Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human mature oocytes and embryonic stem cells reveals overexpression of the proteasome pathway

ABSTRACT: The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation. Therefore, based on a microarray compendium of 205 samples, produced in our laboratory or from public databases, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5) and 18 different zinc finger transcription factors, including ZNF84. Strikingly, a large set of genes was found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declines. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome, resulting in loss of pluripotency and cell growth at doses without any detectable effects on differentiated cells. Taken together, these results suggest that the proteasome pathway may play a role in initiating and maintaining pluripotency during early development and in hESC. Experiment Overall Design: We included in this study 9 samples obtained in our laboratory. These samples were hybridized on U133 Plus 2.0 GeneChips (Affymetrix). All samples were normalized using the MAS5 (GCOS 1.2) algorithm, using the default analysis settings and global scaling as normalization method, with a trimmed mean target intensity value (TGT) of each array arbitrarily set to 100. Experiment Overall Design: Human embryonic stem cells (hESC) samples were compared to somatic tissues, human mature oocytes samples were compared to somatic tissues, then the hESC and the mature oocyte signatures were interesected to define an oocyte/hESC signature.

ORGANISM(S): Homo sapiens

SUBMITTER: John De Vos

PROVIDER: E-GEOD-11450 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A gene expression signature shared by human mature oocytes and embryonic stem cells.

Assou Said S Cerecedo Doris D Tondeur Sylvie S Pantesco Véronique V Hovatta Outi O Klein Bernard B Hamamah Samir S De Vos John J

BMC genomics 20090108

<h4>Background</h4>The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transc ...[more]

PMID: 19128516

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Project description:The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells, holding promise for autologous cell replacement therapy. Though reprogramming of somatic cells by nuclear transfer was first demonstrated more than 60 years ago, only recently have human diploid embryonic stem cells been derived after nuclear transfer of fetal and neonatal fibroblasts. Because of the therapeutic potential of developing diploid embryonic stem cell lines from adult cells of normal and diseased human subjects, we have systematically investigated the parameters affecting efficiency and developmental potential in their derivation. We found that improvements to the oocyte activation protocol, including the use of both a kinase and a translation inhibitor, and cell culture in the presence of histone deacetylase inhibitors enable development of diploid cells to the blastocyst stage. Developmental efficiency varied significantly between oocyte donors, and was inversely related to the number of days of hormonal stimulation required to reach mature oocytes, while the daily dose of gonadotropin or the total number of MII oocytes retrieved did not affect developmental outcome. The use of diluted Sendai virus in calcium-free medium during nuclear transfer improved developmental potential, while the use of concentrated Sendai virus induced an increase in intracellular calcium and caused premature oocyte activation. Using these modifications to the nuclear transfer protocol, we successfully derived diploid pluripotent stem cell lines from both postnatal and adult somatic cells of a type 1 diabetic subject. The goal of this experiment was to determine if human oocytes have the ability to reprogram a somatic cell genome in the absence of the oocyte genome. Our previous research had indicated that human oocytes can reprogram adult somatic cells if the oocyte genome remains present (Noggle et al. Nature 2011, doi:10.1038/nature10397). The data presented here is part of a new series of experiments aimed at obtaining diploid cells after somatic cell nuclear transfer into enucleated oocytes. In this experiment, adult somatic cells were transferred into enucleated oocytes and thereafter cultured in the presence of 240ng/ml scriptaid for 17 hours. Samples were cultured until cleavage stage and then collected for microarray analysis.

Project description:The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. Experiment Overall Design: We generated a database of the human oocyte transcriptome by comparing the transcripts in the oocyte with the reference samples, which contain mRNA from several somatic tissues. Experiment Overall Design: Human oocytes were obtained from three patients undergoing an assisted reproductive treatment (ART) at the Unit of Reproductive Medicine of Clinica Las Condes, Santiago, Chile. It is important to emphasize that the routine in vitro fertilization protocol at Clinica Las Condes calls for fertilizing only those oocytes that will be transferred into the uterus of the patient. Therefore, there is always a surplus of oocytes. We then had the opportunity to use specific criteria to select donors as follows: (i) <35 years old, (ii) reproductively healthy with regular ovulatory cycles, (iii) male factor as the only cause of infertility, and (iv) considerable number of developing follicles that assured spared oocytes. The experimental protocol was reviewed and approved by a local independent Ethics Review Board. All donors signed informed consent. At the time this manuscript was submitted, all three donors had already conceived; two of them got pregnant during the ART cycle in which our samples were collected, and the third one got pregnant after a spontaneous cycle with artificial insemination using donated sperm. Ovarian stimulation, oocyte retrieval, and cell lysis were performed as described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. Experiment Overall Design: Three groups of 10 oocytes each were used. Reference RNA (100 Î¼g) was prepared by mixing 10 Î¼g of total RNA from each of 10 different normal human tissues, including skeletal muscle, kidney, lung, colon, liver, spleen, breast, brain, heart, and stomach (Ambion). Experiment Overall Design: Transcriptional profile of each sample was probed by using Affymetrix Human Genome U133 Plus 2.0 GeneChips.

Dataset Information

Transcription profiling of human mature oocytes and embryonic stem cells reveals overexpression of the proteasome pathway

Publications

A gene expression signature shared by human mature oocytes and embryonic stem cells.

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