Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes
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ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Nikolai Slavov Montserrat Barragan Monasterio
PROVIDER: MSV000093870 | MassIVE | Mon Jan 15 14:38:00 GMT 2024
REPOSITORIES: MassIVE
Publications
Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.
Galatidou Styliani S Petelski Aleksandra A AA Pujol Aïda A Lattes Karinna K Latorraca Lais B LB Fair Trudee T Popovic Mina M Vassena Rita R Slavov Nikolai N Barragán Montserrat M
Molecular human reproduction 20240601 7
Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between imm ...[more]
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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:Background: The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes.<br>Methods: mRNA was isolated for whole genome gene expression microarray analysis from metaphase II (MII) oocytes donated by IVF or ICSI patients (10 women aged <36 years (younger) and 5 women aged 36-39 years (both inclusive) (older)) undergoing controlled ovarian simulation
2009-12-31 | E-MEXP-2347 | biostudies-arrayexpress
Project description:Acetaminophen (APAP) is clinically recommended as analgesic and antipyretic among pregnant women. However, accumulating laboratory evidence shows that the use of APAP during pregnancy may alter fetal development. Since fetal stage is a susceptible window for early oogenesis, we aim to assess the potential effects of maternal administration of APAP on fetal oocytes. Maternal administration and the fetal ovary cultures showed that APAP (50 and 150 mg/kg.bw/day) caused meiotic aberrations in fetal oocytes through its metabolite NAPQI, including meiotic prophase I (MPI) progression delay and homologous recombination defects. Co-treatment with NAM or NRC, NAD+ supplements, efficiently restored the MPI arrest, while the addition of the inhibitor of SIRT7 invalidated the effect of the NAD+ supplement. Additionally, RNA sequencing revealed distorted transcriptomes of fetal ovaries treated with NAPQI. Further, the fecundity of female offspring was affected, exhibiting as delayed primordial folliculogenesis and puberty onset, reduced levels of ovarian hormones, and impaired developmental competence of MII oocytes. Short-term administration of APAP to pregnant mouse caused meiotic aberrations in fetal oocytes by its metabolite NAPQI, while co-treatment with NAD+ supplement efficiently relieves the adverse effects by interacting with SIRT7.
2023-03-18 | GSE227192 | GEO
Project description:TOP2B disturbed the quality of human oocytes with advanced maternal age
| PRJNA515815 | ENA
Project description:The long-standing view of 'immortal germ line versus mortal soma' poses a fundamental question in biology concerning how oocytes age in molecular terms. A mainstream hypothesis is that maternal aging of oocytes has its roots in gene transcription. Investigating the proteins resulting from mRNA translation would reveal how far the levels of functionally available proteins correlate with mRNAs, and would offer novel insight into the changes oocytes undergo during maternal aging. Gene ontology semantic analysis reveals the high similarity of the detected proteome (2,324 proteins) to the transcriptome (22,334 mRNAs), though not all proteins have a cognate mRNA. Concerning their dynamics, 4-fold changes of abundance are more frequent in the proteome (3%) than the transcriptome (0.05%), with correlation. Whereas proteins associated with the nucleus (e.g. structural maintenance of chromosomes, spindle-assembly checkpoints) are largely represented among those that change in oocytes during maternal aging; proteins associated with oxidative stress/damage (e.g. superoxide dismutase) are infrequent. These quantitative alterations are either impoverishing or enriching. Using gene ontology analysis, these alterations do not relate in any simple way to the classic signature of aging known from somatic tissues. We conclude that proteome analysis of mouse oocytes may not be surrogated with transcriptome analysis, given the lack of correlation. Furthermore, we conclude that the classic features of aging may not be transposed from somatic tissues to oocytes in a one-to-one fashion. Overall, there is more to the maternal aging of oocytes than mere cellular deterioration exemplified by the notorious increase of meiotic aneuploidy. Three pools of 20 zona-enclosed B6C3F1 oocytes from each age group were subjected for experiment.
2014-04-15 | E-GEOD-42959 | biostudies-arrayexpress
Project description:The long-standing view of 'immortal germ line versus mortal soma' poses a fundamental question in biology concerning how oocytes age in molecular terms. A mainstream hypothesis is that maternal aging of oocytes has its roots in gene transcription. Investigating the proteins resulting from mRNA translation would reveal how far the levels of functionally available proteins correlate with mRNAs, and would offer novel insight into the changes oocytes undergo during maternal aging. Gene ontology semantic analysis reveals the high similarity of the detected proteome (2,324 proteins) to the transcriptome (22,334 mRNAs), though not all proteins have a cognate mRNA. Concerning their dynamics, 4-fold changes of abundance are more frequent in the proteome (3%) than the transcriptome (0.05%), with correlation. Whereas proteins associated with the nucleus (e.g. structural maintenance of chromosomes, spindle-assembly checkpoints) are largely represented among those that change in oocytes during maternal aging; proteins associated with oxidative stress/damage (e.g. superoxide dismutase) are infrequent. These quantitative alterations are either impoverishing or enriching. Using gene ontology analysis, these alterations do not relate in any simple way to the classic signature of aging known from somatic tissues. We conclude that proteome analysis of mouse oocytes may not be surrogated with transcriptome analysis, given the lack of correlation. Furthermore, we conclude that the classic features of aging may not be transposed from somatic tissues to oocytes in a one-to-one fashion. Overall, there is more to the maternal aging of oocytes than mere cellular deterioration exemplified by the notorious increase of meiotic aneuploidy.
2014-04-15 | GSE42959 | GEO
Project description:Background: Early embryonic development is governed by maternal transcripts stored within the oocyte during oogenesis. Transcriptional activity of the oocyte ultimately dictates its developmental potential and may be influenced by maternal age, resulting in reduced competence of oocytes derived from women of advanced age, compared with the young. In the current study, RNA-Seq was used to perform transcriptome profiling of human GV and MII oocytes derived from young and advanced maternal age women. Participants/Materials and Methods: Cumulus dissection from donated oocytes was performed. GV and MII oocytes underwent deep RNA sequencing using the SMART-Seq v4 Ultra Low Input RNA protocol (Takara-Clontech, USA) and Nextera XT DNA library preparation kit (Illumina, USA). Data processing, quality assessment and bioinformatics analysis were performed using source-software, including FastQC, HISAT2, StringTie, edgeR and DAVID. Results: Following deep single-cell RNA-Seq on GV and MII oocytes, hundreds of transcripts were significantly differentially expressed between young maternal age (YMA) and advanced maternal age (AMA) groups, with the most significant biological processes relating to mitochondrial reserves. The GV to MII transition shares common biological processes between young and AMA groups, however, some genes involved in mitochondria function were altered during ageing. A decrease in mitochondrial-related transcripts was also observed during the GV to MII transition. However, there was a much greater reduction of mitochondrial-related transcripts in MII oocytes of AMA. This observation was confirmed when YMA MII oocytes were compared with the AMA MII group with mitochondrial-related transcripts being significantly higher expressed in the YMA group, including biological processes, such as mitochondrial electron transport and ATP biosynthetic process. These results indicate a higher energy potential in YMA MII oocytes that is decreased with age. Other significantly higher biological processes in the YMA MII group include transcripts involved in the regulation of ubiquitin-dependent degradation. Lack of these transcripts could lead to a non-appropriate removal of oogenesis remnants following fertilisation in the AMA MII group. Discussion: Understanding reproductive ageing effects at the RNA level in human oocytes may reveal differences in the mechanisms regulating the GV to MII transition that impact on oocyte quality in YMA and AMA patients. Further investigations of the up-/down-regulated transcripts during ageing could guide and improved IVF outcomes for AMA patients.
2022-01-11 | GSE164371 | GEO
Project description:The mean age of women seeking infertility treatment has gradually increased over recent years. This has coincided with the emergence of in vitro maturation (IVM), a method used in assisted reproductive technology for patients with special requirements. However, when compared with conventional in vitro fertilization, IVM is associated with poor embryonic development potential and low live birth rates, thus limiting the widespread application of this technique. In this study, we performed RNA-sequencing transcriptomic assays and identified a total of 2,627 significant differentially expressed genes (DEGs) between IVM oocytes and in vivo matured oocytes from mice of advanced reproductive age. Next, Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to identify the potential functions of the DEGs. The most significantly enriched pathway was oxidative phosphorylation (OXPHOS). In addition, we constructed a protein-protein interaction network to identify key genes and determined that most of the hub genes were mtDNA-encoded subunits of respiratory chain complex I. Antioxidant supplementation lead to an increase in ATP production and reduced the gene expression profile of the OXPHOS pathway in the IVM group. Moreover, alternative splicing (AS) events were identified during in vivo or in vitro oocyte maturation; data showed that skipped exons were the most frequent type of AS event. A number of genes associated with the OXPHOS pathway exhibited alterations in AS events, including Ndufa7, Ndufs7, Cox6a2, Ndufs5, Ndufb1, and Uqcrh. Furthermore, the process of IVO promoted the skipping of exon 2 in Ndufa7 and exon 3 in Ndufs7 compared with the IVM oocytes, as determined by semi‑quantitative RT‑PCR. Collectively, these findings provide potential new therapeutic targets for improving IVM of aged women who undergo infertility treatment.
2022-01-13 | GSE182711 | GEO
Project description:While the oocytes’ transcriptome is predominantly determined by maturation stage, transcripts related to specific pathways such as chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.
2021-03-24 | GSE158802 | GEO