Apposition to endometrial epithelial cells activates mouse blastocysts for implantation

Ruane, Peter T; Berneau, Stéphane; Koeck, Rebekka; Watts, Jessica; Kimber, Susan J.; Brison, Daniel R.; Westwood, Melissa; Aplin, John D.

doi:10.1093/molehr/gax043

Cited by 47 publications

(61 citation statements)

References 55 publications

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“…This supports a mechanism of TE maturation driven by contact with the ICM. Polar initiation of human TE maturation is consistent with the observation that the majority of human blastocysts attached to endometrial cells by the polar side with subsequent spreading of TE maturation to mural cells [20][21][22][23][24][25][26][27] . Analysis of most enriched signaling pathways pointed out prime candidates potentially driving maturation of EPI and TE: TGFβ, IGF1, BMP2, IL6 and FGF4 (Extended Data Fig.…”

supporting

confidence: 86%

Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

Meistermann

Loubersac

Reignier

et al. 2019

Preprint

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Recent technological advances such as single-cell RNAseq 1-3 and CRISPR-CAS9-mediated knock-out 4 have allowed an unprecedented access into processes orchestrating human preimplantation development 5 . However, the sequence of events which occur during human preimplantation development are still unknown. In particular, timing of first human lineage specification, the process by which the morula cells acquire a specific fate, remains elusive. Here, we present a human preimplantation development model based on transcriptomic pseudotime modelling of scRNAseq biologically validated by spatial information and precise time-lapse staging. In contrast to mouse, we show that trophectoderm (TE) / inner cell mass (ICM) lineage specification in human is only detectable at the transcriptomic level at the blastocyst stage, just prior to expansion. We validated the expression profile of novel markers enabling precise staging of human preimplantation embryos, such as IFI16 which highlights establishment of epiblast (EPI) and NR2F2 which appears at the transition from specified to mature TE. Strikingly, mature TE cells arise from the polar side, just after specification, supporting a model of polar TE cells driving TE

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supporting

confidence: 86%

Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

Meistermann

Loubersac

Reignier

et al. 2019

Preprint

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“…CD-1 (Charles River, Tranent, UK) mice were maintained, superovulated and housed overnight with stud males for mating as previously described by Ruane et al (2017). Denuded fail to fertilize M-II oocytes (n=8) were obtained 36h post-ovulation and cultured briefly (2-4h) in 50µl KSOM medium (Millipore) containing 0.4% BSA at 37°C, 5% CO2 under ovoil (Vitrolife, Göteborg, Sweden) prior to assay in the XFP in a single group of 8.…”

Section: Preparation Of Mouse Oocytesmentioning

confidence: 99%

Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos

Muller

Lewis

Adeniyi

et al. 2019

Preprint

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Background: Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific molecular events remains lacking.Results: Here, extracellular flux analysis (EFA) was applied to small groups of bovine, equine, mouse and human oocytes and bovine early embryos to measure OCR. Using EFA, we report the changes in mitochondrial activity during the processes of oocyte maturation, fertilization, and pre-implantation development to blastocyst stage in response to physiological demands in mammalian embryos. Crucially, we describe the real time partitioning of overall OCR to spare capacity, proton leak, non-mitochondrial and coupled respirationshowing that while there are alterations in activity over the course of development to respond to physiological demand, the overall efficiency is unchanged.Conclusion: EFA is shown to be able to measure mitochondrial function in small groups of mammalian oocytes and embryos in a manner which is robust, rapid and easy to use. EFA is non-invasive and allows real-time determination of the impact of compounds on OCR, facilitating an assessment of the parameters of mitochondrial activity. This provides proof-ofconcept for EFA as an accessible system with which to study oocyte and embryo metabolism.

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“…Confluent endometrial cells were incubated in serum-free medium (DMEM, 2mM 105 L-glutamine, 100µg/ml streptomycin and 100IU/ml penicillin) for 24 hrs prior to 106 co-culture with hatched E4.5 mouse blastocysts (3 per well), as previously described 107 (Ruane et al, 2017). 108…”

Section: In-vitro Attachment Assay 104mentioning

confidence: 99%

“…Transfer of hatched E4.5 mouse blastocysts to Ishikawa epithelial cell monolayers is 177 followed by an initial period (28 hrs) of weak and reversible attachment that initiates 178 the activation required for embryos to progress, over the next 20 hrs, from stable 179 attachment to breaching and displacement of the underlying cells (Ruane et al, 2017). 180 Figure 2A shows 5F12 reactivity in cells surrounding an attachment site with no 181 trophoblast invasion, whereas in Figure 2B, the trophoblast has breached the 182 subjacent cell layer and is beginning to laterally invade, with CD44-positive epithelial 183 cells crowded together in adjacent locations.…”

Section: Cd44 Immunoreactivity In Attachment Sites In Vitro 176mentioning

confidence: 99%

“…We have used Ishikawa cells as a model endometrial epithelium for examining 67 interaction with blastocyst stage embryos (Ruane et al, 2017, Ruane et al, 2018, 68 Singh et al, 2010. When embryos are transferred to confluent Ishikawa cell 69 monolayers, initial attachment to the apical surface is followed by breaching and 70 trophoblast outgrowth.…”

Section: Introduction 38mentioning

confidence: 99%

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Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model

Berneau

Ruane

Brison

et al. 2019

MHR: Basic Science of Reproductive Medicine

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21Implantation failure is an important impediment to increasing success rates in assisted 22 reproductive technologies (ART). Knowledge of the cascade of morphological and 23 molecular events at implantation remains limited. Cell surface CD44 and hyaluronate 24 (HA) have been reported in the uterus, but a role in intercellular interaction at 25 implantation remains to be evaluated. Mouse embryos were co-cultured with human 26 Ishikawa endometrial epithelial monolayers over two days. Attachment was tenuous 27 during the first 24 hrs, after which it became stable, leading to breaching of the 28 monolayer. The effects of enzymatically reducing the density of HA, or introducing a 29 function-blocking antibody to CD44, were monitored during progression from weak to 30 stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the 31 epithelial cells enhanced the speed of attachment, while a similar treatment of 32 embryos had no effect. The antibody to CD44 caused retardation of initial attachment. 33These results suggest that CD44-HA binding could be employed by embryos during 34 initial docking, but the persistence of HA in epithelial cells might be detrimental to later 35 stages of implantation by retarding attainment of stable attachment. 36

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Apposition to endometrial epithelial cells activates mouse blastocysts for implantation

Abstract: This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared.

Cited by 47 publications

References 55 publications

Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

Application of extracellular flux analysis for determining mitochondrial function in mammalian oocytes and early embryos

Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model

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