Project description:There is massive destruction of transcripts during maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using the microarray approach. A system for oocyte transcript amplification using both internal and 3'-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among those stables. Experiment Overall Design: Comparison immature GV-stage oocyte (3 biological replicates) with mature MII-stage oocytes (3 biological replicates)
Project description:There is massive destruction of transcripts during maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using the microarray approach. A system for oocyte transcript amplification using both internal and 3’-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among those stables. Keywords: Developmental stage (mature vs. immature) comparison
Project description:Purpose: The goals of this study are to study the function of Cnot6l during oocyte maturation and the differences between loss of Cnot6l and Btg4 during oocyte maturation. Methods:Comparing the degration of transcripts at different stage in WT, Btg4-/- and Cnot6l-/- oocytes by RNA sequencing. Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads per sample to the mouse genome (build mm9) and identified 23236 transcripts with TopHat workflow. Conclusions: CNOT6L stimulated degradation of maternal transcriptsto oocyte meiotic maturation.
Project description:Oocyte maturation is accompanied by a transition from mRNA stability to instability. We investigated the role of DCP1A and DCP2, proteins responsible for mRNA decapping, in mRNA destabilization during mouse oocyte maturation. siRNA-mediated knockdown of both Dcp1a and Dcp2 transcripts prior to initiation of maturation inhibited the maturation-associated increase of DCP1A and DCP2, stabilized a set of maternal mRNAs that are normally degraded during maturation, and inhibited development beyond the 2-cell stage, likely a consequence of failure to activate fully the zygotic genome.
Project description:Oocyte maturation is accompanied by a transition from mRNA stability to instability. We investigated the role of DCP1A and DCP2, proteins responsible for mRNA decapping, in mRNA destabilization during mouse oocyte maturation. siRNA-mediated knockdown of both Dcp1a and Dcp2 transcripts prior to initiation of maturation inhibited the maturation-associated increase of DCP1A and DCP2, stabilized a set of maternal mRNAs that are normally degraded during maturation, and inhibited development beyond the 2-cell stage, likely a consequence of failure to activate fully the zygotic genome. Total RNA from 30 MII eggs was used in each sample. Three independent biological replicates were analyzed for each condition.
Project description:Purpose: The goals of this study are to study the function of Cnot6l during oocyte maturation . Methods: Comparing the polysome-bounded transcripts at GV, MI and MII stage in WT and Cnot6l-/- oocytes by RNA sequencing. Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads per sample to the mouse genome (build mm9) and identified 23236 transcripts with TopHat workflow. Conclusions: CNOT6L stimulated degradation of maternal transcriptsto oocyte meiotic maturation.
Project description:Oocyte maturation is the foundation for developing healthy individuals of mammals. Upon germinal vesicle breakdown, oocyte meiosis resumes and the synthesis of new transcripts ceases. To quantitatively profile the transcriptomic dynamics after meiotic resumption throughout the oocyte maturation, we generated transcriptome sequencing data with individual mouse oocytes at three main developmental stages: germinal vesicle (GV), metaphase I (MI), and metaphase II (MII). When clustering the sequenced oocytes, results showed that isoform-level expression analysis outperformed gene-level analysis, indicating isoform expression provided extra information that was useful in distinguishing oocyte stages. Comparing transcriptomes of the oocytes at the GV stage and the MII stage, in addition to identification of differentially expressed genes (DEGs), we detected many differentially expressed transcripts (DETs), some of which came from genes that were not identified as DEGs. When breaking down the isoform-level changes into alternative RNA processing events, we found the main source of isoform composition changes was the alternative usage of polyadenylation sites. With detailed analysis focusing on the alternative usage of 3'-UTR isoforms, we identified, out of 3810 tested genes, 512 (13.7%) exhibiting significant switches of 3'-UTR isoforms during the process of moues oocyte maturation. Altogether, our data and analyses suggest the importance of examining isoform abundance changes during oocyte maturation, and further investigation of the pervasive 3'-UTR isoform switches in the transition may deepen our understanding on the molecular mechanisms underlying mammalian early development.
Project description:As an important post-transcriptional regulatory mechanism, asymmetric localization of mRNAs is essential for cell polarity and cell fate determination during early development. In an effort to understand localization of mRNAs encoding proteasome components during Xenopus oocyte-to-embryo transition, we discovered that the endoplasmic reticulum (ER), which is thought to mainly serve “house-keeping” roles in the cell, plays a multifaceted role in controlling localization of maternal RNAs. Our RNA-seq analysis of fractionated transcripts reveals that more than 40% of RNAs, including proteasome mRNAs, are present on the ER in Xenopus oocytes. After meiotic maturation, a large fraction of ER-associated RNA is released into the cytosol. The release of proteasome RNAs from the ER into the cytosol is conserved during mouse oocyte maturation. Our comparative proteomic analysis and ribonucleoprotein immunoprecipitation demonstrate that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation, providing a mechanistic explanation for the dynamic regulation of RNA-ER association. We further show that the ER is remodeled massively during Xenopus oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of proteasome mRNAs in mature eggs. To our knowledge, our findings demonstrate for the first time that dynamic regulation of RNA-ER association and remodeling of the ER are fundamentally important for asymmetric localization of RNAs during development.