Depletion of double homeobox proteins in bovine zygotes abolishes blastocyst formation

International audienceIt remains unclear which factors—maternal or embryonic—are responsible for reprogramming the mammalian epigenome following fertilisation. Recently, overexpression of double homeobox (DUX) transcription factors—a family of proteins conserved among placental mammals—was found to induce a totipotent-like state in human and murine stem cells. In humans and mice, DUX transcription is activated in zygotes, suggesting it could be an early regulator of embryonic genome activation (EGA). Moreover, DUX4 has been reported to interact with histone acetyltransferases (HATs) in human cells, suggesting it could also be involved in epigenetic reprogramming. However, it is unclear whether DUX proteins are essential for development, as knockout studies in mice have found variable effects on EGA and blastocyst formation. Moreover, the function of DUX in embryos has yet to be established in any other mammal, despite recent reports that mice may not be the most appropriate model for human development. Here, we evaluate the function of DUX during the first divisions of the developing bovine embryo. Public annotations of DUX4 in cattle were largely based on predictions from the analogous human locus, so bovine DUX4 transcripts were manually reconstructed from published short-read RNA-seq data. The predicted bovine DUX4 protein resembled human DUX4 (E-value 2e-42), including homology at the HAT-binding domain. Using qPCR and immunofluorescence, we find that DUX4 is absent in the oocyte but present in zygotes by 22 h post-fertilisation (hpf), similar to what has been described in humans. Notably, this is well before minor EGA in both species, suggesting that DUX4 may be the first activated gene in the developing embryo. Conversely, the only other DUX family member present in the bovine genome, DUXA, is not appreciably expressed until the 8-cell stage (major EGA). To determine the function of DUX proteins in bovine development, we depleted zygotes for either DUX4 or DUXA through electroporation with siRNA at 10–14 hpf. The knock-down was verified by RT-qPCR and immunofluorescence. We find that DUX4 and DUXA depletion significantly reduced blastocyst formation from 44% (56/128) for controls to 2.5% (3/122) and 5.5% (7/126), respectively. Moreover, DUX4 depletion causes downregulation of EGA-associated genes, suggesting that DUX4 is a major regulator of EGA, as in the mouse. Considering the interaction of DUX4 with HATs in human cells, it is possible that DUX4 activates target gene expression through histone acetylation. Using CUT&Tag, we are currently investigating the effect of DUX4 depletion on genome-wide histone acetylation. Overall, these data indicate that zygotic expression of DUX is conserved among placental mammals, and that these factors serve as key regulators of EGA

Extracellular vesicles from early pregnant uterine fluids trigger expression of implantation-related markers in ovine endometrium

Extracellular vesicles from early pregnant uterine fluids trigger expression of implantation-related markers in ovine endometrium. 3. Cellfit annual meeting 201

Extracellular vesicles from early pregnant uterine fluids promote expression of implantation-related markers in ovine endometrium

Theme: Early Development and PregnancyExtracellular vesicles (EVs) are crucial for intercellular communications and they may play important role in the delivery of molecular messages during the pre-implantation period of pregnancy. EVs have been isolated from uterine luminal fluid (ULF) in human and sheep. Recent data have provided evidence that EVs contained in ovine ULF can penetrate endometrial epithelial cells after a 6-days infusion in vivo. Nevertheless, embryo implantation involves rapid and dynamic changes in molecular interactions with the endometrium. Our present work aims to determine whether EVs collected from ULF interact with endometrial epithelial cells and modify cell physiology after a short time of in vitro and in vivo incubation conditions.Primary cultures of endometrial epithelial cells were derived from ovine uteri on day 12 post-oestrus. EVs were purified from ovine ULFs on Day 14 of pregnancy (2 days before conceptus implantation). The presence of EVs in ULF was confirmed by transmission electron microscopic observation. ULF EVs were labeled with lipophilic PKH26 fluorescent dye and then incubated with primary cultures of epithelial cells during 30 min to 24h. Confocal microscopy analyses revealed an uptake of EVs as early as 30 minutes after incubation, followed by a progressive increase of intracellular fluorescence up to 6 hours.For the in vivo study, ovine ULF EVs isolated on Day 14 of pregnancy were labeled with PKH26 fluorescent dye and infused into the uterine lumen of cyclic ewes on Day 12 post-oestrus. After 24h, numerous epithelial cells from the luminal layer and superficial glands exhibited an intensive fluorescence signal, whereas deep endometrial glands displayed few fluorescent cells. No signal was detectable in the stroma. The impact of EVs on endometrial function was investigated by quantifying transcript expression of a selection of endometrial genes. First data pointed out that expression of two genes, including the Myxovirus-Resistance Protein (MX1) was up-regulated following EVs uptake by the endometrial epithelium. This work provides first evidence that ovine EVs from pregnant ULF can (i) enter in endometrial epithelial cells within 30 min in vitro or 24h in vivo (ii) modulate expression of endometrial gene expression known to be critical for embryo implantation. These results suggest a critical role for EVs in the preparation of endometrium when implantation iniates

Implantation-related genes in the ovine endometrium are regulated by extracellular vesicles from earlypregnant uterine fluids

National audienc

Les vésicules extra-cellulaires (EVs) de fluide utérin stimulent l’expression de marqueurs de l’implantation dans l’endomètre ovin in vivo

National audienc

DICER : Dialogue pendant l'Implantation entre le Conceptus et l'Endomètre : implication des microARN

Crédits Incitatifs 2014Champ thématique: AdaptationDICER : Dialogue pendant l'Implantation entre le Conceptus et l'Endomètre : implication des microARN. Journées d’Animation des Crédits Incitatifs du Département de Physiologie Animale et Systèmes d’Elevage (JACI Phase 2016