44 research outputs found
Effect of oxygen tension on the amino acid utilisation of human embryonic stem cells
Background/aims: human embryonic stem cells (hESCs) are a potential source of cells for treatment of many degenerative diseases, but in culture have a propensity to spontaneously differentiate, possibly due to suboptimal conditions. Culture at low oxygen tensions improves hESC maintenance and regulates carbohydrate metabolism. Hence, a greater understanding of the nutrient requirements of hESCs will allow production of more appropriate culture media. This study aims to investigate the effect of environmental oxygen tension on the amino acid metabolism of hESCs. Methods: the production or depletion of amino acids by hESCs cultured at 5% or 20% oxygen in the presence or absence of FGF2 was measured by reversephase HPLC. Results: atmospheric oxygen, or removal of FGF2 from hESCs cultured at 5% oxygen, perturbed the uptake or release of individual amino acids and the total amino acid turnover compared to hESCs cultured at 5% oxygen. In particular, serine uptake was reduced at 20% oxygen and by removal of FGF2. Conclusions: highly pluripotent hESCs, cultured at 5% oxygen, demonstrate a greater amino acid turnover than hESCs cultured at 20% oxygen, or without FGF2. These data suggest that amino acid turnover could be used as a measure of the self-renewal capacity of hESC
Hypoxic regulation of preimplantation embryos: lessons from human embryonic stem cells
Development of the preimplantation embryo is reliant on nutrients present in the milieu of the reproductive tract. While carbohydrates, amino acids, lipids, and micronutrients are often considered when discussing preimplantation embryo nutrition, environmental oxygen is frequently overlooked. Although oxygen is not classically considered a nutrient, it is an important component of the in vitro culture environment and a critical regulator of cellular physiology. Oxygen is required to sustain an oxidative metabolism but when oxygen becomes limited, cells mount a physiological response driven by a family of transcription factors termed 'hypoxia inducible factors' which promote expression of a multitude of oxygen sensitive genes. It is this hypoxic response that is responsible not only for the switch to a glycolytic metabolism but also for a plethora of other cellular responses. There has been much debate in recent years over which environmental oxygen tension is preferential for the culture of preimplantation embryos. The review will evaluate this question and highlights how research using human embryonic stem cells can inform our understanding of why culturing under physiological oxygen tensions may be beneficial for the development of embryos generated through clinical in vitro fertilisation
Identification of viable embryos by noninvasive measurement of amino acids in culture media
This chapter highlights many important roles of amino acids for human preimplantation embryos. Amino acids are not only beneficial to embryo development but their utilisation by the embryo is also predictive of future viability, genetic health, DNA damage and trophectoderm integrity. These findings were remarkable and highlight how integral amino acids are to the physiology of the embryo. Thus, it is important that much consideration is given to the media used in clinical IVF. This will require suppliers to provide details of media formulations so that informed choices can be made. The use of amino acid profiling in a clinical setting offers the exciting prospect to nonsubjectively select the most developmentally competent embryo for transfer with the greatest chance of producing a live birth
Preimplantation embryo metabolism
Preimplantation embryos are remarkably adaptable, being able to develop in a variety of culture media containing different energy sources. Routinely, embryos are cultured in a simple balanced salt solution supplemented with pyruvate, glucose, lactate, amino acids and a protein source; any of which may be metabolised as a potential energy source for the embryo. Energy metabolism results in the generation of ATP and is integral for successful embryo development. The best global indication of the ability of an embryo to produce energy is oxygen consumption, which together with the amount of lactate produced can be used to calculate ATP production. Prior to the blastocyst stage ATP formation remains low but cavitation is associated with a large increase in ATP production. Using immunosurgery to isolate inner cell mass (ICM) cells, it can be calculated that the trophectoderm produces approximately 80% of the ATP generated at the blastocyst stage with the ICM contributing 20%. It is likely that the majority of this energy is used to fuel Na+ pumping via the Na+, K+, ATPase. Energy metabolism is intrinsic to embryo health and may be used to predict developmental capacity. In vitro fertilisation has permitted many thousands of couples to conceive but is limited by a low pregnancy rate and high multiple birth rate due to the transfer of more than one embryo. At present, embryos are selected for transfer based on their morphology, but this is subjective and a poor predictor of viability. Amino acid profiling offers the potential to predict non-invasively the developmental capacity of single human embryos as early as day 2 post-insemination, obviating the need for prolonged culture to the blastocyst stage
Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst
Mammalian pre-implantation development culminates in the formation of the blastocyst consisting of two distinct cell lineages, approximately a third of the cells comprise the pluripotent inner cell mass (ICM) and the remainder the differentiated trophectoderm (TE). However, the contribution made by these two cell types to the overall energy metabolism of the intact blastocyst has received relatively little attention. In this study, the metabolism of the intact mouse blastocyst and isolated ICMs were determined in terms of total ATP formation (calculated from oxygen consumption and lactate formation), mitochondrial distribution and amino acid turnover to provide an indication of protein synthesis. The TE consumed significantly more oxygen, produced more ATP and contained a greater number of mitochondria than the ICM. Amino acid turnover was significantly greater (p<0.001) in the TE compared with the ICM. Specifically, there was a significant difference in the utilization of aspartate (p=0.020), glutamate (p=0.024), methionine (p=0.037), and serine (p=0.041) between the cells of the ICM and TE. These data suggest that the TE produces approximately 80% of the ATP generated and is responsible for 90% of amino acid turnover compared with the ICM. The major fate of the energy produced by the TE is likely to be the Na+, K+ATPase (sodium pump enzyme) located on the TE basolateral membrane. In conclusion, the pluripotent cells of the ICM display a relatively quiescent metabolism in comparison with that of the TE
Media composition: amino acids and cellular homeostasis
Amino acids are beneficial for the developing preimplantation embryo and therefore form an important component of culture media. This chapter will critically review the importance of amino acids for preimplantation embryos and the impact of this research for the development of sequential culture media used in many assisted conception units. The advantages of culturing embryos in a full complement of amino acids, at close to physiological concentrations will be considered. Moreover, the noninvasive measurement of amino acid turnover by individual embryos, a method which holds great promise to assess developmental competency prior to transfer, will also be discussed. Thus, this chapter highlights the fundamental role of amino acids for the metabolic and homeostatic regulation of the preimplantation embry
GLUT3 and PKM2 regulate OCT4 expression and support the hypoxic culture of human embryonic stem cells
Human embryonic stem cells (hESCs) have the capacity to differentiate into all cell types and thus have great potential for regenerative medicine. hESCs cultured at low oxygen tensions are more pluripotent and display an increased glycolytic rate but how this is regulated is unknown. This study therefore aimed to investigate the regulation of glucose metabolism in hESCs and whether this might impact OCT4 expression. In contrast to the glucose transporter GLUT1, GLUT3 was regulated by environmental oxygen and localised to hESC membranes. Silencing GLUT3 caused a reduction in glucose uptake and lactate production as well as OCT4 expression. GLUT3 and OCT4 expression were correlated suggesting that hESC self-renewal is regulated by the rate of glucose uptake. Surprisingly, PKM2, a rate limiting enzyme of glycolysis displayed a nuclear localisation in hESCs and silencing PKM2 did not alter glucose metabolism suggesting a role other than as a glycolytic enzyme. PKM2 expression was increased in hESCs cultured at 5% oxygen compared to 20% oxygen and silencing PKM2 reduced OCT4 expression highlighting a transcriptional role for PKM2 in hESCs. Together, these data demonstrate two separate mechanisms by which genes regulating glucose uptake and metabolism are involved in the hypoxic support of pluripotency in hESCs
Ca2+ -linked upregulation and mitochondrial production of nitric oxide in the mouse preimplantation embryo
Previous studies have demonstrated a role for the signalling agent nitric oxide in regulating preimplantation embryo development. We have now investigated the biochemical mode of action of nitric oxide in mouse embryos in terms of mitochondrial function and Ca2+ signalling. DETA-NONOate, a nitric oxide donor, decreased day 4 blastocyst cell number and oxygen consumption, consistent with a role for nitric oxide in the inhibition mitochondrial cytochrome c oxidase. Using live cell imaging and the nitric-oxide-sensitive probe DAF-FM diacetate, nitric oxide was detected at all stages of preimplantation development and FRET analysis revealed a proportion of the nitric oxide to be colocalised with mitochondria. This suggests that mitochondria of preimplantation embryos produce nitric oxide to regulate their own oxygen consumption. Inhibiting or uncoupling the electron transport chain induced an increase in nitric oxide and [Ca2+]i as well as disruption of Ca2+ deposits at the plasma membrane, suggesting that mitochondrial disruption can quickly compromise cellular function through Ca2+ -stimulated nitric oxide production. A link between antimycin-A-induced apoptosis and nitric oxide signalling is proposed
Glycolysis regulates human embryonic stem cell self-renewal under hypoxia through HIF-2α and the glycolytic sensors CTBPs
Glycolysis and hypoxia are key regulators of human embryonic stem cell (hESC) self-renewal, but how changes in metabolism affect gene expression is poorly understood. C-terminal binding proteins (CTBPs) are glycolytic sensors that through NADH binding link the metabolic state of the cell to its gene expression, by acting as transcriptional corepressors, or coactivators. However, the role of CTBPs in hESCs has not previously been investigated. A direct interaction between hypoxia-inducible factor 2α (HIF-2α) and the CTBP proximal promoters in hESCs cultured only under hypoxia was demonstrated. Decreasing the rate of flux through glycolysis in hESCs maintained under hypoxia resulted in a reduction of CTBPs, OCT4, SOX2, and NANOG, but also in the expression of HIF-2α. Silencing CTBP expression resulted in the loss of pluripotency marker expression demonstrating that CTBPs are involved in hESC maintenance. These data suggest that under hypoxia, glycolysis regulates self-renewal through HIF-2α and the induction of the metabolic sensors CTBPs