TOP2B disturbed the quality of human oocytes with advanced maternal age
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PROVIDER: PRJNA515815 | ENA |
REPOSITORIES: ENA
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Project description:STUDY QUESTION: Does maternal age affect the maturated oocyte quality and the fol-lowing development after fertilization in human? SUMMARY ANSWER: Maternal age affects the quality of maturated oocytes by altering the stored mRNA levels in human, such as TOP2B. WHAT IS KNOWN ALREADY: Intracellular mRNAs in maturated oocytes are tran-scripted from the maternal genome during oogenesis and important for the zygotic genome activation (ZGA) after fertilization. Microarray data showed that maternal age affected polyadenylated transcript abundance in human oocytes. These genes are involved in in signaling pathway related to cell cycle regulation, chromosome alignment. However, which genes are the key genes affected by maternal age and important for the development after fertilization had not been reported. Therefore, single-cell RNA sequencing (scRNA-Seq) technology is employed in this study to screen the key genes affected by maternal age in human maturated oocytes. STUDY DESIGN, SIZE, DURATION: We isolated mRNA from maturated (MII) oo-cytes donated by IVF or ICSI patients (three oocytes from young (≤ 30 years) and three oocytes from advanced maternal age (≥ 40 years) patients) undergoing controlled ovarian stimulation. Thus, a total of six maturated oocytes were individually processed for scRNA-seq analysis. The key genes screened from scRNA-seq analysis are confirmed using mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients undergoing infertility treatment at the Yuhuangding Hospital of Yantai underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ul-trasound guided oocyte retrieval. We isolated RNA, generated single cell RNA-seq librar-ies (Smart-Seq2) and sequenced by Illumina Hiseq X-ten platform with 150 bp paired-end. Bioinformatics analysis of the sequencing data was done to find the biological processes and key genes that led to the decline in the quality of oocytes with advanced maternal age. To validate the findings, we used mouse model and validated candidate genes by RT-PCR and knockdown experiments. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 1439 genes differentially expressed between older and younger women's maturated oocytes (|foldchange|>2, P < 0.05). These genes are significantly enriched with annotations related to transporter activity, cytoskeleton, oxidative stress, catalytic activity, immune function, cellular senescence and biosynthesis. The key candidate gene TOP2B was found by protein interaction network analysis, and knockdown verification on young mouse maturated oocytes showed that TOP2B was a key gene affecting the oocyte quality and disturbing early embryo development. LARGE SCALE DATA Raw data from this study can be accessed through GSE. LIMITATIONS, REASONS FOR CAUTION: The human maturated oocytes used in this study were from patients with different causes of infertility and may affect oocyte gene expression. In addition, the study was based on a lim-ited number of patients, and there are possible natural biological variance existed in human samples. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, we used scRNA-seq to detect global gene transcriptome of maturated oocytes in young and older women. These results are useful to indicate the molecular mechanisms of female ovary aging and establishing a criterion to evaluate the quality of oocytes in women with advanced maternal age. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Key Research and Development Program of China (2018YFC1004304, 2016YFA0100203), Medical and Health Science Technology Development Plan Project of Shandong Province (Grant#. 2017WS566). There are no competing interests.
2019-01-19 | GSE125300 | GEO
Project description:Study Question: What effects do maternal age and oocyte maturation stage have on the human oocyte transcriptome that may be associated with oocyte developmental potential? Summary Answer: Although polyadenylated transcript abundance changes during human oocyte maturation irrespective of age, young (YNG) and advanced maternal age (AMA) metaphase II (MII) oocytes exhibit divergent transcriptomes. What is known already: Maternal age and maturation stage affect oocyte polyadenylated transcript abundance in human oocytes. Although RNA-Seq analysis of single human MII oocytes has been conducted, comparison of the germinal vesicle (GV) and MII oocyte transcriptomes has not been investigated using RNA-Seq, a technique that could provide novel insight into oocyte maturation and age-associated aberrations in gene expression. Participants / materials, settings, methods: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided egg retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 hour incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads in HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, Weighted Gene Correlation Network Analysis (WGCNA), 3’UTR motif analysis, Ingenuity Pathway Analysis) were performed. Main results and the role of chance: Within the 12,770 expressed genes in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least 3 of 5 replicates for a minimum of one sample type), 458 and 3,506 genes significantly (adjusted p < 0.05 and log2 fold change > 1) increased and decreased in polyadenylated transcript abundance during oocyte maturation, respectively. The differentially expressed genes were enriched (FDR < 0.05) for biological functions and canonical pathways related to cell cycle and mitochondrial function. The majority (76%) and minority (25%) of up- and down-regulated transcripts with a complete 3’UTR were predicted to be targets of cytoplasmic polyadenylation (910 total genes), respectively. Differential gene expression analysis between young and advanced maternal age oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted p < 0.1 and log2 fold change > 1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5, and PRDX1, which have been reported to affect oocyte developmental potential and be markers of oocyte quality. Despite similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle, and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were determined to be correlated (p < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. Limitations, reasons for caution: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by in vitro maturation of patient oocytes, which has the benefit of identifying intrinsic differences between samples, but may not be completely representative of in vivo matured oocytes. Thus, these factors should be considered when interpreting the results. Wider Implications of the findings: Transcriptome profiles of young and advanced maternal age oocytes, particularly at the MII stage, suggest aberrant transcript abundance contributes to the age-associated decline in fertility.
2018-04-12 | GSE95477 | GEO
Project description:Advanced maternal age, defined as 35 years or older, is associated with a decline in both ovarian reserve and oocyte quality, which leads to the female infertility, pregnancy loss, fetal anomalies, stillbirth, and obstetric complications. At present, the effective approaches to counteract the maternal age-related decay of oocyte quality are still not fully determined. Here, we report that in vivo supplementation of nicotinamide mononucleotide (NMN) efficaciously ameliorates the quality of oocytes from naturally aged mice by recovering nicotinamide adenine dinucleotide (NAD + ) levels in oocytes. NMN supplementation increases the number of antral follicles, ovulated oocytes and matured oocytes from aged mice. Specifically, NMN supplementation maintains the normal spindle/chromosome structure and dynamics of cortical granule component ovastacin to ensure the meiotic competency and fertilization ability of aged oocytes. Moreover, single cell transcriptome analysis shows that the beneficial effect of NMN on the aged oocytes is mediated by the restoration of the mitochondrial function, thereby reducing the accumulated ROS to suppress the occurrence of apoptosis. To sum up, our data reveal that supplementation of NMN is a feasible approach to prevent oocyte quality from advanced maternal age-related deterioration, contributing to improve the reproductive outcome of aged women and the assisted reproductive technology.
2023-07-01 | GSE153920 | GEO
Project description:TOP2B is involved in transcriptional initiation in response to nuclear hormone ligands and plays a role in transcriptional elongation. Whole genome TOP2B ChIP-seq was carried out on human MCF7 cells in the presence and absence of the nuclear hormone estradiol. Three peak calling methods were used and the peaks identified by at least two methods were analyzed further. Approximately half of the peaks fell either within a gene or within 5Kb of a transcription start site. The coincidence of TOP2B peaks and gene promoters was analyzed; TOP2B peaks were less frequently associated with promoters in estradiol treated than in control cells, suggesting a role of TOP2B in repression of transcription or a transient role in estradiol induced transcriptional changes. Whole genome TOP2B ChIP-seq was carried out on human MCF7 cells in the presence (30 mins exposure) and absence of the nuclear hormone estradiol. "Input" control samples were also sequenced for background detection and comparison.
2015-09-21 | E-GEOD-66753 | biostudies-arrayexpress
Project description:To compare expression profiles in the cardiomyocytes with wild type top2b and those with top2b deletion after in vivo treatment of mice with doxorubicin or drug vehicle Doxorubicin is widely used in modern cancer treatments, despite the advent of targeted therapy. However, a dose-dependent cardiotoxicity often limits its clinical use. The prevailing theory hypothesizes that doxorubicin-induced cardiotoxicity is the result of reactive oxygen species (ROS) generation due to redox-cycling of doxorubicin. Here we showed that cardiomyocyte-specific deletion of Topoisomerase II beta (Top2b) markedly reduced DNA double-strand breaks, apoptosis, and functional damages in doxorubicin-treated hearts. To investigate transcriptomic changes after doxorubicin treatment in wild type mouse and mouse with cardiac specific deletion of Top2b, we examined the expression profiles in 4 groups of mice (3/group), ie. wildtype mice with or without doxorubicin treatment and mice with Top2b deletion in the cardiomyocytes with or without doxorubicin treatment. Mice were treated with doxorubicin (25mg/kg, i.p.) or PBS (drug vehicle) for 16 hr or 72 hr. The heart was removed and cardiomyocytes were isolated by using a Langendorff apparatus. After purification, total RNA was extracted from the cardiomyocytes, purified, and used for gene expression analysis. Compared with that in control cardiomyocytes or cardiomyocytes with Top2b deletion, doxorubicin caused a significant expression change in the genome of cardiomyocytes from the wildtype mice. Among the changes, multiple genes encoding mitochondrial structural protein and components of the respiratory chain complexes were down-regulated 72 hr after treatment while multiple genes in the p53 pathway were up-regulated 16 hr after treatment in the wildtype cardiomyocytes. Expression changes were examined in 2 groups of mice (wild type and conditional knockout of top2b in the cardiomyocytes) treated with doxorubicin or PBS for 16 or 72 hours
2012-10-28 | E-GEOD-40289 | biostudies-arrayexpress
Project description:Purpose: Identify Top2b-dependent genes by comparing retinal transcriptome profiling (RNA-seq) Methods: Retinal mRNA profiles of neonatal (postnatal day 0 and 6) wild-type (WT) and Topoisomerase IIbeta (Top2b) conditional knockout (Top2b−/−, or cKO) mice were generated using the SOLiD System (Applied Biosystems). Data analysis was performed according to published protocols (Trapnell et al., 2012) with minor modifications. Briefly, colorspace data of raw 50 bp reads was aligned to the mouse genome (mm9 or GRCm37) using Bowtie (Langmead et al., 2009). Gene expression levels were analyzed with Cufflinks, with differentially expressed genes determined by Cuffdiff (Trapnell et al., 2010). Results: After sequencing, ~120 million 50 bp reads for each time point and each group were obtained and mapped to the mouse genome (MGI, as of Feb 15, 2012). Over 62,000 transcripts were identified; these include ~22,000 annotated genes (including alternative-spliced variants) which cover 76% of the whole mouse genome. Among all the annotated genes, 8.80% (1,935/22,000) for P0 and 1.25% (274/22,000) for P6 showed differential expression between the control and cKO samples (p-value ≤ 0.05, q-value ≤ 0.05). Conclusions: All genes with differential expression levels in Top2b cKO samples are direct or indirect dependent to Top2b.
2017-02-12 | GSE86187 | GEO
Project description:Topoisomerase IIb (TOP2B) is essential for neural development and function, yet its precise molecular roles remain poorly understood. Here we trapped catalytically engaged TOP2B in DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that chromosome compartments set the threshold of TOP2B activity within the genome, while specific nucleosome configurations stimulate TOP2B activity within strongly transcribed regions and enhancers. Highly expressed genes that are devoid of usually associated chromatin marks, such as H3K36me3, are deficient in TOP2B activity, indicating that TOP2B may be inefficient at sensing transcription-generated torsional stress directly. Active promoters and the transcription start sites (TSS) with high RNA polymerase II (RNAPII) occupancy show elevated TOP2B ChIP-seq signals but are depleted in TOP2Bccs, indicating that TOP2B is held inactive at active promoters. Surprisingly, TOP2B inhibition with etoposide increased nascent transcription at highly expressed genes and enhancers. While ETP treatment reduced nascent transcription within long genes, these effects were independent of transcript length and instead correlated with the presence of intragenic enhancers. Together these results provide insights into the epigenetic regulation of topoisomerase activity and reveal new roles for TOP2B in neuronal transcriptional regulation.
2024-01-29 | GSE227458 | GEO
Project description:We performed array-based expression profiling to determine genes regulated by Chd7 and Top2b in CGNs. Our data show Chd7 and Top2b coregulate a common set of neuronal genes. Furthermore, we compared the gene expession in proliferating and postmitotic granule cells.
2017-01-20 | GSE76975 | GEO
Project description:Topoisomerase IIb (TOP2B) is essential for neural development and function, yet its precise molecular roles remain poorly understood. Here we trapped catalytically engaged TOP2B in DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that chromosome compartments set the threshold of TOP2B activity within the genome, while specific nucleosome configurations stimulate TOP2B activity within strongly transcribed regions and enhancers. Highly expressed genes that are devoid of usually associated chromatin marks, such as H3K36me3, are deficient in TOP2B activity, indicating that TOP2B may be inefficient at sensing transcription-generated torsional stress directly. Active promoters and the transcription start sites (TSS) with high RNA polymerase II (RNAPII) occupancy show elevated TOP2B ChIP-seq signals but are depleted in TOP2Bccs, indicating that TOP2B is held inactive at active promoters. Surprisingly, TOP2B inhibition with etoposide increased nascent transcription at highly expressed genes and enhancers. While ETP treatment reduced nascent transcription within long genes, these effects were independent of transcript length and instead correlated with the presence of intragenic enhancers. Together these results provide insights into the epigenetic regulation of topoisomerase activity and reveal new roles for TOP2B in neuronal transcriptional regulation.
2024-01-29 | GSE227462 | GEO
Project description:Topoisomerase IIb (TOP2B) is essential for neural development and function, yet its precise molecular roles remain poorly understood. Here we trapped catalytically engaged TOP2B in DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that chromosome compartments set the threshold of TOP2B activity within the genome, while specific nucleosome configurations stimulate TOP2B activity within strongly transcribed regions and enhancers. Highly expressed genes that are devoid of usually associated chromatin marks, such as H3K36me3, are deficient in TOP2B activity, indicating that TOP2B may be inefficient at sensing transcription-generated torsional stress directly. Active promoters and the transcription start sites (TSS) with high RNA polymerase II (RNAPII) occupancy show elevated TOP2B ChIP-seq signals but are depleted in TOP2Bccs, indicating that TOP2B is held inactive at active promoters. Surprisingly, TOP2B inhibition with etoposide increased nascent transcription at highly expressed genes and enhancers. While ETP treatment reduced nascent transcription within long genes, these effects were independent of transcript length and instead correlated with the presence of intragenic enhancers. Together these results provide insights into the epigenetic regulation of topoisomerase activity and reveal new roles for TOP2B in neuronal transcriptional regulation.
2024-01-29 | GSE227460 | GEO