In vitro oocyte maturation (IVM) holds great promise as a tool for enhancing clinical treatment of infertility, enhancing availability of non human primates for development of disease models, and facilitating endangered species preservation. However, IVM outcomes have remained significantly below success rates obtained using in vivo matured (VVM) oocytes from humans and non human primates. A cDNA array based analysis is presented, comparing the transcriptomes of VVM oocytes with IVM oocytes. We observe a small set of just 59 mRNAs that are differentially expressed between the two cell types. These mRNAs are related to cellular homeostasis, cell-cell interactions including growth factor and hormone stimulation and cell adhesion, and other functions such as mRNA stability and translation. Additionally, we observe in IVM oocytes overexpression of PLAGL1 and MEST, two maternally imprinted genes, indicating a possible interruption or loss of correct epigenetic programming. These results indicate that, under certain IVM conditions, oocytes that are molecularly highly similar to VVM oocytes can be obtained, however the interruption of normal oocyte-somatic cell interactions during the final hours of oocyte maturation may preclude the establishment of full developmental competence.
Effects of in vitro maturation on gene expression in rhesus monkey oocytes.
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View SamplesThe oocytes of many species, both invertebrate and vertebrate, contain a large collection of localized determinants in the form of proteins and translationally inactive maternal mRNAs. However, it is unknown whether mouse oocytes contain localized MmRNA determinants and what mechanisms might be responsible for their control. We collected intact MII oocytes, enucleated MII oocyte cytoplasts (with the spindle removed), and spindle-chromosome complexes which had been microsurgically removed. RNA was extracted, amplified, labeled, and applied to microarrays to determine if any MmRNA determinants were localized to the SCC.
Association of maternal mRNA and phosphorylated EIF4EBP1 variants with the spindle in mouse oocytes: localized translational control supporting female meiosis in mammals.
Sex, Specimen part, Disease
View SamplesTranscriptional activation in mammalian embryos occurs in a stepwise manner. In mice, it begins at the late one-cell stage, followed by a minor wave of activation at the early two-cell stage, and then the major genome activation (MGA) at the late two-cell stage. Cellular homeostasis, metabolism, cell cycle, and developmental events are orchestrated before MGA by time-dependent changes in the array of maternal transcripts being translated (i.e., the translatome). Despite the importance of maternal mRNA and its correct recruitment for development, neither the array of recruited mRNA nor the regulatory mechanisms operating have been well cheracterized. We present the first comprehensive analysis of changes in the maternal component of the zygotic translatome during the transition from oocyte to late one-cell stage embryo, revealing global transitions in the functional classes of translated maternal mRNAs, and apparent changes in the underlying cis-regulatory mechanisms.
Analysis of polysomal mRNA populations of mouse oocytes and zygotes: dynamic changes in maternal mRNA utilization and function.
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View SamplesCumulus oophorus cells play an essential role in oocyte development. CBX4 is a member of the Polycomb complex, which plays a role in regulating cellular differentiation.
Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos.
Sex, Specimen part
View SamplesWhile the reprogramming factors OCT4, SOX2, KLF4, and MYC (OSKM) can reactivate the pluripotency network in terminally differentiated cells, they also regulate expression of non-pluripotency genes in other contexts, such as the mouse primitive endoderm. The primitive endoderm is an extraembryonic lineage established alongside the pluripotent epiblast in the blastocyst, and is the progenitor pool for extraembryonic endoderm stem (XEN) cells. Several studies have shown that endodermal genes are upregulated in fibroblasts undergoing reprogramming, although whether endodermal genes promote or inhibit acquisition of pluripotency is unclear. We show that, in fibroblasts undergoing conventional reprogramming, OSKM-induced expression of endodermal genes leads to formation of induced XEN (iXEN) cells, which possess key properties of blastocyst-derived XEN cells, including morphology, transcription profile, self-renewal, and multipotency. Our data show that iXEN cells arise in parallel to iPS cells, indicating that OSKM are sufficient to drive cells to two distinct fates during reprogramming. Overall design: Sequence-based mRNA transcriptional profiling of three different cell lines (MEF, XEN, iXEN) with multiple biological replicates, under two different growth medium conditions (ESC medium, XEN medium) for XEN and iXEN cells.
OSKM Induce Extraembryonic Endoderm Stem Cells in Parallel to Induced Pluripotent Stem Cells.
Specimen part, Treatment, Subject
View SamplesGlobal gene experssion study of the HAEC transcriptional response to artificial chlyomicron remnant-like particles (A-CRLPs) prepared with triglycerides extracted from four natural dietary oils: fish, DHASCO, corn and palm oils. We hypothesised that A-CRLPs could differentially regulate HAEC gene expression according to thier triglyceride content. These data provide an important starting point for investigations into the effects of A-CRLPs on endothelial cells, particulary genes involved in redox balance and inflammatory processes.
Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway.
Specimen part
View SamplesFollicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.
Rhesus monkey cumulus cells revert to a mural granulosa cell state after an ovulatory stimulus.
Specimen part
View SamplesCloned embryos produced by somatic cell nuclear transfer (SCNT) display a plethora of phenotypic characteristics that make them different from fertilized embryos, indicating defects in the process of nuclear reprogramming by the recipient ooplasm. To elucidate the extent and timing of nuclear reprogramming, we used microarrays to analyze the transcriptome of mouse SCNT embryos during the first two cell cycles. We identified a large number of genes mis-expressed in SCNT embryos. We found that genes involved in transcription and regulation of transcription are prominent among affected genes, and thus may be particularly difficult to reprogram, and these likely cause a ripple effect that alters the transcriptome of many other functions, including oxidative phosphorylation, transport across membrane, and mRNA transport and processing. Interestingly, we also uncovered widespread alterations in the maternal (i.e. non transcribed) mRNA population of SCNT embryos. We conclude that gene expression in early SCNT embryos is grossly abnormal, and that this is at least in part the result of incomplete reprogramming of transcription factor genes.
Tough beginnings: alterations in the transcriptome of cloned embryos during the first two cell cycles.
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View SamplesThe elaboration of a quality oocyte is integrally linked to the correct developmental progression of cumulus cell phenotype. In humans and non-human primates, oocyte quality is diminished with in vitro maturation. To determine the changes in gene expression in rhesus monkey cumulus cells (CC) that occur during the final day prior to oocyte maturation and how these changes differ between in vitro and in vivo maturation (IVM and VVM), we completed a detailed comparison of transcriptomes using the Affymetrix gene array. We observe a large number of genes differing in expression when comparing IVM-CC and VVM-CC directly, but a much larger number of differences comparing the transitions from the pre-oocyte maturation to post- IVM and post-VVM state. We observe a truncation or delay in the normal pattern of gene regulation, but also remarkable compensatory changes in gene expression during IVM. Among the genes affected in cumulus cells by IVM are those that contribute to productive cell-cell interactions between cumulus cell and oocyte and between cumulus cells. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. We identify a panel of 24 marker genes, the expression of which should provide the foundation for understanding how IVM can be improved, for monitoring IVM conditions, and for diagnosing oocyte quality.
Extensive effects of in vitro oocyte maturation on rhesus monkey cumulus cell transcriptome.
Specimen part
View SamplesThe function of Structural maintenance of chromosome flexible domain containing 1 (Smchd1) was examined during mouse preimplantation development using an siRNA knockdown approach. Transient SMCHD1 deficiency during the period between fertilization and morula/early blastocyst stage compromised embryo viability and resulted in reduced cell number, reduced embryo diameter, and reduced nuclear volumes at the morula stage. RNAseq analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of mRNAs related to the trophoblast lineage, indicating SMCHD1 inhibits trophoblast lineage gene expression and promotes inner cell mass formation. siRNA knockdown also reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 (Skp2). Smchd1 expression was elevated in Caudal type homeobox transcription factor 2 (Cdx2)-/- blastocysts, indicating enriched expression, and further indicating a role in inner cell mass development. These results indicate that Smchd1 plays dual roles in the preimplantation embryo, promoting a lineage-appropriate pattern of gene expression supporting inner cell mass formation, whilst controlling lineage formation and gene expression in the trophectoderm. Overall design: Effects of SMCHD1 siRNA knockdown were tested in mouse embryos
Novel key roles for structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development.
Treatment, Subject
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