Early Embryo Metabolism Research Articles

P–154 The role of the X Chromosome in early human embryo metabolism

Abstract Study question What role does the X chromosome play in early embryo metabolism? Does X chromosome copy number contribute to sex differences in early embryonic metabolism? Summary answer Chromosome X contains several metabolism-related genes that are expressed prior to X-inactivation, suggesting that their dosage plays a role in sex-biased regulation of embryo metabolism. What is known already Published reports indicate that sex differences in preimplantation embryo metabolism exist across mammalian species, including humans. Two observations supporting this are that male embryos reach blastocyst stage earlier than their female counterparts, and that glucose uptake and processing is thought to be higher in female compared to male embryos. It has been hypothesized that these differences reflect the location of the metabolism gene G6PD, the rate limiting enzyme in the Pentose Phosphate Pathway, on Chromosome X. Study design, size, duration This study is a reanalysis of publicly available RNA-seq data, including 1176 single cells from 59 blastocysts (24 E5, 18 E6, 17 E7) published in one study (Petropoulos et al 2016). Participants/materials, setting, methods Cells were subjected to a digital karyotype inference algorithm and aneuploid samples were removed from the dataset. Sex differential gene expression analyses (DE) were then performed in euploid trophectoderm cells (TE; 233 XY from 16 embryos and 180 XX cells from 12 embryos). Cell numbers from ICM were too sparse to compare. Main results and the role of chance Analysis of XX and XY TE revealed 618 significantly differentially expressed genes (DEGs; 507 upregulated in XX cells, and 111 upregulated in XY cells). These genes are spread across autosomes and sex chromosomes. Interestingly, G6PD is not significantly more highly expressed in XX cells. Gene Ontology (GO) analysis of the XX-biased DEGs revealed a transcriptional sex bias in metabolism-related GO categories, including “mitochondrial ATP synthesis coupled electron transport”, and “respiratory chain complex I”. Gene-level assessment revealed that the drivers of these enrichments are spread across the genome, but 28/64 reside on Chromosome X (hypergeometric p-value = 5.984473e–27), including NDUFA1, NDUFB11, and COX7B (components of the electron transport chain), and SLC25A5 (an ATP/ADP transporter involved in maintaining mitochondrial membrane potential). This indicates a direct role for multiple X-linked genes in sex-biased regulation of embryo metabolism. Metabolic genes that are not sex-biased are distributed across the genome, with no significant enrichment on Chromosome X (76/266, hypergeometric p-value=0.607). Together, these data indicate that GO metabolic term X enrichment is a feature of sex-biased expression and not due to an accumulation of metabolism-related genes on the X. Limitations, reasons for caution This analysis draws on publicly available data, and thus we are unable to perform orthogonal validation of karyotype calls. Additionally, while the initial dataset is large, the quality-filtered dataset (euploid XX and XY TE) is small, and single cell data is infamously variable. Further data collection is required. Wider implications of the findings: Our analysis of sex-biased gene expression in early human embryos suggests a more important role for the X chromosome in modulating sex biases in early embryo metabolism than previously recognized. This study provides insight into the mechanisms underlying the development of metabolic sex differences throughout the lifespan. Trial registration number NA

Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

Exposure to elevated glucose concentrations alters the metabolomic profile of bovine blastocysts.

Chronically high blood glucose concentrations are a characteristic of diabetes mellitus. Maternal diabetes affects the metabolism of early embryos and can cause a delay in development. To mimic maternal diabetes, bovine in vitro fertilization and embryo culture were performed in fertilization medium and culture medium containing 0.5, 2, 3, and 5 mM, glucose whereas under control conditions, the medium was glucose free (0 mM). Compared to control conditions (0 mM, 31%), blastocyst development was decreased to 23% with 0.5 and 2 mM glucose. Presence of 3 or 5 mM glucose in the medium resulted in decreased blastocyst rates (20% and 10% respectively). The metabolomic profile of resulting day 8 blastocysts was analysed by UPLC-MS/MS, and compared to that of blastocysts cultured in control conditions. Elevated glucose concentrations stimulated an increase in glycolysis and activity of the hexosamine pathway, which is involved in protein glycosylation. However, components of the tricarboxylic acid cycle, such as citrate and alpha-ketoglutarate, were reduced in glucose stimulated blastocysts, suggesting that energy production from pyruvate was inefficient. On the other hand, activity of the polyol pathway, an alternative route to energy generation, was increased. In short, cattle embryos exposed to elevated glucose concentrations during early development showed changes in their metabolomic profile consistent with the expectations of exposure to diabetic conditions.

The effect of progesterone on early embryo metabolism

The effect of progesterone on early embryo metabolism

History of oocyte and embryo metabolism.

The basic pattern of metabolism in mammalian oocytes and early embryos was established in the 1960s and 1970s, largely in terms of the consumption of oxygen and the utilisation of nutrients present in culture media at the time, mainly glucose, pyruvate and lactate. The potential importance of endogenous fuels was also recognised but was largely ignored, only to be rediscovered quite recently. The 1980s and 1990s saw the arrival of a 'new generation' of culture media, characterised metabolically by the addition of amino acids, an initiative driven strongly by the need to improve embryo culture and selection methods in assisted reproductive technologies. This trend has continued alongside some basic metabolic studies and the general recognition of the importance of metabolism in all aspects of biology. A framework for future studies on oocyte and early embryo metabolism has been provided by: (1) the developmental origins of health and disease concept and recognition of the relationship between development, epigenetics and metabolism; (2) the need to understand cell signalling within, and between the cells of, the early embryo; and (3) the importance of identifying the mechanisms underlying dialogue between the oocyte and early embryo and the female reproductive tract.

Parallels between embryo and cancer cell metabolism

A key characteristic of cancer cells is the ability to switch from a predominantly oxidative metabolism to glycolysis and the production of lactate even when oxygen is plentiful. This metabolic switch, known as the Warburg effect, was first described in the 1920s, and has fascinated and puzzled researchers ever since. However, a dramatic increase in glycolysis in the presence of oxygen is one of the hallmarks of the development of the early mammalian embryo; a metabolic switch with many parallels to the Warburg effect of cancers. The present review provides a brief overview of this and other similarities between the metabolism in tumours and early embryos and proposes whether knowledge of early embryo metabolism can help us to understand metabolic regulation in cancer cells.

138 FATTY ACID β-OXIDATION REGULATES BOVINE EMBRYO OXYGEN METABOLISM

Mammalian oocytes are rich in endogenous lipid, which provides a potential source of metabolic energy during oocyte maturation and early embryo development after fertilisation. Despite this, studies on early embryo metabolism have focussed on consumption of substrates from the culture medium, with comparatively little consideration for endogenous stores. Additionally, the interaction between fatty acid β-oxidation and oxygen consumption during pre-implantation development has not been investigated. This study has investigated the relationship between embryo fatty acid metabolism and oxygen consumption rate (OCR), a marker of overall oxidative metabolic activity. The amount of oxygen consumption by bovine blastocysts coupled to ATP synthesis was assessed by measuring embryo OCR following acute treatment with oligomycin, which inhibits ATP synthase. Bovine embryos were then produced in vitro and cultured from zygote to blastocyst stage (8 days) in the presence of β-mercaptoacetate (BMA), a competitive inhibitor of fatty acid β-oxidation, or L-carnitine, a cofactor that promotes β-oxidation. OCR of individual embryos was measured using a noninvasive, highly sensitive oxygen probe (Unisense), whereas OCR of embryos in groups was determined using the Oxygen Biosensor (OBS) fluorimetric assay (BD Biosciences, Erembodegem, Belgium). Mean OCR measured by each technique correlated well. Data are presented as means ± SEM. The effect of acute inhibition were compared by a paired t-test, with pre-treated embryos acting as their own control, whereas the effect of L-carnitine and BMA on OCR was compared across treatment groups using ANOVA with Dunn’s test for unequal groups post hoc, using Sigmaplot (Systat Software Inc., San Jose, CA, USA). Treatment with oligomycin caused blastocyst OCR to fall from 23.5 ± 3.3 to 7.9 ± 2.5 pmol/embryo/h (n = 3; P = 0.03). Inhibition of β-oxidation by BMA led to a modest, but consistent increased OCR in cleavage stage embryos: 4-cell: 14.8 ± 7.2 (n = 12) v. 8.9 ± 1.1 pmol/embryo/h (n = 3) control; 8-cell: 15.8 ± 2.2 (n = 6) v. 11.9 ± 2.3 pmol/embryo/h (n = 3) control. At the morula stage, BMA led to a significant rise in OCR (26.8 ± 3.6 (n = 7) v. 7.8 ± 1.7 pmol/embryo/h (n = 3) control; P = 0.003) and at the blastocyst stage, OCR was significantly elevated in the presence of BMA; 41.3 ± 2.6 (n = 19) v. 16.9 ± 1.3 pmol/embryo/h (n = 14) control (P = 0.01). By contrast, when groups of embryos were cultured in the presence of L-carnitine, blastocyst OCR was reduced by 45% from 24.7 ± 5.1 (group control) to 15.9 ± 3.7 pmol/embryo/h (n = 30; P < 0.06). We report that 66.5% of oxygen consumption by bovine blastocysts is oligomycin sensitive, meaning that 33.5% of oxygen consumption is used for processes other than ATP synthesis. Surprisingly, inhibition of fatty acid β-oxidation caused total bovine embryo OCR to rise, although the reasons for this are unclear. It is possible that inhibiting β-oxidation leads to an accumulation of free fatty acids in the mitochondrion, which can disrupt the inner membrane and lead decoupling of ATP synthesis from oxygen consumption. Thus, manipulating fatty acid oxidation in the early embryo dysregulates oxidative metabolism; the implications of this are under further investigation.

Growth hormone inhibits apoptosis in in vitro produced bovine embryos.

Growth hormone (GH) has recently been shown to exert distinct effects on the differentiation and metabolism of early embryos. Up to now, however, it is not clear whether GH is able to modulate apoptosis during early embryogenesis. Differential cell staining of 8-day-old bovine embryos cultured with 100 ng bovine recombinant GH (rbGH) per ml medium (synthetic oviduct fluid-polyvinylalcohol) demonstrated that GH significantly increased the number of inner cell mass (ICM) and trophectoderm cells in bovine expanded blastocysts. As shown by terminal deoxynucleotidyl transferase mediated dUTP labeling (TUNEL) supplementation of bGH decreased the percentage of 8-day-old embryos showing at least one apoptotic cell from 58 to 21%. The percentage of apoptotic cells in one blastocyst was significantly (P < 0.01) reduced from 4.6 to 1.1% by GH treatment. Incubation of the embryos with 150 mM vanillylnonanamide induced apoptosis in all embryos. Whereas in control embryos 14% of the embryonic cells were TUNEL-positive, the percentage of apoptotic cells declined to 2.7% in the GH treated embryos. Expression of immunoreactive bcl-2 in blastocysts was not affected by GH treatment. Synthesis of the bax protein which is known to promote apoptosis was reduced in embryos cultured with GH. Our results suggest that GH acts as survival factor during in vitro culture and reduces apoptosis by altering the bax to bcl-2 ratio during early embryogenesis.

Identification of Drosophila melanogaster RECQE as a member of a new family of RecQ homologues that is preferentially expressed in early embryos

We describe the isolation of a new type of RecQ homologue in Drosophila melanogaster, RECQE. The Recqe gene consists of five exons and four introns, and encodes a protein of 1058 amino acids with a predicted molecular weight of 120,000 Da. The RECQE protein has seven helicase motifs. The helicase domain shows 42% identity overall to that of Escherichia coli RecQ DNA helicase, and is most closely related to Homo sapiens RecQL5 and Caenorhabditis elegans E03A3.2. The C-terminal region of RECQE protein is unique and the longest known among members of the RecQ superfamily. We demonstrate that the RECQE protein has DNA helicase activity and that the C-terminal region is dispensable for this activity. The RECQE mRNA accumulates in oocytes and is expressed at high levels in early embryos. We show for the first time that the expression of a RecQ homologue is developmentally regulated in embryos. These data suggest that the DNA helicase activity of RECQE might be involved in the DNA metabolism of early embryos.

Embryo-derived platelet activating factor: interactions with the arachidonic acid cascade and the establishment and maintenance of pregnancy

Two classes of potent lipid mediators are produced by the mouse and human pre-embryo: platelet activating factor (PAF) and prostaglandins (PGs). This paper reviews the evidence for their production by the pre-embryo and for their role in embryo development and the successful establishment of pregnancy. The biosynthesis of PAF and arachidonic acid may be linked, the synthesis of PAF resulting in the generation of arachidonic acid with its subsequent conversion to prostaglandins. Pharmacological inhibitor studies show that a major site of action of PAF is the embryo itself, acting as an embryonic autocrine growth factor, whereas PGs appear to act primarily on maternal tissues although they do modulate some aspects of early embryo metabolism. It is the maternal tissues that are the primary source of PG production in early pregnancy. Maternal PGs have a variety of functions including involvement in the proinflammatory response of early pregnancy and the control of corpus luteum function. In the ewe, pregnancy is associated with an attenuation of oxytocin-induced production of the luteolysin, prostaglandin F2 alpha (PGF2 alpha). PAF can mimic the effect of pregnancy, preventing the release of PGF2 alpha in response to exogenous oxytocin and, when administered into the uterine lumen, extending the life span of the corpus luteum. Thus, embryo-derived PAF appears to have an essential role in the establishment of pregnancy by acting as an autocrine growth factor for the embryo and by exerting a variety of effects on maternal physiology, including modulating maternal prostaglandin secretion and action.