S věkem spojené rozdíly v translaci proteinů v savčích oocytech

(česky) Samičí zárodečné buňky (známé též jako oocyty) se u savců nacházejí ve vaječnících, kde je jejich vývoj zastaven ve stádiu profáze 1. meiotického dělení (profáze I). Teprve až v průběhu puberty dochází k selekci oocytů, které následně rostou, uvolňují se z výše zmíněného profázního bloku, ovulují a stávají se pak schopnými být oplodněny a dát základ budoucímu embryu. Polovina genetické informace embrya tedy pochází přímo z oocytu a tudíž kvalita oocytu a jeho meiotického zrání hraje v reprodukci klíčovou úlohu. Jedním z faktorů, který může dramaticky snížit kvalitu oocytů u několika druhů savců je pokročilý věk samic. Jak u myší, tak u člověka se snížená kvalita oocytů pocházejících od starších matek projevuje především zvýšením výskytu chromozomální aneuploidie. Důsledkem je přítomnost nesprávného počtu chromozomů v embryo, což snižuje jeho viabilitu a může se projevit i dalšími klinickými příznaky. Práce, která je obsahem této dizertace byla primárně zaměřena na pochopení příčin zvýšeného výskytu chromozomálních aneuploidií v myších oocytech pocházejících od starších samic. Jedním z nejdůležitějších znaků, kterými se oocyty vyznačují, je to, že po překonání profázního bloku a zahájení meiotického dělení je jejich transkripční aktivita prakticky nulová a vlastní regulace průběhu meiózy je...Female germ cells (also known as oocytes) from mammalian species are found in the ovaries in a state of meiotic arrest at prophase I. It is not until puberty that oocytes start to be selected to grow, overcome their meiotic arrest and ovulate, so they become capable of being fertilized and give rise to new individuals. Half of the genetic information from these new individuals comes directly from the oocyte itself, therefore, oocyte and meiosis quality are of great importance for the reproduction. One of the factors, which can drastically reduce oocyte quality in several mammalian species is the advanced age of females. In both mice and humans age-related poor oocyte quality is reflected by a large increase of chromosomal aneuploidy rates. Having an incorrect number of chromosomes reduces embryo viability and may cause severe clinical outcomes. The work of this thesis was primarily directed towards a better understanding of the causes behind age-related aneuploidy in mice oocytes. One of the most characteristic features of oocytes is the fact that they become transcriptionally silent after meiotic resumption, relying heavily on translational control for protein expression. Here we show that after nuclear envelope break down (NEBD), one of the main protein kinases regulating translational...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos

Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased—thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization—and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation—the parthenote—and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research

SGK1 is essential for meiotic resumption in mammalian oocytes

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation. In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes

The translational oscillation in oocyte and early embryo development

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation

Sperm Meiotic Segregation Analysis of Reciprocal Translocations Carriers: We Have Bigger FISH to Fry

Reciprocal translocation (RT) carriers produce a proportion of unbalanced gametes that expose them to a higher risk of infertility, recurrent miscarriage, and fetus or children with congenital anomalies and developmental delay. To reduce these risks, RT carriers can benefit from prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD). Sperm fluorescence in situ hybridization (spermFISH) has been used for decades to investigate the sperm meiotic segregation of RT carriers, but a recent report indicates a very low correlation between spermFISH and PGD outcomes, raising the question of the usefulness of spermFISH for these patients. To address this point, we report here the meiotic segregation of 41 RT carriers, the largest cohort reported to date, and conduct a review of the literature to investigate global segregation rates and look for factors that may or may not influence them. We confirm that the involvement of acrocentric chromosomes in the translocation leads to more unbalanced gamete proportions, in contrast to sperm parameters or patient age. In view of the dispersion of balanced sperm rates, we conclude that routine implementation of spermFISH is not beneficial for RT carriers

Age-related differences in translation of mammalian oocytes

Female germ cells (also known as oocytes) from mammalian species are found in the ovaries in a state of meiotic arrest at prophase I. It is not until puberty that oocytes start to be selected to grow, overcome their meiotic arrest and ovulate, so they become capable of being fertilized and give rise to new individuals. Half of the genetic information from these new individuals comes directly from the oocyte itself, therefore, oocyte and meiosis quality are of great importance for the reproduction. One of the factors, which can drastically reduce oocyte quality in several mammalian species is the advanced age of females. In both mice and humans age-related poor oocyte quality is reflected by a large increase of chromosomal aneuploidy rates. Having an incorrect number of chromosomes reduces embryo viability and may cause severe clinical outcomes. The work of this thesis was primarily directed towards a better understanding of the causes behind age-related aneuploidy in mice oocytes. One of the most characteristic features of oocytes is the fact that they become transcriptionally silent after meiotic resumption, relying heavily on translational control for protein expression. Here we show that after nuclear envelope break down (NEBD), one of the main protein kinases regulating translational..

A bimodal fluorescence and phase microscope for time-lapse imaging of 3D multi-scattering objects

International audienc

Regulation of 4E-BP1 activity in the mammalian oocyte

<p>Fully grown mammalian oocytes utilize transcripts synthetized and stored during earlier development. RNA localization followed by a local translation is a mechanism responsible for the regulation of spatial and temporal gene expression. Here we show that the mouse oocyte contains 3 forms of cap-dependent translational repressor expressed on the mRNA level: 4E-BP1, 4E-BP2 and 4E-BP3. However, only 4E-BP1 is present as a protein in oocytes, it becomes inactivated by phosphorylation after nuclear envelope breakdown and as such it promotes cap-dependent translation after NEBD. Phosphorylation of 4E-BP1 can be seen in the oocytes after resumption of meiosis but it is not detected in the surrounding cumulus cells, indicating that 4E-BP1 promotes translation at a specific cell cycle stage. Our immunofluorescence analyses of 4E-BP1 in oocytes during meiosis I showed an even localization of global 4E-BP1, as well as of its 4E-BP1 (Thr37/46) phosphorylated form. On the other hand, 4E-BP1 phosphorylated on Ser65 is localized at the spindle poles, and 4E-BP1 phosphorylated on Thr70 localizes on the spindle. We further show that the main positive regulators of 4E-BP1 phosphorylation after NEBD are mTOR and CDK1 kinases, but not PLK1 kinase. CDK1 exerts its activity toward 4E-BP1 phosphorylation via phosphorylation and activation of mTOR. Moreover, both CDK1 and phosphorylated mTOR co-localize with 4E-BP1 phosphorylated on Thr70 on the spindle at the onset of meiotic resumption. Expression of the dominant negative 4E-BP1 mutant adversely affects translation and results in spindle abnormality. Taken together, our results show that the phosphorylation of 4E-BP1 promotes translation at the onset of meiosis to support the spindle assembly and suggest an important role of CDK1 and mTOR kinases in this process. We also show that the mTOR regulatory pathway is present in human oocytes and is likely to function in a similar way as in mouse oocytes.</p

CNN-based approach for 3D artifact correction of intensity diffraction tomography images

International audience3D reconstructions after tomographic imaging often suffer from elongation artifacts due to the limited-angle acquisitions. Retrieving the original 3D shape is not an easy task, mainly due to the intrinsic morphological changes that biological objects undergo during their development. Here we present to the best of our knowledge a novel approach for correcting 3D artifacts after 3D reconstructions of intensity-only tomographic acquisitions. The method relies on a network architecture that combines a volumetric and a 3D finite object approach. The framework was applied to time-lapse images of a mouse preimplantation embryo developing from fertilization to the blastocyst stage, proving the correction of the axial elongation and the recovery of the spherical objects. This work paves the way for novel directions on a generalized non-supervised pipeline suited for different biological samples and imaging conditions