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Next Generation Sequencing Facilitates Analysis of The Mouse Oocyte、Cumulus cells and Blastocyst Transcriptomes From PM2.5 Exposure Group And Filtrated Air Group

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare mouse NGS-derived oocyte、cumulus cell and blastocyst transcriptome profiling (RNA-seq) between PM2.5 exposure group and Filtrated air group Methods: mouse oocyte、cumulus cell and blastocyst mRNA profiles were generated by deep sequencing, in triplicate for each stage, using Illumina Hi-Seq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9). Genes with adjusted P < 0.05 and fold-change (FC) ≥ 2 were considered differentially expressed genes (DEGs). The oocyte transcriptome analysis showed that 467 DEGs (260 and 207 up- and down-regulated genes, respectively) were identified in the CAP group compared with the FA group. In the cumulus cell analysis, 592 DEGs (358 and 234 up- and down-regulated genes, respectively) were identified in the CAP group compared with the FA group. The blastocyst transcriptome analysis identified 448 DEGs (254 and 194 up- and down-regulated, respectively) in the CAP group compared with the FA group. Conclusions: Our study represents the first detailed analysis of mouse oocyte、cumulus cell and blastocyst transcriptomes, with biologic replicates, generated by RNA-seq technology between PM2.5 exposure group and Filtrated air group.

ORGANISM(S): Mus musculus

PROVIDER: GSE148563 | GEO | 2020/04/14

REPOSITORIES: GEO

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Project description:The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsin B, S, K and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real time RT-PCR. Elevated transcript abundance for cathepsins B, S and Z was also observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation, versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus oocyte complexes during in vitro oocyte maturation with a cell permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. Results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality. Keywords: Bovine, microarray, cDNA, cumulus cells Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Total RNA from pools of cumulus cells (n = 4) collected from adult and prepubertal animals for microarray experiments was amplified. Two color microarray experiments were conducted using a bovine cDNA array containing expressed sequence tags (ESTs) representing approximately 15200 unique genes. Hybridizations were performed on duplicate slides (prepubertal versus adult) and incorporated a dye swap. The total number of slides used is eight.

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Next Generation Sequencing Facilitates Analysis of The Mouse Oocyte、Cumulus cells and Blastocyst Transcriptomes From PM2.5 Exposure Group And Filtrated Air Group

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