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How living systems achieve precision in form and function despite their intrinsic stochasticity is a fundamental yet open question in biology. Here, we establish a quantitative morphomap of pre-implantation embryogenesis in mouse, rabbit and monkey embryos, which reveals that although blastomere divisions desynchronise passively without compensation, 8-cell embryos still display robust 3D structure. Using topological analysis and genetic perturbations in mouse, we show that embryos progressively change their cellular connectivity to a preferred topology, which can be predicted by a simple physical model where noise and actomyosin-driven compaction facilitate topological transitions lowering surface energy. This favours the most compact embryo packing at the 8- and 16-cell stage, thus promoting higher number of inner cells. Impairing mitotic desynchronisation reduces embryo packing compactness and generates significantly more cell mis-allocation and a lower proportion of inner-cell-mass-fated cells, suggesting that stochasticity in division timing contributes to achieving robust patterning and morphogenesis.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.

Breast Cancer Anti-estrogen Resistance 4 (BCAR4) was previously characterised in bovine species as a gene preferentially expressed in oocytes, whose inhibition is detrimental to in vitro embryo development. But its role in oogenesis, folliculogenesis and globally fertility in vivo remains unknown. Because the gene is not conserved in mice, rabbits were chosen for investigation of BCAR4 expression and function in vivo. BCAR4 displayed preferential expression in the ovary compared to somatic organs, and within the ovarian follicle in the oocyte compared to somatic cells. The transcript was detected in follicles as early as the preantral stage. Abundance decreased throughout embryo development until the blastocyst stage. A lineage of genome-edited rabbits was produced; BCAR4 expression was abolished in follicles from homozygous animals. Females of wild-type, heterozygous and homozygous genotypes were examined for ovarian physiology and reproductive parameters. Follicle growth and the number of ovulations in response to hormonal stimulation were not significantly different between genotypes. Following insemination, homozygous females displayed a significantly lower delivery rate than their heterozygous counterparts (22 ± 7% vs 71 ± 11% (mean ± SEM)), while prolificacy was 1.8 ± 0.7 vs 6.0 ± 1.4 kittens per insemination. In conclusion, BCAR4 is not essential for follicular growth and ovulation but it contributes to optimal fertility in rabbits.

As sport career is a priority in most of equine breeds, mares are frequently bred for the first time at an advanced age. Both age and first gestation were shown to have a deleterious effect on reproduction outcomes, respectively on fertility and offspring weight but the effect mare’s parity in older mares on embryo quality has never been considered. The aim of this project was to determine the effect of old mare’s nulliparity on gene expression in embryos. Day 8 post ovulation embryos were collected from old (10-16 years old) nulliparous (ON, N=5) or multiparous (OM, N=6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner cell mass enriched (ICMandTE) trophoblast were obtained by embryo bisection and paired end, non-oriented RNA sequencing (Illumina, NextSeq500) was performed on each hemi-embryo. To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the ICMandTE dataset. Differential expression was analyzed (DESeq2) with embryo sex and diameter as cofactors using a false discovery rate Summary sentenceMare’s parity in old mares impacts the expression of genes related to development and molecule exchanges in ICM and TE of blastocysts suggesting an adaptation to an altered environment.

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit pre-implantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic, and metabolic map of the pluripotency continuum in rabbit preimplantation embryos and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with that of humans and non-human primates.Summary StatementRabbit preimplantation embryos share characteristics with human and monkey embryos with respect to timing of early lineage segregation and expression of marker genes for naive and primed pluripotency.

An increased incidence in early embryo loss has been observed in aged mares. Moreover, the first foal born to a mare is lighter than her subsequent foals, with reported impaired placental function at term. Because trophoblast function may be affected from the embryo stage, the aim of this project was to determine the effect of parity in aged mares on gene expression in Day-8.5 embryos. Middle-aged (13.5±2.2 years) nulliparous (never foaled) (ON) or multiparous (1.8±1.6 foals) (OM) Saddlebred, non-nursing mares were inseminated with the semen of one unique stallion. At 8 days post-ovulation (10 days post-hCG), embryos were recovered by uterine flushing and bisected to obtain samples of pure (trophectoderm, TE) or inner cell mass enriched (ICM) trophoblast. Paired end, non-oriented RNA sequencing was performed with Illumina (NextSEqn 500) on 5 and 6 TE and ICM collected from ON and OM, respectively. Differential expression was analysed with DESEqn 2. Embryo size was included in the model and a P

The success of embryo development and implantation depends in part on the environment in which the embryo evolves. However, the composition of the uterine fluid surrounding the embryo in the peri-implantation period remains poorly studied. In this work, we aimed to develop a new strategy to visualize, collect, and analyze both blastocoelic liquid and juxta-embryonic uterine fluid from in vivo peri-implantation rabbit embryos. Using high-resolution ultrasound biomicroscopy, embryos were observed as fluid-filled anechoic vesicles, some of which were surrounded by a thin layer of uterine fluid. Ultrasound-guided puncture and aspiration of both the blastocoelic fluid contained in the embryo and the uterine fluid in the vicinity of the embryo were performed. Using nuclear magnetic resonance spectroscopy, altogether 24 metabolites were identified and quantified, of which 21 were detected in both fluids with a higher concentration in the uterus compared to the blastocoel. In contrast, pyruvate was detected at a higher concentration in blastocoelic compared to uterine fluid. Two acidic amino acids, glutamate and aspartate, were not detected in uterine fluid in contrast to blastocoelic fluid, suggesting a local regulation of uterine fluid composition. To our knowledge, this is the first report of simultaneous analysis of blastocoelic and uterine fluids collected in vivo at the time of implantation in mammals, shedding new insight for understanding the relationship between the embryo and its local environment at this critical period of development.

Authors are members of RGB-Net (TD 1101) and Epiconcept (FA 1201) COST actions - Remerciements aux membres de l'UCEA pour leur responsabilité sur leur lapin - Reconnaissance à Hélène Jammes pour formation d'E. Canon aux protocoles de traitement au bisulfite - Reconnaissance à Christian Poirier pour son aide en préparation des chiffres - Remerciements à Pierre Adenot et la MIMA2 pour accès à l'ApoTome microscopy et aussi à la Région Ile de France pour son financement; International audience; The POU5F1 gene encodes one of the "core" transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with POU5F1 expression and function in in vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends on de novo methylation of its promoter. However, little is known about the epigenetic regulation of POU5F1 expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the POU5F1 5'upstream region, relative to its regulated expression in different compartments of the blastocyst over a two-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation, and the gradual repression of POU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naive pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

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International audience; Mono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. The current study examined whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. Bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A proteomic analysis was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes (Data are available via ProteomeXchange with identifier PXD012092). The study explores, for the first time, the risk associated with exposing oocytes to low concentration (i.e., environmentally relevant concentration) of MEHP to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality

International audience; BACKGROUND: The placenta controls exchanges between the mother and the fetus and therefore fetal development and growth. The maternal environment can lead to disturbance of placental functions, with consequences on the health of the offspring. Since the rabbit placenta is very close to that of humans, rabbit models can provide biomedical data to study human placental function. Yet, to limit the use of animal experiments and to investigate the mechanistic aspects of placental function, we developed a new cell culture model in which rabbit trophoblast cells are differentiated from rabbit trophoblast stem cells. METHODS: Rabbit trophoblast stems cells were derived from blastocysts and differentiated onto a collagen gel and in the presence of a flow of culture medium to mimic maternal blood flow. Transcriptome analysis was performed on the stem and differentiated cells. RESULTS: Our culture model allows the differentiation of trophoblast stem cells. In particular, the fluid shear stress enhances microvilli formation on the differentiated cell surface, lipid droplets formation and fusion of cytotrophoblasts into syncytiotrophoblasts. In addition, the transcriptome analysis confirms the early trophoblast identity of the derived stem cells and reveals upregulation of signaling pathways involved in trophoblast differentiation. CONCLUSION: Thereby, the culture model allows mimicking the in vivo conditions in which maternal blood flow exerts a shear stress on trophoblast cells that influences their phenotype. GENERAL SIGNIFICANCE: Our culture model can be used to study the differentiation of trophoblast stem cells into cytotrophoblasts and syncytiotrophoblasts, as well as the trophoblast function in physiological and pathological conditions.

Preimplantation development in mammals : how relevant is the mouse model ? Knowledge of the molecular and cellular events of the early stages of mammalian embryo development is essential for biomedical research. The mechanisms involved, sometimes very specific to this period, are also the subject of much basic research. The mouse has long been used as a unique model for the analysis of these stages of development. Its features and the tools available to study it make it a very interesting model, but it is insufficient to represent all mammals and in particular the human embryo., La connaissance des évènements moléculaires et cellulaires des premières étapes du développement de l’embryon des Mammifères est indispensable à la recherche biomédicale. Les mécanismes mis en jeu, parfois très spécifiques de cette période, font aussi l’objet de nombreuses recherches fondamentales. La souris a longtemps été utilisée comme modèle unique pour l’analyse de ces stades de développement. Les mécanismes qui les régissent et les outils disponibles pour les étudier en font un modèle très intéressant mais insuffisant pour représenter les embryons de tous les mammifères et en particulier l’embryon humain., Duranthon Véronique. La période préimplantatoire du développement des mammifères: la souris est-elle un bon modèle ?. In: Bulletin de l'Académie Vétérinaire de France tome 172, 2019. pp. 77-82.

In mammals, male embryos grow faster. Equine embryo size is highly variable for same developmental stage, with no reported relation to embryo sex nor gene expression. Our objective was to evaluate gene expression sexual dimorphism on equine D8 blastocysts. Non-nursing, Saddlebred mares located on 2 experimental farms were inseminated with the semen of one unique stallion per farm. At 8 d post ovulation, 17 and 11 embryos from each farm, each from a different dam, were recovered by uterine flushing and bissected to obtain samples of pure (TE) or inner cell mass enriched (TE-ICM) trophoblast. RNA expression was analyzed separately in the TE-ICM and the TE of 16 female and 12 male embryos using paired end, non-oriented RNA sequencing (Illumina, NextSeq500). To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the TE-ICM data. Differential expression was analyzed (DESeq2) with farm and embryo size as cofactors using a false discovery rate (FDR)

Study question In comparison to in vivo development, how do different conditions of in vitro culture ('one step' versus 'sequential medium') impact DNA methylation and hydroxymethylation in preimplantation embryos? Summary answer Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used. What is known already Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations. Study design size, duration The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coitum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference. Participants/materials, setting, methods Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing differences or tendencies toward differences between the different conditions in the first round of quantification. Main results and the role of chance The kinetics of DNA methylation and hydroxymethylation were modified in in vitro cultured embryos, and the observed differences depended on the type of medium used. These differences were statistically significant. In addition, the expression of TET1 and TET2 was significantly reduced in post-embryonic genome activation (EGA) embryos after in vitro culture in both tested conditions. Finally, the expression of two retroviral sequences was analyzed and found to be significantly affected by in vitro culture. Limitations reasons for caution Our study remains mostly descriptive as no direct link can be established between the epigenetic changes observed and the expression changes in both effectors and targets of the studied epigenetic modifications. The results we obtained suggest that gene expression could be affected on a large scale, but this remains to be confirmed. Wider implications of the findings Our results are in agreement with the literature, showing that DNA methylation is sensitive to in vitro culture. As we observed an effect of both tested culture conditions on the tested epigenetic marks and on gene expression, we cannot conclude which medium is potentially closest to in vivo conditions. However, as the observed effects are different, additional studies may provide more information and potential recommendations for the use of culture media in assisted reproductive technology. Study funding/competing interests This work was supported by an 'AMP diagnostic prenatal et diagnostic genetique' 2012 grant from the French Agence de la Biomedecine. This study was performed within the framework of ANR LABEX 'REVIVE' (ANR-10-LABX-73). Authors are members of RGB-Net (TD 1101) and Epiconcept (FA 1201) COST actions. The authors declare that there is no competing interest.

International audience; The preconceptional and/or periconceptional periods (before and just after fertilization until the embryo blastocyst stage) are critical for the developmental origins of health and disease. Major epigenetic modifications occur during gametogenesis, fertilization, and the early stages of embryonic development. These modifications can be altered by the environment in vivo, particularly through maternal and/or paternal nutrition, but also in vitro, including during procedures such as assisted reproduction. Female gametes, but also male, are involved as targets of the epigenetic modifications and also as vectors of modified epigenetic marks, leading to long-term effects on the offspring. Physiological and epigenetic effects observed vary depending on fetal gender. Although this review focuses on new developments in epigenomics in the horse, most of the mechanism information was derived from mice, men, and cattle. Indeed, although the long-term impact of equine embryo technologies is yet to be evaluated, data from other species already indicate that the nutritional status of both gamete donors and of the recipient mare should not be overlooked. Finally, as opposed to most other domestic species, and closer to the situation in humans, older horses are used for reproduction, and this may also affect the quality of gametes and subsequently offspring to be born.

The pre- and peri-conceptional periods (before and just after fertilization, until the blastocyst stage) are critical in the context of the Developmental Origins of Health and Disease (DOHaD). Maternal in vivo environment, in particular nutrition, can disturb the apposition of epigenetic marks throughout gametogenesis, fertilization and the first steps of embryonic development, which are times during which major epigenetic changes take place. The in vitro environment, in the case of assisted reproduction techniques, also affects epigenetic marks. Whilst the embryo is a target of these changes, female and male gametes are both target and vector of these epigenetic changes, thus leading to multigenerational effects. Long term consequences on the phenotype of offspring vary according to the sex of the vector parent, the sex of the individual and the generation.

We recently developed and validated a methodology based on early morphokinetics to predict 6 categories of bovine embryos, including 4 categories of blastocysts: early hatching blastocysts (EHB), hatching blastocysts (HB), low hatching blastocysts (LHB), and arrhythmic blastocysts (AB) (Reis et al. 2018 Anim. Reprod. 15, 601). We hypothesised that different early morphokinetic pathways could (1) be accompanied by different biological patterns (EGA, etc.) and (2) influence interaction with environmental constraints (cryopreservation, etc.). The objective of this study was (1) to investigate whether transcriptomic differences between the predicted EHB, HB, LHB, or AB could be observed immediately after the fourth embryonic cycle (just after EGA); (2) to assess cryotolerance of these morphokinetic categories. Time lapses were produced during 4 days post invitro insemination (dpi) (Reis et al. 2018). Study 1: 128 embryos having finished the fourth embryonic cycle were individually dry frozen at 4.7 dpi, classified as EHB, HB, LHB, or AB, and pooled into four batches of eight embryos/category (total 16 samples). Total RNA was extracted, amplified using the SMARTseq V4 ultralow input kit (Clontech), libraries were prepared using the Nextera XT Illumina library preparation kit, and sequenced (paired-end 50-34bp) on an Illumina NextSEqn 500 instrument. Identification of differentially expressed genes was achieved using Limma package (R). The P-values were adjusted using the Benjamini and Hochberg false discovery rate (Saenz-de-Juano et al. 2014). Fold change >2 or 135µm and −2µm at zona pellucida) were slow frozen at 6.2 or 7 dpi, classified as EHB (n=8), HB (n=29), LHB (n=32), or AB (n=21), and further thawed and cultured invitro for 72h. Cryotolerance was evaluated at 24 and 72h post-thawing. Statistical analysis was performed using chi-square or Fisher test. Study 1: the AB category presented 258, 699, and 899 upregulated genes and 40, 62, and 60 downregulated in comparison to LHB, HB, and EHB, respectively. The functional comparison of the two extremes (EHB vs. AB), at 4.7 dpi, showed that AB embryos present intense transcription regulation activity. The monocarboxylic acid binding pathway was upregulated in EHB. Study 2: the post-thawing invitro survival was similar between EHB, HB, LHB, and AB (50%, 34.5%, 31.3%, and 42.9%, respectively). The hatching/alive ratio in EHB category tended to be lower than AB (1/4 vs. 8/9; P=0.052). Frozen/thawed EHB and HB presented reduced hatching rates, whereas LHB and AB significantly increased the hatching potential after thawing compared with their not-frozen counterparts of the learning database (25% vs. 100%; 50% vs. 100%; 70% vs. 11%; 88.9% vs. 18%, respectively). Morphokinetic prediction highlighted a new classification of bovine embryos with different biological features. This could contribute to improve practice on IVF embryo transfer taking into account these differences.

Single-cell transcriptomics and gene editing on human pre-implantation embryos have proved that mechanisms previously identified and well characterised in mouse pre-implantation development may not hold true in the human embryo. However, ethical and legal concerns limit the availability of surplus human embryos for research, resulting in the need for novel animal models. Bovine embryos share morphological and temporal resemblance with human early development; still, key molecular and cellular developmental mechanisms need to be further explored. In the present study, we performed a comparative single-cell RNA sequencing analysis across multiple pre-implantational developmental stages of invitro-derived bovine, human (Petropoulos et al. 2016 Cell 165, 1012-1026; https://doi.org/10.1016/j.cell.2016.03.023), and mouse embryos (Cheng et al. 2019 Cell Rep. 26, 2593-2607, https://doi.org/10.1016/j.celrep.2019.02.031; Posfai et al. 2017 eLife 6, e22906, https://doi.org/10.7554/eLife.22906; Chen et al. 2016 Genome Res. 26, 1342-1354). In total 497 blastomeres corresponding to embryonic days (E) E4-E8 were prepared using Smart sEqn 2, Illumina HiSEqn 2500 sequencing with 50-bp single-end reads. Embryonic stage was defined by cell numbers and morphology as multicellular stage (Mu, ~E4), morulae (M; E5), early blastocysts (EB, ~6), mid-expanded blastocysts, (MB, ~E7), and late expanded/hatched blastocysts (LB, ~E8). We found top differentially expressed genes elucidating how lineage specification and pluripotency is controlled in the early bovine embryo. Our transcriptomic data showed that the first lineage segregation in the pre-implantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and trophectoderm (TE) profiles. Different from the mouse, where the first lineage segregation of TE-ICM is suggested to occur at the morula stage due to CDX2-OCT4 antagonism, we found that the expression of OCT4 protein cattle expanded blastocysts is not restricted to the ICM, similar to what has been reported to occur in the human embryo. Interestingly, we also found that the second lineage segregation, which segregates the epiblast from the primitive endoderm within the ICM, starts in bovine at E8 expanded hatched late blastocysts, suggesting a potential difference with humans, where TE-ICM and epiblast-primitive endoderm has previously been reported to occur almost concurrently. We are now complementing our findings with genome editing in bovine embryos by generating knockout embryos of different target genes part of an evolutionarily conserved signaling pathway to study their effects in the early pre-implantation embryo. We aim to elucidate how early lineage specification and pluripotency establishment occurs while offering theoretical support for efficient derivation and culture of bovine embryonic stem cells.

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Changes to the spatial organization of specific chromatin domains such as constitutive heterochromatin have been studied extensively in somatic cells. During early embryonic development, drastic epigenetic reprogramming of both the maternal and paternal genomes, followed by chromatin remodeling at the time of embryonic genome activation (EGA), have been observed in the mouse. Very few studies have been performed in other mammalian species (human, bovine, or rabbit) and the data are far from complete. During this work, we studied the three-dimensional organization of pericentromeric regions during the preimplantation period in the rabbit using specific techniques (3D-FISH) and tools (semi-automated image analysis). We observed that the pericentromeric regions (identified with specific probes for Rsat I and Rsat II genomic sequences) changed their shapes (from pearl necklaces to clusters), their nuclear localizations (from central to peripheral), as from the 4-cell stage. This reorganization goes along with histone modification changes and reduced amount of interactions with nucleolar precursor body surface. Altogether, our results suggest that the 4-cell stage may be a crucial window for events necessary before major EGA, which occurs during the 8-cell stage in the rabbit. Electronic supplementary material The online version of this article (10.1007/s00412-018-0671-z) contains supplementary material, which is available to authorized users.

Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones., Summary: A unique quantitative approach based on complete reconstruction of the cell lineage that unveils an unknown mechanism of size control in cell populations of rabbit blastocysts, wild types or clones.

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Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3&ndash, 8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.

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Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones.

International audience; Epidemiological studies in humans and animal models (including ruminants and horses) have highlighted the critical role of nutrition on developmental programming. Indeed, it has been demonstrated that the nutritional environment during the periconceptional period and foetal development can altered the postnatal performance of the resultant offspring. This nutritional programming can be exerted by maternal and paternal lineages and can affect offspring beyond the F1 generation. Alterations in epigenetic mechanisms have been proposed as the causative link behind the programming trajectories observed in the offspring. Although a clear cause-effect relationship between epigenetic modifications during early development and later offspring phenotype has not been demonstrated in livestock species, strong associations have been reported for some epigenetic marks (e.g. messenger RNA) that are worth exploring as possible predictors of future offspring phenotype. In this review, we shortly describe the main epigenetic mechanisms studied so far in mammals (i.e. mainly in the mouse) thought to be associated with developmental programming, and discuss the few studies available in mammalian herbivores (e.g. cattle) showing the effect of nutrition on epigenetic marks and the associated phenotype. Clearly, there is a need to develop research on nutritional strategies capable of modulating the epigenetic machinery with positive influence on the phenotype of livestock herbivores. This type of research is needed to alleviate the challenges currently faced by the livestock industry (e.g. impaired fertility of high-yielding dairy cows). This in turn will have a positive influence on animal welfare and productivity of livestock enterprises.

Maternal exposure to diluted diesel engine exhaust alters placental function of the first and second generation in rabbit model. 8. International Symposium on Nanotechnology, Occupational and Environmental Health

Remerciements à l'Unité UCEA de Bressonvilliers, à V. Gelin et A. Neveu pour leur aide.; Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2. Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2. Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo–uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Cette notice sera modifiée dès parution définitive de l'article; International audience; Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Here we analyze HDGF expression in day-8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein free medium containing polyvinyalcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. The HDGF is synthesized by the endometrium and embryo alike, and it may exert embryotrophic effects by autocrine and/or paracrine mechanisms.

Rabbits softly swept to domestication When people domesticate animals, they select for tameness and tolerance of humans. What else do they look for? To identify the selective pressures that led to rabbit domestication, Carneiro et al. sequenced a domestic rabbit genome and compared it to that of its wild brethren (see the Perspective by Lohmueller). Domestication did not involve a single gene changing, but rather many gene alleles changing in frequency between tame and domestic rabbits, known as a soft selective sweep. Many of these alleles have changes that may affect brain development, supporting the idea that tameness involves changes at multiple loci. Science , this issue p. 1074 ; see also p. 1000

In a previous work, we proposed an algorithm to select a subset of discriminant morphokinetical parameters within a larger predefined set (116 parameters) and to predict 6 major bovine embryo profiles of in vitro development. The algorithm relies on flexible combinations of the discriminant parameters selected within the four first cleavage cycles. The retained profiles were arrested embryos (embryos without mitotic activity, showing signs of life); dead embryos (embryos with all cells dead); anarchic embryos (embryos with abnormal morphological and/or kinetical development: some of these embryos can result in a blastocyst); not hatched blastocysts (blastocysts not hatching by 8 dpi); hatching blastocysts (blastocysts hatching in vitro from 7.3 to 8 dpi); and early hatching blastocysts (blastocysts hatching from 6 to 7.2 dpi) (Reis et al. 2018 Anim. Reprod.). The aim of the present work was to develop a ready-to-use software (EasyPickAndPredict) allowing the extension of this methodology to other embryologists. The software of high portability was built with the JAVA language and embedded with the classifier (R language, R Foundation for Statistical Computing, Vienna, Austria) and a user’s help for annotation (Adobe Premiere Pro CS5, San Jose, CA, USA). The predictive software is easy to handle, fast to load, and has high portability. The “manual annotation” function is based on click actions to annotate the discriminant parameters. The “prediction” function calls the embedded classifier. The “report” function creates customised reports including the embryo classification, the summary of the measures, and the accuracy of the prediction (vote system). The “help” function calls an audiovisual guideline with annotation specifications for all the morphokinetic parameters currently described (including the discriminant subset of parameters). This document includes help to produce a good time-lapse video (16min 51s); annotation specifications for the cleavage cycles 1 to 4 (36 min 26s); and a summary with examples of the 6 major embryonic morphokinetical profiles (16min 47s). The predictive graphical interface is easy to manipulate and automatically extracts the value of each discriminant parameter on the time-lapse picture as validated by the user click. The functions “manual annotation”, “automatic prediction”, “help”, and “report” supply embryologists with a standard approach to predict and analyse morphokinetical profiles of their embryos. This standardised approach is necessary to improve the capacity of comparison of morphokinetical works in different laboratories and enhance knowledge about in vitro-produced bovine embryos.

Reproductive effects of gestational exposure to diesel exhaust in a rabbit model. XIV. International Congress of Toxicology

Artemin a member of the glial cell line-derived neurotrophic factor (GDNF) family is present in mice and human preimplantation embryos, and reproductive tract, during early pregnancy promoting embryo development in vitro. The presence of artemin in cattle embryos and reproductive tract, however, is unknown. In the present work we identified for first time artemin in bovine uterine fluid (UF) (Western blot), endometrium (RT-PCR, Western blot and immunohistochemistry) and embryos (RT-PCR and immunohistochemistry) during early preimplantation development. In addition, GFRalpha3, a component of the artemin receptor was localized in blastocysts produced in vitro. Individually developing embryos released ARTEMIN in culture medium and triggered ARTEMIN mRNA down-regulation in epithelial cells from endometrial cell cultures. Our results suggest that ARTEMIN derived from early embryos and maternal reproductive tract may exert important roles during early development in cattle. © 2016 Elsevier Inc.

International audience; The effect of the Developmental Origins of Health and Disease on the spread of non-communicable diseases is recognised by world agencies such as the United Nations and the World Health Organization. Early environmental effects on offspring phenotype also apply to domestic animals and their production traits. Herein, we show that maternal nutrition not only throughout pregnancy, but also in the periconception period can affect offspring phenotype through modifications of gametes, embryos and placental function. Because epigenetic mechanisms are key processes in mediating these effects, we propose that the study of epigenetic marks in gametes may provide additional information for domestic animal selection.