Ca2+ dynamics in oocytes from naturally-aged mice

The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca2+ concentration ([Ca2+]i) in the ooplasm as a result of sperm-egg fusion. We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca2+ responses at fertilisation. To test this hypothesis we performed the first examination of Ca2+ dynamics in eggs from young and naturally-aged mice. Strikingly, we find that Ca2+ stores and resting [Ca2+]i are unchanged with age. Although eggs from aged mice feature a reduced ability to replenish intracellular Ca2+ stores following depletion, this difference had no effect on the duration, number, or amplitude of Ca2+ oscillations following intracytoplasmic sperm injection or expression of phospholipase C zeta. In contrast, we describe a substantial reduction in the frequency and duration of oscillations in aged eggs upon parthenogenetic activation with SrCl2. We conclude that the ability to mount and respond to an appropriate Ca2+ signal at fertilisation is largely unchanged by advancing maternal age, but subtle changes in Ca2+ handling occur that may have more substantial impacts upon commonly used means of parthenogenetic activation

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes

Ultrafast imaging of cell elasticity with optical microelastography

International audienceElasticity is a fundamental cellular property that is related to the anatomy, functionality and pathological state of cells and tissues. However, current techniques based on cell deformation, acoustic force microscopy or Brillouin scattering are rather slow and do not always accurately represent cell elasticity. Here, we have developed an alternative technique by applying shear wave elastography to the micrometer scale. Elastic waves were mechanically induced in live mammalian oocytes using a vibrating micropipette. These audible frequency waves were observed optically at 200,000 frames per second and tracked with an optical flow algorithm. Whole cell elasticity was then mapped using an elastography method inspired by the seismology field. Using this approach, we show that the elasticity of mouse oocyte is decreased when the oocyte cytoskeleton is disrupted with cytochalasin B. The technique is fast (less than 1 ms for data acquisition), precise (spatial resolution of a few micrometers), able to map internal cell structures, robust, and thus represents a tractable novel option for interrogating biomechanical properties of diverse cell types

Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development

Cells with high ploidy content are common in mammalian extraembryonic and adult tissues. Cell-to-cell fusion generates polyploid cells during mammalian development and tissue regeneration. However, whether increased ploidy can be occasionally tolerated in embryonic lineages still remains largely unknown. Here, we show that pluripotent, fusion-derived tetraploid cells, when injected in a recipient mouse blastocyst, can generate diploid cells upon ploidy reduction. The generated diploid cells form part of the adult tissues in mouse chimeras. Parental chromosomes in pluripotent tetraploid cells are segregated through tripolar mitosis both randomly and nonrandomly and without aneuploidy. Tetraploid-derived diploid cells show a differentiated phenotype. Overall, we discovered an unexpected process of controlled genome reduction in pluripotent tetraploid cells. This mechanism can ultimately generate diploid cells during mouse embryo development and should also be considered for cell fusion-mediated tissue regeneration approaches.This work was supported by the Ministerio de Ciencia, Innovación y Universidades, (BFU2017-86760-P to M.P.C.) (AEI/FEDER, UE) and the AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya, (2017 SGR 689 to M.P.C.). Work in M.P.C. lab was supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 686637 (CellViewer). We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme / Generalitat de Catalunya. KU Leuven C1 funds (C14/16/078 to F.L.), FWO national grants (G097618N to F.L.), Instituto de Salud Carlos III (CP10/00445 to F.L.), a “CRG-Severo Ochoa” predoctoral fellowship (to P.C.), and a grant from the Fundação para a Ciência e a Tecnologia (FCT) (SFRH/BD/76068/2011 to J.F.)

Generation of a familial hypercholesterolemia model in non-human primate

Abstract Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients

Müller glia fused with adult stem cells undergo neural differentiation in human retinal models

Background: Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons. Methods: We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation. Findings: We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids. Interpretation: We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.Funding: This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. Profiling the chromatin-bound proteome (chromatome) by genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos lead to developmental arrest, reduction of H3K9me3 and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme (CellViewer No 686637 to M.P.C.); Ministerio de Ciencia e Innovación, grant BFU2017-86760-P (AEI/FEDER, UE); and an AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa I Coneixement de la Generalitat de Catalunya (2017 SGR 689 to M.P.C.); National Natural Science Foundation of China (No 31971177 to M.P.C.); Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PID2019-108322GB-100 to L.D.C.), and from AGAUR (L.D.C.). We acknowledge the support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme. The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) and it is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013-2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. R.S.-P. was supported by a FI-AGAUR PhD fellowship from the Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and the co-finance of Fondo Social Europeo (2018FI_B_00637 and FSE). X.T. is supported by a FPI PhD fellowship from the Ministerio de Ciencia e Innovación (PRE2018-085107). S.A. is funded by the Ramon y Cajal program of the Ministerio de Ciencia, Innovación y Universidades and the European Social Fund under the reference number RYC-2018-025002-I, and the Instituto de Salud Carlos III-FEDER (PI19/01814). L.M. is supported by a grant for the recruitment of early-stage research staff FI-2020 (Operational Program of Catalonia 2014-2020 CCI grant no. 2014ES05SFOP007 of the European Social Fund) and La Caixa Foundation fellowship (LCF/BQ/DR20/11790016). M.P. was supported by a Severo Ochoa PhD fellowship from the Subprograma Estatal de Formación del Ministerio de Economía y Competitividad (BES-2015-072802). M.V.N. is funded by FP7/2007–2013 under an REA grant (608959) and Juan de la Cierva-Incorporación 2017