Cytoplasmic lattices are not linked to mouse 2-cell embryos developmental arrest

Cytoplasmic lattices are important regulators of oocyte maturation. They store components of the protein synthesis machinery including ribosomes and, among others, they are involved in the regulation of microtubule dynamics in both mouse and human. Cytoplasmic lattices undergo dramatic reorganizations at crucial stages of oocyte maturation, where they are abundantly present in the cytoplasm of developmentally competent oocytes named SN (Surrounded Nucleolus) while they are rare in the cytoplasm of 2-cell stage-arresting NSN (Not Surrounded Nucleolus) oocytes, suggestive of a requirement of cytoplasmic lattices for development past the 2-cell stage. Here, to elucidate this requirement, 2-cell mouse embryos derived from SN and NSN oocytes were analyzed by transmission electron microscopy. Contrary to what had been proposed hitherto, cytoplasmic lattices are present in 2-cell embryos derived not only from SN, but also from NSN oocytes, irrespective of the embryo production system (intra cytoplasmic sperm injection, parthenogenesis). Hence our conclusion that cytoplasmic lattices do not count among the factor(s) responsible for the embryo arrest at this crucial stage of development

Gene Expression and Chromatin Organization during Mouse Oocyte Growth

AbstractMouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development

Pluripotency and differentiation in embryos and stem cells - Pavia, 17-18 January 2008

Each year many scientific meetings are held on stem cells to appraise the state of knowledge on their potency, differentiation and applications. So why did we hold another meeting? Because we thought one aspect was not adequately addressed in the others. When thinking of how our body is derived from a single fertilized egg, it is self-evident that the embryo is the ‘mother’ of all stem cells. This fact is probably overlooked because it is so remote (decades back in our lives!) and because embryonic stem cells do not exist as such in the embryo. However, this also tends to be ignored on purpose in many stem cell meetings because working on (human) embryos brings up substantial ethical concerns that bear on the scientific undertaking like nothing else. The origin of stem cells has become even more of a sensitive issue since the discovery in 2006 that embryonic stem (ES) cell-like cells can be generated in a petri dish straight from somatic cells by retrovirus-mediated transfer of selected genes. These new cells have been named ‘induced pluripotent stem‘ (iPS) cells and have been obtained without any egg or embryo consumption (Takahashi and Yamanaka, 2006). This leads to the first topic of our meeting: natural and induced pluripotency..

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Chromatin configuration in the oocyte nucleus and genetic aspects of oogenesis and early embryogenesis in the mouse (Mus musculus L.)

Dottorato di ricerca in biologia cellulare animale. 12. ciclo. A.a 1998-99. Tutore Carlo Alberto RediConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Israel et al_supplementary Table S3

Mean immunofluorescence intensities measured in the equatorial region of mouse blastomeres (2-cell stage) that incorporated O-propargyl-puromycin after Trim-away of ZP3 vs. control group (OGDB). </p

Israel et al_supplementary Table S1

iBAQ values of blastocysts after 96 h culture for stable isotope labeling of protein synthesis using the non-radioactive isotopic aminoacids Lys-8 (13C6H1415N2O2) and Arg-10 (13C6H1415N4O2)</p

Israel et al_Supplementary Table S4

Mean ZP3 immunofluorescence intensities measured in the equatorial region of mouse blastomeres (2-cell stage) that were subjected to Trim-away of ZP3 vs. control group (OGDB). </p