Dumeau, C.-E. | Search bioRxiv

bioRxiv2023

Comprehensive comparison of female germ cell development in vitro and in vivo identifies epigenetic gene regulation crucial for oocyte development and embryonic competence

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. HighlightsO_LISingle cell transcriptomics and functional assessment of oocyte development from pluripotent stem cells in culture in a stage-specific manner provides a comprehensive resource for comparisons to oogenesis in vivo. C_LIO_LICulture steps for growth and differentiation of reconstituted follicles are critical for defining embryonic competence of in vitro generated oocytes. C_LIO_LIZygotic genome activation failure and epigenetic impairment are hallmarks of in vitro-generated oocytes that fail to develop after activation or fertilization. C_LIO_LIComputational analysis of gene expression changes and chromatin modification patterns identifies specific gene sets that indicate that Polycomb mediated repression is vulnerable during in vitro folliculogenesis. C_LI

developmental biology↗

bioRxiv2020

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

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for the proper development of embryos. Haploid embryonic stem cells (haESCs) are unique stem cells established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitute of gametic genome, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletions of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed frequently in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs provide high efficiency for semi-cloning when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improvement for development of semi-cloned embryos derived by haESC injection.

developmental biology↗

bioRxiv2020

Tmed2 regulates Smoothened trafficking and Hedgehog signalling

Hedgehog (HH) signalling plays a key role in embryonic pattering and stem cell differentiation. Compounds that selectively bind Smoothened (SMO) can induce cell death in mouse embryonic stem cells (ESCs). Here we perform a genetic screen in haploid ESCs and discover that SMO inhibits a cell death pathway that resembles dissociation induced death of human ESCs and Anoikis. In mouse ESCs, SMO acts through a G-protein coupled mechanism that is independent of GLI activation. Our screen also identifies the Golgi proteins Tmed2 and Tmed10. We show that TMED2 binds SMO and controls its abundance at the plasma membrane. In neural differentiation and neural tube pattering Tmed2 acts as a repressor of HH signalling strength. We demonstrate that the interaction between SMO and TMED2 is regulated by HH signalling suggesting SMO release form the ER-Golgi is critical for controlling G-protein and GLI mediated functions of mammalian HH signalling.

cell biology↗