Adult Body Weight Is Programmed by a Redox-Regulated and Energy-Dependent Process during the Pronuclear Stage in Mouse

In mammals fertilization triggers a series of Ca2+ oscillations that not only are essential for events of egg activation but also stimulate oxidative phosphorylation. Little is known, however, about the relationship between quantitative changes in egg metabolism and specific long-term effects in offspring. This study assessed whether post-natal growth is modulated by early transient changes in NAD(P)H and FAD2+ in zygotes. We report that experimentally manipulating the redox potential of fertilized eggs during the pronuclear (PN) stage affects post-natal body weight. Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity with resulting offspring that are persistently and significantly smaller than controls. Exogenous lactate stimulates NAD+ reduction and impairs mitochondrial activity, and produces offspring that are smaller than controls at weaning but catch up after weaning. Cytosolic alkalization increases NAD(P)+ reduction and offspring of normal birth-weight become significantly and persistently larger than controls. These results constitute the first report that post-natal growth rate is ultimately linked to modulation of NAD(P)H and FAD2+ concentration as early as the PN stage

DNA methylation of the POU5F1 regulatory region in rabbit first embryonic lineages

International audienceThe expression of POU5F1 is controlled by cis-regulatory elements located 5' upstream from the initiation start site. This regulatory region is highly conserved among species, and especially four conserved regions (CR1 to 4) have been identified. The minimal promoter is located in the CR1 region, while the proximal and distal enhancers involved in cell specific regulation of expression are located in CR2- 3 and CR4 regions respectively. In the mouse the two enhancer regions contribute differently to gene expression depending on the developmental stage of the embryo. POU5F1 repression of expression is induced by regulatory factors binding to the 5' upstream region but also by DNA and histone methylation. DNA methylation of POU5F1 has been mostly analyzed in in vitro derived stem cells. However, promotor DNA methylation levels have been shown to differ between embryonic stem cells and the "in embryo" counterparts they are derived from. We thus decided to analyze DNA methylation of the four conserved regions of the POU5F1 upstream region in the first embryonic lineages. Therefore we took benefit from the rabbit embryo whose epiblast, hypoblast and trophoblast can be easily isolated. At that stage, POU5F1 expression is restricted to the epiblast. We evidenced an hypomethylation of the four conserved regions of POU5F1 5' upstream region in the epiblast. Interestingly even the CR4 region which is supposed to be functional in the ICM but not in the epiblast conserved a very low methylation level in this tissue. CR1 methylation was lower in day 6 embryos than in fibroblasts

Progressive establishment of differential methylation on POU5F1 upstream sequence over blastocyst development

International audienceThe POU5F1 gene encodes one of the "core" transcription factors necessary to maintain pluripotency but very few data are available concerning the precise epigenetic regulation of its expression in early embryo. We analysed the progressive modifications of DNA methylation of POU5F1 upstream region in the different compartments of the developing blastocyst. Therefore we used the rabbit embryo as a model for most mammalian blastocysts, where contrarily to the mouse, epiblast differentiates in a plane embryonic disk at the surface of the conceptus and in direct contact with maternal environment. Embryos were micro dissected at Day4,5, and6. POU5F1 expression was quantified by RTqPCR. Methylation profile of four regions encompassing conserved cis-elements (Kobolak et al. 2009 BMC Mol. Biol.10 88.) was determined by bisulfite treatment, cloning and sequencing. POU5F1 is highly expressed in Day4 trophectoderm but significantly enriched in the inner cell mass. At Day5, the POU5F1 relative expression decreased but the difference between embryonic and trophectoderm compartments tends to increase. At Day6, POU5F1 transcripts were restricted to the epiblast, with a very reduced expression in the hypoblast. Noticeably POU5F1 expression progressively decreases in the trophectoderm/trophoblast compartment over the Day4-Day6 period. The four regions were hypomethylated in pluripotent Inner Cell Mass and epiblast. Differences in DNA methylation between pluripotent and differentiated layers occurred as early as Day4 and progressively increased. A principal component analysis segregated pluripotent and highly differentiated samples and identified the CpGs mostly involved in this separation. Interestingly while differentiation-related changes in methylation occurred in the four analyzed regions, theyare more pronounced in the region encompassing the proximal enhancer 1A

Contributions maternelles et zygotiques à la reprogrammation des enhancers chez les embryons bovins pré-implantoires

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DMRT1 is Required For Sex Determination in Rabbits

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DMRT1 is a Sex-Determining Gene in Rabbits

International audienceDMRT1 is a transcription factor containing a DNA-binding domain (DM domain) highly conserved across evolution. It shows gonad-specific expression and it is considered as the Testis Determining Factor in many non-mammalian species. In mice, Dmrt1 disruptions had no impact on testicular differentiation during fetal development, whereas in humans, mutations induce gonadal dysgenesis with XY women phenotype (46, XY DSD). In some others mammals, DMRT1 expression was shown to precede SOX9 up-regulation like in XX sex-reversed goats (FOXL2-/-) or in wild type XY rabbits where SRY and DMRT1 are both expressed in XY genital ridges a few days before testis cords formation. We thus assumed that DMRT1 was involved in sex determination in non-murine species, by directly targeting SOX9 expression. Thanks to CRISPR/Cas9 technology, we generated DMRT1 mutant rabbits. We show that the absence of DMRT1 leads to male-to-female sex reversal in XY homozygous mutants. At 20 days post conception, few days after sex-determination, XY DMRT1-/- gonads are devoid of testis cords structures but rather present a typical ovarian organization with cortical germ cells. Molecular studies confirmed the expressional up-regulation of ovary-driving genes (RSPO1, WNT4, FOXL2 and CYP19A1) and the downregulation of testis-specific ones (SOX9, SOX10 and DHH) while SRY expression is highly maintained in the developing XY ovary (RNA-sequencing and RT-qPCR data). Interestingly, some somatic cells of the XY DMRT1-/- gonads still present SOX9 protein expression. Nevertheless, this is not sufficient to trigger its target genes expression (e.g. AMH) in the developing XY ovary. Our data clearly involved DMRT1 as a key player in sex determination in rabbits. DMRT1 is required to SRY action on SOX9 expression and may also be necessary to SOX9 function on its own target genes. Interestingly, DMRT1 was recently shown to act as a pioneer factor, opening the chromatin and favouring the action of SOX protein such as SOX9. Investigation on DMRT1 targets and epigenetic mark (H3K4me1, H3K27ac and H3K27me3) deposition on these targets are in progress at early stages of rabbit gonadal differentiation

Effects of BOEC and VERO co-culture systems on bovine blastocyst transcriptome

International audienceEarly embryo development is known to be impacted by its environment and especially by oviductal secretions in vivo. In cattle, embryo co-culture with bovine oviduct epithelial cells (BOEC) has thus been developed to mimic the in vivo oviduct/embryo crosstalk. Neverthe-less, to the best of our knowledge, whether BOEC had a specific impact on embryo transcrip-tome hasn’t been investigated yet. To answer this question, we compared bovine blastocysts obtained by co-culture with BOEC to blastocysts obtained with another co-culture system: VERO cells (an epithelial cell line derived from monkey kidney). Control blastocysts were obtained in standard conditions, i.e. at 5% O2 in SOF medium (Minitüb, Tiefenbach, Germa-ny) + 5% Fetal Calf Serum (FCS). Because co-culture systems require 20% O2 to maintain feeder cells alive, embryos cultured at 20% O2 in SOF + 5% FCS were included as an addi-tional control. Cleavage rates and timing of blastocyst appearance were similar in the four culture conditions. A significant decrease in blastocyst rate was observed at 20% O2 without feeder cells. Day 8 blastocysts transcriptome was analyzed on a new customized bovine mi-croarray including more than 26 700 transcripts and 250 retroviral ESTs (GEO platform GPL21734). Hierarchical clustering of the samples revealed very weak differences between culture conditions but a clear clustering of samples depending on the presence or absence of feeder cells. Considering an adjusted P value 2 (Limma test), 36 transcripts were found diffe-rentially expressed between blastocysts obtained in SOF medium in 5% or 20% O2. Compa-ring the two co-culture conditions revealed only 10 differentially expressed transcripts sug-gesting almost no difference induced by the origin of cells used in co-culture systems on bo-vine blastocyst transcriptome. Nevertheless, the presence of BOEC or VERO cells induced differential expression of 192 and 229 transcripts respectively when compared to 5% O2 and 542 and 881 transcripts respectively when compared to 20% O2. A large proportion of the transcripts affected by co-culture with BOEC were also impacted by VERO cells. Several biofunctions relative to cell cycle regulation, free radical scavenging and glucose and lipid metabolism were impacted by both cell types when compared to culture in SOF without fee-der cells. Collectively, co-culture systems, using BOEC or VERO cells, do not improve clea-vage and blastocyst rates and induce weak and closely related modifications of blastocyst transcriptome when compared to 5% O2 culture condition in SOF medium

Effects of BOEC and VERO co-culture systems on bovine early development

International audienceEmbryo development is known to be impacted by its environment and especially by oviductal secretions in vivo. In cattle, embryo co-culture with bovine oviduct epithelial cells (BOEC) has been developed to mimic the in vivo oviduct/embryo crosstalk. However, whether BOEC had a specific impact on embryo transcriptome hasn’t been investigated yet. We thus compa-red bovine embryos co-cultured with BOEC to embryos co-cultured with VERO cells (a kid-ney epithelial cell line from monkey). Control embryos were obtained in SOF medium + 5% Fetal Calf Serum (FCS) at 5% O2. Because co-culture systems require 20% O2, embryos cul-tured at 20% O2 in SOF + 5% FCS were included as an additional control. No impact of co-culture systems was observed on timing and developmental rates. 16-cell stage embryos and day 8 blastocysts' transcriptomes were analyzed on a new customized bovine microarray (GPL21734). Comparing the two co-culture conditions revealed only 14 and 10 differentially expressed transcripts respectively at 16-cell and blastocyst stages suggesting almost no diffe-rence induced by the type of co-culture. Nevertheless, BOEC or VERO cells induced differential expression of 83 and 51 transcripts respectively when compared to 5% O2 and 218 and 309 transcripts respectively when compared to 20% O2 in 16-cell embryos and 192 and 229 transcripts respectively when compared to 5% O2 and 542 and 881 transcripts respectively when compared to 20% O2 in blastocysts. A large proportion of the transcripts affected by BOEC presence were also impacted by VERO cells. Several biofunctions relative to cell cycle regulation and lipid metabolism were impacted by both cell types when compared to culture in SOF without feeder cells. Collectively, co-culture systems, using BOEC or VERO cells, induce weak and closely related modifications of embryos transcriptome without improvement of cleavage and blastocyst rates when compared to standard 5% O2 culture conditions

Investigation of DNA methylation role periconceptional programming in early stage embryos from rabbit diabetic pregnancy model

International audienceDNA methylation is a conservative epigenetic marker. A correct DNA methylation pattern is essential for embryonic development. Mammalian embryos gain a complete de novo DNA methylation design around implantation. DNA methylation is a potential mechanism of periconceptional programming, suggesting that the DNA methylation pattern of developing embryo might be affected in distinct ways, depending on nutritional and hormonal signals. To expand this idea we have investigated the DNA methylation pattern of the promoter region of the POU5F1 (Oct4) pluripotency gene in preimplantation rabbit embryos at day 6 p.c (early gastrulation stage). The blastocysts were collected from healthy and diabetic rabbits. The POU5F1 promoter contains four conservative regions, which are embracing important elements for its transcription: proximal and distal enhancers (PE-1A, PE-1B, DE-1A), a SOX2/Oct4 binding site and hormone responding element (HRE). We have characterized the CpG islands methylation at all mentioned regions in two embryonic tissues: embryoblast and trophoblast, using bisulfite treatment, cloning and sequencing. The analysis has been successfully performed for the trophoblast tissue yet. A higher methylation of the CpG islands for HRE and at the beginning of first exon has been noticed in the trophoblast of diabetic embryos. A hypomethylation in trophoblast from diabetic embryos comparing to trophoblasts from healthy pregnancies was visible for proximal enhancer 1A (PE-1A) in frame of conservative region 3 (CR3). In this context, DNA methylation can be considered as a form of embryo developmental plasticity, which can promote metabolic disorders in adult life

Different co-culture systems have the same impact on bovine embryo transcriptome

International audienceDuring 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