Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos.

BACKGROUND: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. RESULTS: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. CONCLUSIONS: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Endometrial luminal epithelial cells sense embryo elongation in the roe deer independent of interferon-tau†.

Numerous intrauterine changes take place across species during embryo development. Following fertilization in July/August, the European roe deer (Capreolus capreolus) embryo undergoes diapause until embryonic elongation in December/January. Embryonic elongation prior to implantation is a common feature among ungulates. Unlike many other ruminants, the roe deer embryo does not secrete interferon-tau (IFNτ). This provides the unique opportunity to unravel IFNτ-independent signaling pathways associated with maternal recognition of pregnancy (MRP). This study aimed at identifying the cell-type-specific endometrial gene expression changes associated with the MRP at the time of embryo elongation that are independent of IFNτ in roe deer. The messenger RNA (mRNA) expression of genes known to be involved in embryo-maternal communication in cattle, pig, sheep, and mice was analyzed in laser capture microdissected (LMD) endometrial luminal, glandular epithelial, as well as stromal cells. The mRNA transcript abundances of the estrogen (ESR1), progesterone receptor (PGR), and IFNτ-stimulated genes were lower in the luminal epithelium in the presence of an elongated embryo compared to diapause. Retinol Binding Protein-4 (RBP4), a key factor involved in placentation, was more abundant in the luminal epithelium in the presence of an elongated embryo. The progesterone receptor localization was visualized by immunohistochemistry, showing an absence in the luminal epithelium and an overall lower abundance with time and thus prolonged progesterone exposure. Our data show a developmental stage-specific mRNA expression pattern in the luminal epithelium, indicating that these cells sense the presence of an elongated embryo in an IFNτ-independent manner.

Blastocysts exhibit sex-specific signalling of IFNT transcription, translation and activity.

Preimplantation bovine blastocyst supernatants exhibit sex-dependent antiviral activity, due to the ruminant pregnancy recognition signal interferon tau (IFNT). Differing potencies of IFNT variants have been supposed as cause, although evidence remains scarce. Here, we aimed at quantifying the sex-dependent IFNT production on transcriptional, translational and biological activity level in bovine blastocysts, to elucidate the origin of differences in antiviral activity between male and female blastocysts. Day 8 bovine blastocysts were co-cultured with endometrial stroma cells for 48 h. The embryonic IFNT mRNA expression was determined by quantitative reverse transcription followed by polymerase chain reaction (RT-qPCR). Additionally, the IFNT protein concentration was determined using a sensitive in-house developed IFNT-specific enzyme-linked immunosorbent assay (ELISA). The biological activity was assessed by quantifying the response of interferon-stimulated gene (ISG) expression in endometrial stroma cells. While the IFNT-specific ELISA displayed a limit of detection of 7.3 pg/mL, the stroma cell culture system showed to react to as little as 0.1 pg/mL IFNT in RT-qPCR analysis. The female blastocysts had a significant, 5.6-fold, 3.6-fold and 5.2-fold higher IFNT production than male blastocysts as determined by transcript abundance, protein concentration and, protein activity, respectively. Additionally, all parameters correlated positively, and therefore, we conclude that female blastocysts most likely have an increased IFNT gene and protein expression rather than expressing more potent IFNT variants.

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

Embryos burn fat in standby.

Embryonic and adult stem cells enable development and regeneration. Embryonic cells, like adult stem cells, can enter dormancy as part of their lifecycle. Recent evidence suggests that this cellular transition to dormancy requires active rewiring of metabolism. The dormancy-induced metabolic switches in embryonic and adult stem cells are explored here.

Developmental progression continues during embryonic diapause in the roe deer.

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.

FOXO1-mediated lipid metabolism maintains mammalian embryos in dormancy.

Mammalian developmental timing is adjustable in vivo by preserving pre-implantation embryos in a dormant state called diapause. Inhibition of the growth regulator mTOR (mTORi) pauses mouse development in vitro, yet how embryonic dormancy is maintained is not known. Here we show that mouse embryos in diapause are sustained by using lipids as primary energy source. In vitro, supplementation of embryos with the metabolite L-carnitine balances lipid consumption, puts the embryos in deeper dormancy and boosts embryo longevity. We identify FOXO1 as an essential regulator of the energy balance in dormant embryos and propose, through meta-analyses of dormant cell signatures, that it may be a common regulator of dormancy across adult tissues. Our results lift a constraint on in vitro embryo survival and suggest that lipid metabolism may be a critical metabolic transition relevant for longevity and stem cell function across tissues.

TET activity safeguards pluripotency throughout embryonic dormancy.

Dormancy is an essential biological process for the propagation of many life forms through generations and stressful conditions. Early embryos of many mammals are preservable for weeks to months within the uterus in a dormant state called diapause, which can be induced in vitro through mTOR inhibition. Cellular strategies that safeguard original cell identity within the silent genomic landscape of dormancy are not known. Here we show that the protection of cis-regulatory elements from silencing is key to maintaining pluripotency in the dormant state. We reveal a TET-transcription factor axis, in which TET-mediated DNA demethylation and recruitment of methylation-sensitive transcription factor TFE3 drive transcriptionally inert chromatin adaptations during dormancy transition. Perturbation of TET activity compromises pluripotency and survival of mouse embryos under dormancy, whereas its enhancement improves survival rates. Our results reveal an essential mechanism for propagating the cellular identity of dormant cells, with implications for regeneration and disease.

Molecular Regulation of Paused Pluripotency in Early Mammalian Embryos and Stem Cells.

The energetically costly mammalian investment in gestation and lactation requires plentiful nutritional sources and thus links the environmental conditions to reproductive success. Flexibility in adjusting developmental timing enhances chances of survival in adverse conditions. Over 130 mammalian species can reversibly pause early embryonic development by switching to a near dormant state that can be sustained for months, a phenomenon called embryonic diapause. Lineage-specific cells are retained during diapause, and they proliferate and differentiate upon activation. Studying diapause thus reveals principles of pluripotency and dormancy and is not only relevant for development, but also for regeneration and cancer. In this review, we focus on the molecular regulation of diapause in early mammalian embryos and relate it to maintenance of potency in stem cells in vitro. Diapause is established and maintained by active rewiring of the embryonic metabolome, epigenome, and gene expression in communication with maternal tissues. Herein, we particularly discuss factors required at distinct stages of diapause to induce, maintain, and terminate dormancy.

Embryonic diapause in mammals and dormancy in embryonic stem cells with the European roe deer as experimental model.

In species displaying embryonic diapause, the developmental pace of the embryo is either temporarily and reversibly halted or largely reduced. Only limited knowledge on its regulation and the inhibition of cell proliferation extending pluripotency is available. In contrast with embryos from other diapausing species that reversibly halt during diapause, embryos of the roe deer Capreolus capreolus slowly proliferate over a period of 4-5 months to reach a diameter of approximately 4mm before elongation. The diapausing roe deer embryos present an interesting model species for research on preimplantation developmental progression. Based on our and other research, we summarise the available knowledge and indicate that the use of embryonic stem cells (ESCs) would help to increase our understanding of embryonic diapause. We report on known molecular mechanisms regulating embryonic diapause, as well as cellular dormancy of pluripotent cells. Further, we address the promising application of ESCs to study embryonic diapause, and highlight the current knowledge on the cellular microenvironment regulating embryonic diapause and cellular dormancy.

Gestational oral low-dose estradiol-17ß induces altered DNA methylation of CDKN2D and PSAT1 in embryos and adult offspring.

Endocrine disrupting chemicals (EDC) interfere with the natural hormone balance and may induce epigenetic changes through exposure during sensitive periods of development. In this study, the effects of short-term estradiol-17ß (E2) exposure on various tissues of pregnant sows (F0) and on day 10 blastocysts (F1) were assessed. Intergenerational effects were investigated in the liver of 1-year old female offspring (F1). During gestation, sows were orally exposed to two low doses and a high dose of E2 (0.05, 10, and 1000 µg/kg body weight/day). In F0, perturbed tissue specific mRNA expression of cell cycle regulation and tumour suppressor genes was found at low and high dose exposure, being most pronounced in the endometrium and corpus luteum. The liver showed the most significant DNA hypomethylation in three target genes; CDKN2D, PSAT1, and RASSF1. For CDKN2D and PSAT1, differential methylation in blastocysts was similar as observed in the F0 liver. Whereas blastocysts showed hypomethylation, the liver of 1-year old offspring showed subtle, but significant hypermethylation. We show that the level of effect of estrogenic EDC, with the periconceptual period as a sensitive time window, is at much lower concentration than currently presumed and propose epigenetics as a sensitive novel risk assessment parameter.