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Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation

ABSTRACT: Purpose: The goal of this study was to identify differentially expressed genes and pathways between the cumulus of compact/unstimulated cumulus-oocyte-complex (COC) and the cumulus of expanded/stimulated COC. Methods: mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were mapped to the human genome (hg19) using Tophat software. Differential expression analysis was done using DESeq bioconductor package. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 40-80 million sequence reads per sample were mapped to the human genome (hg19). A total of 1746 differentially expressed genes between compact and expanded CCs with fold change > 2, and adjusted p value < 0.05 were identified. Gene ontology analysis of differentially expressed genes revealed a number of cellular processes regulated during the periovulatory interval including cellular movement, inflammatory response, immune cell trafficking, tissue development, lipid metabolism, tissue morphology, DNA replication and cell cycle. A total of 116 of the differentially expressed genes were annotated as long non-coding RNAs, 10 of them coded from introns of genes known to be involved in granulosa cell processes suggesting that unique non coding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Results were validated using qRT-PCR. Conclusions: Using global transcriptome sequencing we identified new important genes and non coding RNAs involved in COC maturation and cumulus expansion, which may contribute to improve the process of in vitro maturation of immature oocytes utilized in IVM cycles

ORGANISM(S): Homo sapiens

PROVIDER: GSE50174 | GEO | 2014/06/04

SECONDARY ACCESSION(S): PRJNA216966

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to identify differentially expressed genes and pathways between the cumulus of compact/unstimulated cumulus-oocyte-complex (COC) and the cumulus of expanded/stimulated COC. Methods: mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were mapped to the human genome (hg19) using Tophat software. Differential expression analysis was done using DESeq bioconductor package. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 40-80 million sequence reads per sample were mapped to the human genome (hg19). A total of 1746 differentially expressed genes between compact and expanded CCs with fold change > 2, and adjusted p value < 0.05 were identified. Gene ontology analysis of differentially expressed genes revealed a number of cellular processes regulated during the periovulatory interval including cellular movement, inflammatory response, immune cell trafficking, tissue development, lipid metabolism, tissue morphology, DNA replication and cell cycle. A total of 116 of the differentially expressed genes were annotated as long non-coding RNAs, 10 of them coded from introns of genes known to be involved in granulosa cell processes suggesting that unique non coding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Results were validated using qRT-PCR. Conclusions: Using global transcriptome sequencing we identified new important genes and non coding RNAs involved in COC maturation and cumulus expansion, which may contribute to improve the process of in vitro maturation of immature oocytes utilized in IVM cycles mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000.

Project description:In cultured cumulus oocyte complexes (COC) FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and identify candidate mRNAs involved. Experiment I: murine COC were cultured 4h in the presence of FSH with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82+/-7%). When DRB was added at 0, 5 or 10 min after culture initiation, oocyte maturation was blocked (17+/-7%, 14+/-6% and 21+/-6% GVBD, respectively). When DRB was added after 15, 20 or 30 min, progressively more COC underwent GVBD (37+/-6%, 39+/-6% and 66+/-6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a 3-fold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P</=0.01 and P</=0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC. Keywords: Analysis of treatment effect using SAGE library comparisons Mouse cumulus oocyte complexes (COC) cultured 25 min in presence (plusDRB) or absence (minusDRB) of 120 uM DRB, a transcriptional inhibitor. SAGE libraries were constructed from 9 to 10 ug RNA from each treatment group. Approx. 48,000 tags seqenced from each library. Comparison of these 2 libraries used to identify potential candidate mRNAs associated with regulation of germinal vesicle breakdown in murine COC.

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Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation

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