluate whether embryo quality is related to an exclusive mitochondrial localization of BCL2L10. Mechanisms mediating the nuclear translocation of BCL2L10 in abnormal embryos and functions of this nuclear pool of BCL2L10 are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: The nuclear localization of BCL2L10 in abnormal embryos suggests a potential role for this protein in pathological conditions resulting in embryo arrest. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. There are no competing interests.

International audience; Insertions or deletions (indels) of amino acids residues have been recognized as an important source of genetic and structural divergence between paralogous Bcl-2 family members. However, these signature sequences have not so far been extensively investigated amongst orthologous Bcl-2 family proteins. Bcl2l10 is an antiapoptotic member of the Bcl-2 family that has evolved rapidly throughout the vertebrate lineage and which shows conserved abundant expression in eggs and oocytes. In this paper, we have unraveled two major sites of divergence between human Bcl2l10 and its vertebrate homologs. The first one provides length variation at the N-terminus (before the BH4 domain) and the second one is located between the predicted α5-α6 pore-forming helices, providing an unprecedented case in the superfamily of helix-bundled pore-forming proteins. These two particular indels were studied phylogenetically and through biochemical and cell biological techniques, including truncation and site-directed mutagenesis. While deletion of the N-terminal extension had no significant functional impact in HeLa cells, our results suggest that the human Bcl2l10 protein evolved a calcium-binding motif in its α5-α6 interhelical region by acquiring critical negatively charged residues. Considering the reliance of female eggs on calcium-dependent proteins and calcium-regulated processes and the exceptional longevity of oocytes in the primate lineage, we propose that this microstructural variation may be an adaptive feature associated with high maternal expression of this Bcl-2 family member.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.