Nathalie Peynot

Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development

X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.

Expression of Pluripotency Master Regulators during Two Key Developmental Transitions: EGA and Early Lineage Specification in the Bovine Embryo

Pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. In bovine, however, little information is available about dynamics of pluripotency genes during these processes. In this study, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, having similar in vitro but different full term development rates. Our findings affirm: firstly, the core triad of pluripotency genes is probably not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Secondly, an earlier ICM specification of transcripts and proteins of SOX2 and NANOG makes them pertinent candidates of bovine pluripotent lineage specification than OCT4. Thirdly, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development.

Hyperlipidic hypercholesterolemic diet in prepubertal rabbits affects gene expression in the embryo, restricts fetal growth and increases offspring susceptibility to obesity

Maternal hypercholesterolemia has been shown to lead to fetal intra-uterine growth retardation (IUGR) in rabbits. The effects of a long term maternal hyperlipidemic and hypercholesterolemic diet on embryo, fetal and post-natal development, have not been addressed so far. Rabbit does were fed either a hypercholesterolemic (0.2%) hyperlipidic (8%) (HH) or a control (C) diet from 10 weeks of age. Sixteen does (N = 8 HH and N = 8 C) were euthanized at 18 weeks to assess the effect of the diet on dams before mating. Embryos from 18 females (N = 9 HH and N = 9 C) were collected from the oviducts at the 16-20 cell stage (embryonic genome activation stage) for gene expression analysis (micro array and quantitative RT-PCR). Thirty females (N = 16 HH and N = 14 C) were mated naturally and fetal growth was monitored by ultrasound. Six of them (N = 4 HH and N = 2 C) were euthanized at D28 of gestation to collect fetuses and placentas. Finally, the remaining 24 does delivered at term and litters were cross fostered and equilibrated in number to create 4 groups according to the biological dam and the foster dam (C-C, C-HH, HH-C, HH-HH). Growth was monitored until weaning. A subset of 26 offspring from the 4 groups was fed the control diet until 25 weeks of age and then fed the HH diet for three weeks. All does had similar growth rates and bodyweight. Transcriptomic analyses evidenced an overexpression of Adipophilin in HH embryos at the stage of embryonic genome activation. This was confirmed by quantitative RT-PCR. During pregnancy, IUGR was observed from D9 by ultrasound and subsequently, fetal weight at 28 days, birthweight and fat deposition in newborn offspring were significantly decreased in HH (P < 0.05). After weaning, there was no significant difference for weight between HH-HH and HH-C offspring and both groups became significantly heavier (P < 0.0001) than C-C and C-HH offspring. During the 3 weeks when offspring were fed the HH diet, the differences in feed intake were no longer significant between groups but the differences in body weight remained. At post-mortem, offspring from HH does had significantly more abdominal and inter-scapular fat than offspring from C does (P < 0.05). These data illustrate the importance of maternal nutrition before and during gestation in the establishment and control of the growth trajectory of the conceptus and in the onset of disease in adult life.

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P ≤ 0.01). On the contrary to what was found for Cx37, Cx43 was weakly expressed in preantral follicles. Concomitant with antrum formation, the level of Cx43 expression increased significantly (P ≤ 0.01). A further increase was correlated with antral follicular size (P ≤ 0.01). Cx43 immunoreactivity declined significantly in morphologically atretic follicles (P ≤ 0.01). A comparative analysis showed that Cx37 and Cx43 expression patterns were differentially regulated and could reflect specific physiological roles for each gap junction protein throughout folliculogenesis in cow. Mol. Reprod. Dev. 57:60–66, 2000.

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro.

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).

Sexual Dimorphism of the Feto-Placental Phenotype in Response to a High Fat and Control Maternal Diets in a Rabbit Model

Maternal environment during early developmental stages plays a seminal role in the establishment of adult phenotype. Using a rabbit model, we previously showed that feeding dams with a diet supplemented with 8% fat and 0.2% cholesterol (HH diet) from the prepubertal period and throughout gestation induced metabolic syndrome in adult offspring. Here, we examined the effects of the HH diet on feto-placental phenotype at 28 days post-coïtum (term = 31days) in relation to earlier effects in the blastocyst (Day 6). At 28 days, both male and female HH fetuses were intrauterine growth retarded and dyslipidemic, with males more affected than females. Lipid droplets accumulated in the HH placentas’ trophoblast, consistent with the increased concentrations in cholesteryl esters (3.2-fold), triacylglycerol (2.5-fold) and stored FA (2.12-fold). Stored FA concentrations were significantly higher in female compared to male HH placentas (2.18-fold, p<0.01), whereas triacylglycerol was increased only in HH males. Trophoblastic lipid droplet accumulation was also observed at the blastocyst stage. The expression of numerous genes involved in lipid pathways differed significantly according to diet both in term placenta and at the blastocyst stage. Among them, the expression of LXR-α in HH placentas was reduced in HH males but not females. These data demonstrate that maternal HH diet affects the blastocyst and induces sex-dependent metabolic adaptations in the placenta, which appears to protect female fetuses from developing severe dyslipidemia.

Alteration of DNA demethylation dynamics by in vitro culture conditions in rabbit pre-implantation embryos

Alterations to DNA methylation have been attributed to in vitro culture and may affect normal embryo development. We chose to analyze DNA methylation reprogramming in the rabbit which, of the species with delayed transcriptional activation of the embryonic genome, allows easy comparisons between in vivo-developed (IVD) and in vitro-cultured (IVC) embryos. In this species, variations in DNA methylation had not previously been quantified, even in IVD embryos. IVD and IVC embryos were recovered at the 2, 4, 8 and 16-cell, morula and blastocyst stages. Immunostaining for 5-methyl-cytidine and normalization of the quantity of methylated DNA vs. the total DNA content were then performed. Our quantitative results evidenced DNA demethylation during pre-implantation development in both IVD and IVC embryos, but with different kinetics. Demethylation occurred earlier in vitro than in vivo between the 2 and 8-cell stages in IVC embryos, reaching its lowest level, while it only started at the 4-cell stage and ended at the 16-cell stage in IVD embryos. We also showed that an absence of serum from the culture medium significantly altered the degree of DNA demethylation. Finally, at the blastocyst stage, ICM was more methylated than the trophectoderm in all cases. Despite a morphological delay observed in in vitro cultured blastocysts, the difference in DNA methylation between ICM and trophectoderm cells appeared at the same time post-fertilization in IVD and IVC embryos, which may reflect another difference in the dynamics of DNA methylation during blastocyst formation. Our data thus clearly establish an effect of embryonic environment on DNA methylation reprogramming during pre-implantation development in a non-rodent species.

Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization.

The reprogramming of DNA methylation in early embryos has been considered to be essential for the reprogramming of differentiated parental genomes to totipotency, the transcription of embryonic genome activation (EGA) and subsequent development. However, its degree appears to differ as a function of species and it may be altered by the in vitro environment. While the rabbit is a pertinent model for species with a delayed EGA because both in vivo and in vitro developed embryos are easily available, the status of DNA methylation levels in both parental genomes after fertilization remains controversial. In order to generate precise data on the DNA methylation status in rabbit zygotes, we first of all defined five pronuclear (PN) stages during the first cell cycle and then classified in vivo and in vitro developed rabbit zygotes according to these PN stages. Using this classification we precisely quantified both methylated DNA and the total DNA content during the one cell stage. The quantification of methylated DNA, normalized for the total DNA content, showed that both pronuclei displayed distinct patterns of DNA methylation reprogramming. In the maternal pronucleus (MP) the methylation level remained constant throughout the one cell stage, thanks to maintenance methylation during the S phase. Conversely, in the paternal pronucleus (PP) partial demethylation occurred before replication, probably as a result of active DNA demethylation, while maintenance methylation subsequently occurred during the S phase. Interestingly, we showed that PP DNA methylation reprogramming was partially altered by the in vitro environment. Taken together, our approach evidenced that rabbit is one of the species displaying partial DNA demethylation in the PP, and for the first time demonstrated maintenance methylation activity in both pronuclei during the first S phase.

Revealing the dynamics of gene expression during embryonic genome activation and first differentiation in the rabbit embryo with a dedicated array screening

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.

Expression and localization of interleukin 1 beta and interleukin 1 receptor (type I) in the bovine endometrium and embryo

The interleukin-1 (IL1) system likely mediates mammalian embryo-maternal communication. In cattle, we have reported that the uterine fluid of heifers carrying early embryos shows downregulated IL1 beta (IL1B), which could lead to reduced NFkB expression and dampening of maternal innate immune responses. In this work, we assessed the expression of IL 1 beta (IL1B) and its receptor, interleukin 1 receptor type I (IL1R1) in the bovine endometrium and embryos by RT-PCR, immunohistochemistry and Western blot at the time of blastocyst development. Day 8 endometrium, both collected from animals after transfer of day 5 embryos (ET) and sham transferred (ST), showed IL1B and IL1R1 mRNA transcription and protein co-localization. Similarly, day 8 blastocyst, from ET animals and entirely produced in vitro, showed IL1R1 mRNA transcription and IL1B and IL1R1 protein co-localization. IL1B mRNA was detected in the analyzed blastocysts, but at very low levels that precluded its quantification. IL1B and IL1R1 immunostaining was observed in luminal epithelial cells, glandular epithelium and stromal cells. The presence of embryos increased endometrial IL1B protein locally, while no differences regarding IL1R1 protein and IL1B and IL1R1 mRNA were detected. These results suggest that the early preimplantation bovine embryo in the maternal tract might interact with the maternal immune system through the IL1 system. Such a mechanism may allow the embryo to elicit local endometrial responses at early stages, which are required for the development of a receptive endometrium.