Single-cell analysis of transcriptome and DNA methylome in human oocyte maturation

Oocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and used our previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CpG methylation. DNMT3B, a gene encoding a key non-CpG methylation enzyme, is one of the 1,077 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CpG methylation as oocytes mature. Non-CpG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CpG methylation. Over 98% of non-CpG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CpG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors.

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Single-cell analysis of Non-CG methylation dynamics and gene expression in human oocyte maturation

10.1101/651141 ◽

2019 ◽

Author(s):

Bo Yu ◽

Naresh Doni Jayavelu ◽

Stephanie L. Battle ◽

Thomas H. Smith ◽

Samuel E Zimmerman ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Single Cell ◽

Oocyte Maturation ◽

Gene Expression Regulation ◽

Single Cell Analysis ◽

Reproductive Potential ◽

Fertility Treatment ◽

Human Oocyte

ABSTRACTOocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and use previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CG methylation. DNMT3B, a gene encoding a key non-CG methylation enzyme, is one of the 1000 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CG methylation as oocytes mature. Non-CG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CG methylation. Over 98% of non-CG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors.

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Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation

Stem Cell Reports ◽

10.1016/j.stemcr.2017.05.026 ◽

2017 ◽

Vol 9 (1) ◽

pp. 397-407 ◽

Cited By ~ 39

Author(s):

Bo Yu ◽

Xiao Dong ◽

Silvia Gravina ◽

Önder Kartal ◽

Timothy Schimmel ◽

...

Keyword(s):

Single Cell ◽

Oocyte Maturation ◽

Cpg Methylation ◽

Human Oocyte ◽

Genome Wide

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Changes in the Mitochondria-Related Nuclear Gene Expression Profile during Human Oocyte Maturation by the IVM Technique

Cells ◽

10.3390/cells11020297 ◽

2022 ◽

Vol 11 (2) ◽

pp. 297

Author(s):

Zhi-Yong Yang ◽

Min Ye ◽

Ya-Xin Xing ◽

Qi-Gui Xie ◽

Jian-Hong Zhou ◽

...

Keyword(s):

Oocyte Maturation ◽

Single Cell Analysis ◽

Nuclear Gene ◽

Mature Stage ◽

Human Oocyte ◽

Acid Oxidation ◽

Developmental Needs ◽

Nuclear Gene Expression

To address which mitochondria-related nuclear differentially expressed genes (DEGs) and related pathways are altered during human oocyte maturation, single-cell analysis was performed in three oocyte states: in vivo matured (M-IVO), in vitro matured (M-IVT), and failed to mature in vitro (IM-IVT). There were 691 DEGs and 16 mitochondria-related DEGs in the comparison of M-IVT vs. IM-IVT oocytes, and 2281 DEGs and 160 mitochondria-related DEGs in the comparison of M-IVT vs. M-IVO oocytes, respectively. The GO and KEGG analyses showed that most of them were involved in pathways such as oxidative phosphorylation, pyruvate metabolism, peroxisome, and amino acid metabolism, i.e., valine, leucine, isoleucine, glycine, serine, and threonine metabolism or degradation. During the progress of oocyte maturation, the metabolic pathway, which derives the main source of ATP, shifted from glucose metabolism to pyruvate and fatty acid oxidation in order to maintain a low level of damaging reactive oxygen species (ROS) production. Although the immature oocytes could be cultured to a mature stage by an in vitro technique (IVM), there were still some differences in mitochondria-related regulations, which showed that the mitochondria were regulated by nuclear genes to compensate for their developmental needs. Meanwhile, the results indicated that the current IVM culture medium should be optimized to compensate for the special need for further development according to this disclosure, as it was a latent strategy to improve the effectiveness of the IVM procedure.

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Single-cell transcriptome and cell-specific network analysis reveal the reparative effect of neurotrophin-4 in preantral follicles grown in vitro

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00818-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Yingchun Guo ◽

Peigen Chen ◽

Tingting Li ◽

Lei Jia ◽

Yi Zhou ◽

...

Keyword(s):

Network Analysis ◽

Single Cell ◽

Fertility Preservation ◽

Oocyte Maturation ◽

Human Oocyte ◽

Preantral Follicle ◽

Preantral Follicles ◽

Reparative Effect ◽

Human And Mouse

Abstract Background In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth. Methods We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect. Results We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG. Conclusions Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

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Fabrication of high-aspect-ratio microstructures in polymer microfluid chips for in vitro single-cell analysis

Technical Physics ◽

10.1134/s106378421610008x ◽

2016 ◽

Vol 61 (10) ◽

pp. 1566-1571 ◽

Cited By ~ 10

Author(s):

A. S. Bukatin ◽

I. S. Mukhin ◽

E. I. Malyshev ◽

I. V. Kukhtevich ◽

A. A. Evstrapov ◽

...

Keyword(s):

Aspect Ratio ◽

Single Cell ◽

High Aspect Ratio ◽

Single Cell Analysis ◽

Cell Analysis

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Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

The Journal of Immunology ◽

10.4049/jimmunol.164.6.3047 ◽

2000 ◽

Vol 164 (6) ◽

pp. 3047-3055 ◽

Cited By ~ 193

Author(s):

Dragana Jankovic ◽

Marika C. Kullberg ◽

Nancy Noben-Trauth ◽

Patricia Caspar ◽

William E. Paul ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cytokine Profile ◽

Cell Analysis ◽

In Vitro Development ◽

Th2 Cytokine ◽

Vitro Development ◽

Cd4 Lymphocytes

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Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

Scientific Reports ◽

10.1038/srep34373 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 11

Author(s):

Tomislav Sarenac ◽

Martin Trapecar ◽

Lidija Gradisnik ◽

Marjan Slak Rupnik ◽

Dusica Pahor

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis ◽

Smad Pathway ◽

Human Keratocytes

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Single-cell analysis revealed that IL4I1 promoted ovarian cancer progression

Journal of Translational Medicine ◽

10.1186/s12967-021-03123-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hongyu Zhao ◽

Yu Teng ◽

Wende Hao ◽

Jie Li ◽

Zhefeng Li ◽

...

Keyword(s):

Ovarian Cancer ◽

Single Cell ◽

Cancer Progression ◽

Cox Regression ◽

Single Cell Analysis ◽

Cell Types ◽

In Vitro Assays ◽

Migration And Invasion ◽

Dominant Type

Abstract Background Ovarian cancer was one of the leading causes of female deaths. Patients with OC were essentially incurable and portends a poor prognosis, presumably because of profound genetic heterogeneity limiting reproducible prognostic classifications. Methods We comprehensively analyzed an ovarian cancer single-cell RNA sequencing dataset, GSE118828, and identified nine major cell types. Relationship between the clusters was explored with CellPhoneDB. A malignant epithelial cluster was confirmed using pseudotime analysis, CNV and GSVA. Furthermore, we constructed the prediction model (i.e., RiskScore) consisted of 10 prognosis-specific genes from 2397 malignant epithelial genes using the LASSO Cox regression algorithm based on public datasets. Then, the prognostic value of Riskscore was assessed with Kaplan–Meier survival analysis and time-dependent ROC curves. At last, a series of in-vitro assays were conducted to explore the roles of IL4I1, an important gene in Riskscore, in OC progression. Results We found that macrophages possessed the most interaction pairs with other clusters, and M2-like TAMs were the dominant type of macrophages. C0 was identified as the malignant epithelial cluster. Patients with a lower RiskScore had a greater OS (log-rank P < 0.01). In training set, the AUC of RiskScore was 0.666, 0.743 and 0.809 in 1-year, 3-year and 5-year survival, respectively. This was also validated in another two cohorts. Moreover, downregulation of IL4I1 inhibited OC cells proliferation, migration and invasion. Conclusions Our work provide novel insights into our understanding of the heterogeneity among OCs, and would help elucidate the biology of OC and provide clinical guidance in prognosis for OC patients.

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HIV-1 provirus transcription and translation in macrophages differs from pre-integrated cDNA complexes and requires E2F transcriptional programs

10.1101/2021.04.21.440655 ◽

2021 ◽

Author(s):

Albebson L. Lim ◽

Philip Moos ◽

Christopher D. Pond ◽

Erica C. Larson ◽

Laura J. Martins ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Immunological Response ◽

Virus Production ◽

Cell Analysis ◽

Gene Transcripts ◽

Nuanced Understanding ◽

New Perspective ◽

Hiv 1

AbstractHIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. While previous work has focused on the transcription and translation of HIV-1 genes; our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes (PIC) and those containing integrated provirus and actively making late HIV proteins. These are also compared to control cells, not exposed to virus.Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, cells harboring PIC cDNA have transcript loads comparable to cells transcribing from provirus and making p24, mCherry, and vpu proteins. Second, all HIV-1 transcripts are easily detectable in abundance from PIC cDNA transcription, as is the case with cells transcribing from provirus, although the frequency of PIC cells with detectable gag-pol, tat, env, and nef transcripts is higher than the corresponding frequencies observed for “Provirus cells”. Third, the background transcriptomes of cells harboring pre- integrated HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV- 1 transcript. Fourth, integration and production of p24, mCherry, and Vpu proteins is accompanied by a switch from transcriptomes characterized by NFkB and AP-1 promoted transcription to a transcriptome characterized by E2F family transcription products. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of PIC cDNA transcription and the transcriptomic changes that support HIV-1 protein production from integrated provirus.Author SummarySingle cell analysis is able to distinguish between HIV-1 infected macrophage cells that are transcribing pre-integrated HIV-1 cDNA and those transcribing HIV-1 provirus. Only cells transcribing HIV-1 provirus are making p24, marker mCherry and Vpu proteins, which corresponds with a change in the host cell’s background transcriptome from one expressing viral restriction and immunological response genes to one that is expressing genes associated with cell replication and oxidative phosphorylation.

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Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells

Human Reproduction Update ◽

10.1093/humupd/dmaa021 ◽

2020 ◽

Vol 26 (5) ◽

pp. 670-688 ◽

Cited By ~ 1

Author(s):

Lin Li ◽

Risako Yang ◽

Chenghong Yin ◽

Kehkooi Kee

Keyword(s):

Stem Cells ◽

Single Cell ◽

Germ Cells ◽

Regulatory Networks ◽

Single Cell Analysis ◽

Reproductive Development ◽

In Vitro Differentiation ◽

Gene Markers ◽

Human Reproductive

Abstract BACKGROUND Understanding the molecular and cellular mechanisms of human reproductive development has been limited by the scarcity of human samples and ethical constraints. Recently, in vitro differentiation of human pluripotent stem cells into germ cells and single-cell analyses have opened new avenues to directly study human germ cells and identify unique mechanisms in human reproductive development. OBJECTIVE AND RATIONALE The goal of this review is to collate novel findings and insightful discoveries with these new methodologies, aiming at introducing researchers and clinicians to the use of these tools to study human reproductive biology and develop treatments for infertility. SEARCH METHODS PubMed was used to search articles and reviews with the following main keywords: in vitro differentiation, human stem cells, single-cell analysis, spermatogenesis, oogenesis, germ cells and other key terms related to these subjects. The search period included all publications from 2000 until now. OUTCOMES Single-cell analyses of human gonads have identified many important gene markers at different developmental stages and in subpopulations of cells. To validate the functional roles of these gene markers, researchers have used the in vitro differentiation of human pluripotent cells into germ cells and confirmed that some genetic requirements are unique in human germ cells and are not conserved in mouse models. Moreover, transcriptional regulatory networks and the interaction of germ and somatic cells in gonads were elucidated in these studies. WIDER IMPLICATIONS Single-cell analyses allow researchers to identify gene markers and potential regulatory networks using limited clinical samples. On the other hand, in vitro differentiation methods provide clinical researchers with tools to examine these newly identify gene markers and study the causative effects of mutations previously associated with infertility. Combining these two methodologies, researchers can identify gene markers and networks which are essential and unique in human reproductive development, thereby producing more accurate diagnostic tools for assessing reproductive disorders and developing treatments for infertility.

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