Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells

Aizawa, Eishi; Dumeau, Charles-Étienne; Freimann, Remo; Minin, Giulio Di; Wutz, Anton

doi:10.1371/journal.pone.0233072

Cited by 4 publications

(4 citation statements)

References 37 publications

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“…C57BL/6J females were mated with 129S6/SvEvTac males to obtain hybrid embryos to establish ESC lines. After genotyping of sex (Aizawa et al, 2020), a female ESC line was co-transfected with a piggyBac vector carrying a CAG-EGFP-IRES-hygro transgene and a hyperactive piggyBac transposase expression plasmid (Yusa et al, 2011) using lipofectamine 2000 by following a manufacturer’s protocol. Subsequently, single cells with EGFP expression were sorted by FACS.…”

Section: Methodsmentioning

confidence: 99%

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Comprehensive comparison of female germ cell developmentin vitroandin vivoidentifies epigenetic gene regulation crucial for oocyte development and embryonic competence

Aizawa

Ozonov

Kawamura

et al. 2023

Preprint

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Germ cells are the origin of new individuals. Hence, specifying germ cell identity is crucial for reproduction. The recent establishment of in vitro culture systems for generating oocytes from mouse pluripotent stem cells provides a basis for progress in studies of oogenesis and reproductive technology. However, currently the developmental competence of in vitro generated oocytes is low compared to in vivo grown oocytes. The causes underlying poor oocyte quality remain to be determined. By reconstituting germ cell development in culture from different developmental starting points within gametogenesis, we show that the differentiation of primordial germ cells (PGCs) and primordial germ cell-like cells (PGCLCs) to growing oocytes (GROs), as well as the subsequent growth of follicles are critical culture steps for specifying competence of fully-grown oocytes (FGOs) for preimplantation development. A systematic comparison of transcriptomes of single oocytes having undergone different in vitro culture trajectories identifies genes normally upregulated during oocyte growth to be susceptible for mis-regulation during in vitro oogenesis. Many of such genes have been described as targets of Polycomb repressive complexes (PRCs). Deregulation of Polycomb repression therefore likely perturbs the accumulation of cytoplasmic factors and/or setting of chromatin states in FGOs that are required for embryonic development after fertilization. Conversely, in vitro derived oocytes often displayed failure of zygotic genome activation (ZGA) and abnormal acquisition of 5-hydroxymethylcytosine (5hmC) on maternal chromosomes after activation. In addition, subcellular delocalization of pyruvate dehydrogenase (PDH) and of STELLA were observed suggesting new molecular markers for defective oocyte development. Our study identifies epigenetic regulation at an early stage of oogenesis as crucial for developmental competence and suggests specific in vitro culture steps as targets for improving oocyte quality.

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Section: Methodsmentioning

confidence: 99%

“…DNA extraction of cells was performed as previously described (Aizawa et al ., 2020). PCR was performed using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific) following the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive comparison of female germ cell developmentin vitroandin vivoidentifies epigenetic gene regulation crucial for oocyte development and embryonic competence

Aizawa

Ozonov

Kawamura

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Cell lines C57BL/6J females were mated with 129S6/SvEvTac males to obtain hybrid embryos to establish ESC lines. After genotyping of sex (Aizawa et al, 2020), a female ESC line was co-transfected with a piggyBac vector carrying a CAG-EGFP-IRES-hygro transgene and a hyperactive piggyBac transposase expression plasmid (Yusa et al, 2011) using lipofectamine 2000 by following a manufacturer's protocol. Subsequently, single cells with EGFP expression were sorted by FACS.…”

Section: Methods and Protocolsmentioning

confidence: 99%

“…Genotyping DNA extraction of cells was performed as previously described (Aizawa et al, 2020). PCR was performed using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific) following the manufacturer's protocol.…”

Section: Assessment Of Follicle Developmentmentioning

confidence: 99%

Epigenetic regulation limits competence of pluripotent stem cell‐derived oocytes

Aizawa,

Ozonov,

Kawamura

et al. 2023

The EMBO Journal

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Recent studies have reported the differentiation of pluripotent cells into oocytes in vitro. However, the developmental competence of in vitro‐generated oocytes remains low. Here, we perform a comprehensive comparison of mouse germ cell development in vitro over all culture steps versus in vivo with the goal to understand mechanisms underlying poor oocyte quality. We show that the in vitro differentiation of primordial germ cells to growing oocytes and subsequent follicle growth is critical for competence for preimplantation development. Systematic transcriptome analysis of single oocytes that were subjected to different culture steps identifies genes that are normally upregulated during oocyte growth to be susceptible for misregulation during in vitro oogenesis. Many misregulated genes are Polycomb targets. Deregulation of Polycomb repression is therefore a key cause and the earliest defect known in in vitro oocyte differentiation. Conversely, structurally normal in vitro‐derived oocytes fail at zygotic genome activation and show abnormal acquisition of 5‐hydroxymethylcytosine on maternal chromosomes. Our data identify epigenetic regulation at an early stage of oogenesis limiting developmental competence and suggest opportunities for future improvements.

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In vitro gametogenesis: Towards competent oocytes

Aizawa,

Peters,

Wutz

2024

BioEssays

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Production of oocytes from pluripotent cell cultures in a dish represents a new paradigm in stem cell and developmental biology and has implications for how we think about life. The spark of life for the next generation occurs at fertilization when sperm and oocyte fuse. In animals, gametes are the only cells that transmit their genomes to the next generation. Oocytes contain in addition a large cytoplasm with factors that direct embryonic development. Reconstitution of mouse oocyte and embryonic development in culture provides experimental opportunities and facilitates an unprecedented understanding of molecular mechanisms. However, the application of in vitro gametogenesis to reproductive medicine or infertility treatment remains challenging. One significant concern is the quality of in vitro‐derived oocytes. Here, we review the current understanding and identify limitations in generating oocytes in vitro. From this basis, we explore opportunities for future improvements of the in vitro approach to generating high‐quality oocytes.

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Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells

Cited by 4 publications

References 37 publications

Comprehensive comparison of female germ cell developmentin vitroandin vivoidentifies epigenetic gene regulation crucial for oocyte development and embryonic competence

Comprehensive comparison of female germ cell developmentin vitroandin vivoidentifies epigenetic gene regulation crucial for oocyte development and embryonic competence

Epigenetic regulation limits competence of pluripotent stem cell‐derived oocytes

In vitro gametogenesis: Towards competent oocytes

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