Functional analysis of microRNA-130b in bovine oocyte maturation and preimplantation embryo development

MicroRNAs (miRNAs) are well known to regulate the proteins involved in various biological processes including development. The expression pattern of miRNAs is believed to vary between immature and in vitro matured bovine oocytes. Among these, miR-130b was reported to upregulated in immatured compared to matured oocytes. However, its functional role in cell viability, proliferation or transcription during bovine oocyte maturation and preimplantation embryo development is not known. Therefore, this experiment was aimed to investigate the functional role of miR-130b in oocyte maturation and oocyte surrounding cells and its involvement in preimplantation embryo development. For this, the spatiotemporal expression pattern of miR-130 family was performed throughout the bovine preimplantation stage embryos. Accordingly, miR-130b was found to be highly expressed in cumulus and granulosa cells, immature oocyte, morula and blastocyst stage embryos. Once the expression pattern of miR-130b was evaluated, its target genes were in silico analyzed and experimentally validated. Accordingly, MSK1, SMAD5, MEOX2, DOC1R and EIF2C4 were found to be the real targets of miR-130b. To investigate the involvement of miR-130b during oocyte maturation, immatured oocytes were microinjected with pre-miR-130b (precursor) or sequence specific antisense (inhibitor) of miR-130b, while scramble miRNA injected and uninjected oocytes were used as controls. The maturational status of the oocytes and the level of miR-130b target genes expression were assessed 22 hours post microinjection. The result showed that the first polar body extrusion was 86.3, 73, 85 and 84.6% in oocytes injected with pre-miR-130b, anti-miR-130b, scramble and uninjected controls, respectively. Similarly, mitotic staining showed that majority of oocytes injected with anti-miR-130b remains arrested at the telephase I stage (22%) and significantly reduced to reach Metaphase II compared to other oocyte groups. In addition, the mitochondrial activity was higher in pre-mir-130b and lower in anti-miR-130b injected oocytes compared to scramble and uninjected oocytes. This was associated with the reduction of miR-130b and increase of its target genes SMAD5 and MSK1 expression. Furthermore, oocyte surrounding cells are required for oocyte maturation, the involvement of miR-130b in cumulus and granulosa cell proliferation, lactate production and cholesterol level was assessed after transfection of pre-miR-130b or anti-miR-130b in both cell types. The inhibition of miR-130b resulted in reduction of cell proliferation and lactate production. However, knockdown of miR-130b did not change the cholesterol level in the granulosa or cumulus cells. Apart from oocyte maturation and oocyte companion cell function, the role of miR-130b was investigated during preimplantation embryo development by microinjecting zygotes with pre-miR-130b or anti-miR-130b. The result has shown that the first cleavage rate was unaffected by knockdown or ectopic expression of miR-130b, but the rate of morula and blastocyst were significantly reduced in anti-miR-130b injected zygotes. Therefore this study provides the significant evidence that miR-130b may be required during bovine oocyte in vitro maturation and granulosa cell proliferation, morula and blastocyst formation, further functional in depth studies are necessary to understand whether miR-130b is involved in bovine oocyte in vivo maturation or embryo implantation.Funktionelle Analyse der microRNA miR-130b während der bovinen Oozytenmaturation und der preimplantativen Embryonalentwicklung MicroRNAs (miRNAs) sind dafür bekannt, dass sie eine regulatorische Rolle in verschiedenen biologischen Prozessen, wie in der Embryonalentwicklung spielen. Es wir angenommen, dass das Expressionsmuster von miRNAs zwischen immaturen und in vitro maturierten bovinen Oozyten variiert, wobei gezeigt wurde, da miR-130b in inmaturierten Oozyten hoch reguliert ist. Allerdings ist seine funktionelle Rolle in der Zellvitalität, Proliferation und Transkription während der bovinen Oozytenmaturation und der Präimplantiationsembryoentwicklung noch nicht bekannt. Daher war das Ziel dieser Studie die Bedeutung von miR-130b in der Maturation von Oozyten-, Granulosa- und Kumuluszellen und in der präimplantativen Embryoentwicklung zu untersuchen. Dafür wurde das Expressionsmuster der miR-130-Familie im bovinen embryonalen Präimplantationsstadium erstellt. MiR-130b war in Kumulus- und Granulosazellen, in immaturen Oozyten sowie im Morula- und Blastozystenstadium höher exprimiert. Die miR-130b Zielgen Identifizierung erfolgte mittels der In silico Analyse und der experimentellen Validierung durch den Luciferase-Assay. Dementsprechend konnten MSK1, SMAD5, MEOX2, DOC1R und EIF2C4 als Zielgene von miR-130b ermittelt werden. Um den Einfluss der miR-130b während der Maturation der Oozyten zu untersuchen, wurden in immaturierten Oozyten pre-miR-130b und sequenz-spezifische miR-130b Antisense (Inhibitor) mikroinjiziert, während mit scrambled miRNA injizierte und nicht injizierte Oozyten als Kontrolle dienten. 24 Stunden nach der Mikroinjektion wurde der Reifungsstatus der Oozyten mittels der ersten Polkörper-Extrusion, festgestellt. Sie betrug 86,3, 73, 85, und 84,6% bei Oozyten mit injizierter pre-miR-130b, anti-miR-130b, scramble RNA und nicht injizierte Kontrolle. Die Mehrheit der anti-miR-130b injizierten Oozyten blieb in der Telophase 1 (22%) stehen. Darüber hinaus konnte eine höhere mitrochendriale Aktivität in pre-miR-130b und eine niedrigere in anti-miR-130b injizierten Oozyten im Vergleich zu scramble RNA und nicht injizierte Oozyten gefunden werden. Dies konnte mit der Zunahme der Proteinexpression der miR-130b Zielgene SMAD5 und MSK1 assoziiert werden. Oozyten Companionzellen sind für die Oozytenmaturation erforderlich. Der Einfluss der miR-130b konnte bei der Zellproliferation, Laktatproduktion und beim Cholesterinspiegel durch die Transfektion der miR-130b Precursor RNA oder anti-miR-130b RNA in Kumulus- und Granulosazellen beobachtet werden. Mit der Reduktion der miR-130b folgte einen Reduzierung in der Zellproliferation und der Laktatproduktion, allerdings keine Änderungen im Cholesterinspiegel in Granulosa- oder Kumuluszellen. Neben der Maturation der Oozyten und der Oozyten Companionzellfunktion wurde die Rolle der miR-130b während der präimplantations Embryoentwicklung nach einer Mikroinjektion der miR-130b Precursor oder – Inhibitor in Zygoten untersucht. Das Ergebnis zeigte, dass die erste Teilungsrate unbeeinflusst vom Knockdown oder der Überexpression der miR-130b war, jedoch war die Morula/Blastozysten rate der anti-miR-130b injizierten Eizellen signifikant reduziert. Diese Studie liefert Hinweise dafür, dass die miR-130b während der bovinen Oozytenmaturation, der Granulosazellproliferation und der Morula- und Blastozystenformation funktionell beteiligt ist

Multiplicity dependence of light (anti-)nuclei production in p–Pb collisions at sNN=5.02 TeV

The measurement of the deuteron and anti-deuteron production in the rapidity range −1 < y < 0 as a function of transverse momentum and event multiplicity in p–Pb collisions at √sNN = 5.02 TeV is presented. (Anti-)deuterons are identified via their specific energy loss dE/dx and via their time-of- flight. Their production in p–Pb collisions is compared to pp and Pb–Pb collisions and is discussed within the context of thermal and coalescence models. The ratio of integrated yields of deuterons to protons (d/p) shows a significant increase as a function of the charged-particle multiplicity of the event starting from values similar to those observed in pp collisions at low multiplicities and approaching those observed in Pb–Pb collisions at high multiplicities. The mean transverse particle momenta are extracted from the deuteron spectra and the values are similar to those obtained for p and particles. Thus, deuteron spectra do not follow mass ordering. This behaviour is in contrast to the trend observed for non-composite particles in p–Pb collisions. In addition, the production of the rare 3He and 3He nuclei has been studied. The spectrum corresponding to all non-single diffractive p-Pb collisions is obtained in the rapidity window −1 < y < 0 and the pT-integrated yield dN/dy is extracted. It is found that the yields of protons, deuterons, and 3He, normalised by the spin degeneracy factor, follow an exponential decrease with mass number

Higher harmonic non-linear flow modes of charged hadrons in Pb-Pb collisions at sNN\sqrt{s_{\rm{NN}}} = 5.02 TeV

International audienceAnisotropic flow coefficients, v$_{n}$, non-linear flow mode coefficients, χ$_{n,mk}$, and correlations among different symmetry planes, ρ$_{n,mk}$ are measured in Pb-Pb collisions at $\sqrt{s_{\mathrm{NN}}}$ = 5.02 TeV. Results obtained with multi-particle correlations are reported for the transverse momentum interval 0.2 < p$_{T}$< 5.0 GeV/c within the pseudorapidity interval 0.4 < |η| < 0.8 as a function of collision centrality. The v$_{n}$ coefficients and χ$_{n,mk}$ and ρ$_{n,mk}$ are presented up to the ninth and seventh harmonic order, respectively. Calculations suggest that the correlations measured in different symmetry planes and the non-linear flow mode coefficients are dependent on the shear and bulk viscosity to entropy ratios of the medium created in heavy-ion collisions. The comparison between these measurements and those at lower energies and calculations from hydrodynamic models places strong constraints on the initial conditions and transport properties of the system.[graphic not available: see fulltext

Measurement of electrons from semileptonic heavy-flavour hadron decays at midrapidity in pp and Pb–Pb collisions at √sNN = 5.02 TeV

The differential invariant yield as a function of transverse momentum (pT) of electrons from semileptonic heavy-flavour hadron decays was measured at midrapidity in central (0–10%), semi-central (30–50%) and peripheral (60–80%) lead–lead (Pb–Pb) collisions at √sNN = 5.02 TeV in the pT intervals 0.5–26 GeV/c (0–10% and 30–50%) and 0.5–10 GeV/c (60–80%). The production cross section in proton–proton (pp) collisions at √s = 5.02 TeV was measured as well in 0.5 < pT < 10 GeV/c and it lies close to the upper band of perturbative QCD calculation uncertainties up to pT = 5 GeV/c and close to the mean value for larger pT. The modification of the electron yield with respect to what is expected for an incoherent superposition of nucleon–nucleon collisions is evaluated by measuring the nuclear modification factor RAA. The measurement of the RAA in different centrality classes allows in-medium energy loss of charm and beauty quarks to be investigated. The RAA shows a suppression with respect to unity at intermediate pT, which increases while moving towards more central collisions. Moreover, the measured RAA is sensitive to the modification of the parton distribution functions (PDF) in nuclei, like nuclear shadowing, which causes a suppression of the heavy-quark production at low pT in heavy-ion collisions at LHC

Multiplicity dependence of K*(892)0 and ϕ(1020) production in pp collisions at t √s=13 TeV

The striking similarities that have been observed between high-multiplicity proton-proton (pp) collisions and heavy-ion collisions can be explored through multiplicity-differential measurements of identified hadrons in pp collisions. With these measurements, it is possible to study mechanisms such as collective flow that determine the shapes of hadron transverse momentum (pT) spectra, to search for possible modifications of the yields of short-lived hadronic resonances due to scattering effects in an extended hadron-gas phase, and to investigate different explanations provided by phenomenological models for enhancement of strangeness production with increasing multiplicity. In this paper, these topics are addressed through measurements of the K∗(892)0 and φ(1020) mesons at midrapidity in pp collisions at √s = 13 TeV as a function of the charged-particle multiplicity. The results include the pT spectra, pT-integrated yields, mean transverse momenta, and the ratios of the yields of these resonances to those of longer-lived hadrons. Comparisons with results from other collision systems and energies, as well as predictions from phenomenological models, are also discussed

Multiplicity dependence of inclusive J/ψ production at midrapidity in pp collisions at √s=13 TeV

Measurements of the inclusive J/ψ yield as a function of charged-particle pseudorapidity density dNch/dη in pp collisions at √s = 13 TeV with ALICE at the LHC are reported. The J/ψ meson yield is measured at midrapidity (|y| < 0.9) in the dielectron channel, for events selected based on the charged-particle multiplicity at midrapidity (|η| < 1) and at forward rapidity (−3.7 < η < −1.7 and 2.8 < η < 5.1); both observables are normalized to their corresponding averages in minimum bias events. The increase of the normalized J/ψ yield with normalized dNch/dη is significantly stronger than linear and dependent on the transverse momentum. The data are compared to theoretical predictions, which describe the observed trends well, albeit not always quantitatively

HΛ3 and H‾Λ‾3 lifetime measurement in Pb–Pb collisions at √sNN=5.02 TeV via two-body decay

An improved value for the lifetime of the (anti-)hypertriton has been obtained using the data sample of Pb–Pb collisions at √sNN = 5.02 TeV collected by the ALICE experiment at the LHC. The (anti-)hypertriton has been reconstructed via its charged two-body mesonic decay channel and the lifetime has been determined from an exponential fit to the dN/d(ct) spectrum. The measured value, τ = 242+34 −38 (stat.) ± 17 (syst.) ps, is compatible with representative theoretical predictions, thus contributing to the solution of the longstanding hypertriton lifetime puzzle