18 research outputs found
Depletion of double homeobox proteins in bovine zygotes abolishes blastocyst formation
No full textInternational 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
No full textExtracellular 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 trigger expression of implantation-related markers in ovine endometrium
No full textExtracellular vesicles from early pregnant uterine fluids trigger expression of implantation-related markers in ovine endometrium. 3. Cellfit annual meeting 201
Implantation-related genes in the ovine endometrium are regulated by extracellular vesicles from earlypregnant uterine fluids
No full textSession 4: Reproduction, Milks, Plants L. Galio: présentation orale sélectionnéeNational audienc
Implantation-related genes in the ovine endometrium are regulated by extracellular vesicles from earlypregnant uterine fluids
No full textSession 4: Reproduction, Milks, Plants L. Galio: présentation orale sélectionnéeNational audienc
Extracellular vesicles from early pregnant uterine fluids promote expression of implantation-related markers in ovine endometrium
No full textExtracellular 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
DICER : Dialogue pendant l'Implantation entre le Conceptus et l'Endomètre : implication des microARN
No full textCrédits 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
Extracellular vesicles from early pregnant uterine fluids promote expression of implantation-related markers in ovine endometrium
No full textTheme: Early Development and PregnancyTheme: 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
Maternal obesity and preconceptional weight loss: high sensitivity of epigenetic regulators gene expression in relation to fetal growth and long-lasting effect in mice
No full textNational audienceMaternal obesity impacts fetal growth and pregnancy outcomes. Epigenetic regulation, that stably modifies the phenotype through chemical changes within the chromatin without alteration in DNA sequence, is one of the potential mechanisms to memorize the parental environment, leading to disease in adulthood. To counteract the deleterious effects of obesity on fertility and pregnancy issues, preconceptional weight loss is recommencled to obese women. Whether this weight loss is beneficial or detrimental to the offspring remains poorly explored. Epigenetic mechanisms could be affected by maternal weight changes, perturbing expression of key devel opmental genes in the placenta or the fetus. Our aim was to investigate the effects of chronic maternal obesity on feto-placental growth at term of gestation and in adulthood, along with the underlying epigenetic mechanisms. We also testecl whether preconceptional weight loss could· alleviate these effects. Female mice were fed either a control diet (CTRL group), a high-fat diet (obese group, OB) for 4 months, or a high-fat diet switched to a control diet 2 months before conception (weight loss, WL) group). At mating, OB females exhibited an obese phenotype while WL females normalizecl their weight and metabolic parameters. At embryonic day 18.5, fetuses from OB females showed fetal growth restriction and the odd-ratio for small for gestational age phenotype (SGA) was 3.2. These parameters were normalized for WL fetuses. The expression of 60 epigenetic machinery genes and 32 metabolic genes was measured in the fetal liver, placental labyrinth and junctional zone. One third of the epigenetic machinery genes were differentially expressed between at least two maternal groups, especially the histone acetylation pathway. Maternal weight loss normalized the expression of only a subset of these genes. A second cohort of offspring was followed from birth until 6 months for weight gain, food intake, glucose metabolism and cholesterol. During lactation, OB offspring rapidly caught up in weight and after weaning the diet-induced obesity was enhanced in OB males compared to CTRL. No obvious phenotype was observed in female offspring. Maternal weight loss rescued this phenotype. These results show that expression of epigenetic machinery genes and in particular histone acety lation regulators, is highly sensitive to preconceptional maternal metabolic features. The obesity induced transcriptional changes could alter the placental and hepatic epigenomes, contributing to SGA phenotype. Preconceptional weight loss alleviates the effects of obesity on fetal and adult weight but even if obesity was cured before conception, some effects were retained in the offspring phenotype at term. This study is an important step toward understanding immediate and long-term mechanisms linking maternal nutrition to fetal growth and adult healt
Les vésicules extra-cellulaires (EVs) de fluide utérin stimulent l’expression de marqueurs de l’implantation dans l’endomètre ovin in vivo
No full textNational audienc
