Gene Expression
Nebulas
Gene Expression NebulasSummary: Aim: The mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro matured (IVM) oocytes, remains to be elucidated. Here, we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression. Results: Three key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Further, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP+ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy. Innovation: The present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence. Conclusions: In vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.
Overall Design: Investigated the transcriptome characteristics of human oocytes matured in vitro and in vivo to gain a transcriptome-level understanding of how oocytes mature and to illuminate the differences between human IVM and in vivo (IVO) matured oocytes at the transcript level.
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| Growth Protocol: | - |
| Treatment Protocol: | growing environment: In vitro; growing environment: In vivo |
| Extract Protocol: | Oocytes were rinsed in Ca2+/Mg2+-free PBS solution and individually placed in 0.2-ml PCR tubes containing a 5-μl mixture of lysis buffer and RNase inhibitor. RNA from a single oocyte was amplified using the SMARTer Ultra Low RNA kit for Illumina sequencing. |
| Library Construction Protocol: | The cDNA library was generated as follows. The quantified cDNA (20 ng) was fragmented into 200-bp lengths by ultrasonic waves using a Bioruptor® Sonication System. The cDNA fragments then underwent end repair, adaptor addition and purification using AMPure XP Beads. The products with adaptors were amplified by PCR. The different samples were marked with different index tags. PCR amplification products were electrophoresed on 2% agarose gels, and 200–300-bp DNA fragments were recovered using the CWBIO Gel Extraction Kit (CW2302A, Cwbiotech, Inc., China) and dissolved in EB buffer. cDNA library quality was tested using an Agilent 2100 Bioanalyzer, and the effective concentration was quantified by quantitative real-time PCR. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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