The causal mechanisms relating embryo environmental sensitivity with abnormal fetal and postnatal development are unlikely to reside exclusively at the epigenetic level. We need to consider how embryos interact with their normal in vivo environment; how this interaction may alter under suboptimal in vitro or in vivo conditions; and what type of responses may occur in embryos to compensate, a measure of their developmental plasticity, essential for survival. Disturbance in developmental and/or nutrient cues across the maternal-embryonic interface may have long-lasting effects given the evolutionarily favorable concept that the setting of fetal growth trajectory may derive from a perception of maternal nutrient supply gained at an early stage of development. We also need to evaluate the contribution of egg quality to this assessment of plasticity and potential, an issue covered elsewhere in recent reviews [55– 57]. In this context, diets high in energy or protein applied in ruminants prior to conception reduce oocyte fertility and developmental potential due to exposure to elevated urea levels [58].
The ionic and nutrient composition of the oviduct and uterus experienced by cleavage-stage embryos has been reviewed recently [8, 59]. There is some evidence that energy substrate levels may vary between human oviduct and uterine fluids [60] and that nutrient levels are relatively low compared with those routinely present within culture media [61, 62]. However, data are scarce with regard to metabolite composition experienced by embryos in vivo at different stages of cleavage. In contrast, we know from extensive in vitro studies with human and different animal models that metabolic activity and substrate preferences appear to change between early and late cleavage with elevated glucose and oxygen consumption evident as embryos approach cavitation [63, 64; reviewed in 62]. However, nutrient consumption data vary dependent on in vitro culture composition and conditions, and the extent of exchange between the embryo and its environment is further complicated by the extent of endogenous stores [8, 62].
In vivo, other factors regulating nutrient exchange will derive from the maternal hormonal status responsive to dietary intake. Thus, progesterone level during the first 3 days of development in sheep has been shown to enhance subsequent fetal growth [65, 66]. Recently, ghrelin, the growth hormone secretogogue receptor (GHSR) ligand, implicated in modulating feeding behavior and energy metabolism, has been identified in mouse uterine fluid and endometrium as well as in morula and blastocyst stages [67]. Embryos also express GHSR, and ghrelin is inhibitory to blastocyst development in vitro; uterine levels of ghrelin increase during fasting, suggesting it may modulate embryo metabolic demands in line with maternal nutrient availability [67]. Although such environmental conditions will influence nutrient exchange kinetics, from the perspective of the embryo, it has been proposed that low metabolite consumption rates (quiet metabolism) more closely equate with developmental competence and viability than do high consumption rates (active metabolism), which are characteristic of metabolic stress [62, 68]. Embryo metabolic parameters may therefore be a significant contributor to the programming of future growth and physiological activity.
Hyperglycemia and Metabolic Stress
Hyperglycaemic conditions illustrate the association between embryo metabolism, stress, and future potential. Although glucose consumption rate does increase in late cleavage, oxidative phosphorylation and not glycolysis is the primary source of energy production in blastocysts across the species due to the corresponding elevation in oxygen consumption [62, 64]. In fact, elevated glucose metabolism, particularly via glycolysis, can be viewed as a stress response in embryos [15, 68], and mouse blastocysts showing high glycolytic rates have a lower capacity for implantation [69]. Sheep embryos collected from ewes fed a high-protein diet during the periconceptional period exhibit a higher metabolic rate and consume more glucose, associated with reduced developmental potential, than do embryos from control diets [27].
While elevated glycolysis is an indicator of metabolic stress within embryos, the extent of glucose uptake itself has been shown to be a positive indicator of fetal developmental potential after transfer [70]. However, in rodent diabetic models where a hyperglycemic embryo environment with significantly elevated glucose levels are utilized, embryo potential is reduced. Here, high external glucose acts to deplete glucose transporter expression at both mRNA and protein levels [71], leading to glucose starvation and activation of apoptotic pathways, chromatin degradation, and nuclear fragmentation [72], which culminate in reduced blastocyst cell numbers [73]. Similar responses to high glucose have been identified in bovine embryos [74]. These detrimental effects in embryos can also derive from perturbation in oocyte maturation in response to high glucose levels [75]. Following embryo transfer, embryos derived from a diabetic environment show increased resorptions and pregnancy loss, illustrating the link between embryo stress response and future developmental capacity. Similar reduction in longer term potential has been shown to occur in response to inhibition in glucose transporter activity during mouse cleavage [76]. A deficient inner cell mass (ICM) cell number has also been identified as a potential causative component of fetal growth retardation and large placenta, which are characteristic of the inherited BB/ E diabetic rat model [73].
The effect of high glucose concentration on the embryo in diabetic models may also disturb later morphogenesis due to a breakdown in normal inductive interactions between blastocyst ICM and trophectoderm lineages by downregulation of ICM Fgf-4 expression required for maintaining trophectoderm proliferative activity [77]. Further support for the importance of embryo glucose/insulin balance has been demonstrated following short-term exposure of preimplantation mouse embryos to insulin, shown to stimulate ICM proliferation [78], which caused a long-term increase in fetal growth rate after transfer [79]. Hyperglycemia is also implicated in the rat low-protein diet models. This diet fed to dams exclusively during the preimplantation period caused transient, mild maternal hyperglycemia associated with blastocyst cell number depletion and abnormal programming of postnatal growth [29]. When the diet is applied 2 wk before mating and during postimplantation development, a transient upregulation of glycolysis is evident in isolated, intact rat conceptuses during 9.5–10.5 days of development coincident with organogenesis [80].
Reactive Oxygen Species
A further stress identified in embryos in response to culture conditions is an increase in hydrogen peroxide production and attendant risk from reactive oxygen species (ROS) [81, 82]. Oxidative stress in the bovine embryo has been shown to lead to DNA damage [83]. ROS exposure will enhance the demand for antioxidant enzymes to maintain homeostatic control, which may further compromise developmental potential [84]. ROS damage in bovine embryos is suppressed by inclusion of vitamin E in culture medium, which stimulated development both before and after embryo transfer [85].
The implications of impaired metabolic activity within embryos for fetal and postnatal development are potentially serious, but direct consequences are yet to be explored mechanistically. Elevated glucose levels can lead to suppression of insulin and glucokinase expression, decreased mitochondrial function, increased ROS formation, and accelerated apoptosis, as well as activation of common stress-activated signaling pathways, which could readily influence proliferative, metabolic, and neuroendocrinal axes during later development [reviewed in 86, 87]. For example, one major intracellular target of hyperglycemia is the transcription factor nuclear factor-kB (NF-kB), which in turn can regulate the expression of diverse growth factors, cytokines, and adhesion molecules, all of which have the potential to modulate the phenotypic response to early embryo environment [87].
Amino Acids
Another and perhaps better example of adverse embryo programming working through metabolic pathways concerns amino acids and their turnover by the embryo. This is not surprising given the multitude of roles ascribed to amino acids in early development; in addition to protein biosynthesis, they also stimulate activation of the embryonic genome, blastocyst formation and hatching, and contribute to energy production, osmoregulation, pH control, cell homeostasis, and, perhaps significantly, to signal transduction cascades [88]. The embryo is equipped with an array of sodium-dependent and -independent transporter systems to regulate amino acid flux and availability [88]. Clear differences in embryo amino acid intracellular content have been demonstrated between in vivo and in vitro developing mouse embryos, indicative of environmental influence on exchange rates [89]. Indeed, amino acid turnover has been shown to vary between individual human embryos assayed noninvasively during early cleavage. Significantly, the pattern of exchange correlated with capacity to form a blastocyst, with those embryos showing lower turnover exhibiting greater viability [90], supporting the concept of a quiet metabolism being linked with future potential [62, 68]. Culture of mouse embryos with amino acids has also been reported to enhance fetal development after transfer [91]. In addition, culture of sheep embryos with amino acids leads to significantly improved fetal development than in medium containing serum [12]. The correct balance of amino acids is important because amino acids, particularly glutamine, may spontaneously break down in culture to produce ammonium ions, which can be deleterious to development in the short- and long-term. Addition of ammonium to mouse culture produced a concentration-dependent reduction in blastocyst and ICM cell numbers, increased apoptosis, and altered the pattern of H19 gene expression; this treatment, after transfer, also reduced the implantation rate, impaired fetal development at Day 15, and caused a higher rate of exencephaly [92, 93].
The importance of amino acid environment during early development is further implicated from in vivo studies. Maternal low-protein diet administered to rats during the preimplantation period induced a transient reduction in maternal serum essential amino acid concentrations, associated with reductions in blastocyst ICM and trophectoderm (TE) cell numbers and leading to abnormal programming of growth and hypertension postnatally [29]. Our current studies reveal, using the mouse model, that maternal protein undernutrition alters the profile of amino acids present within the uterine fluid during the period of morula and blastocyst occupancy, prior to implantation [94]. These studies further indicate that specific amino acids, notably the branch-chain group of leucine, isoleucine, and valine, are significantly depleted in both serum and uterine fluid in response to low-protein diet [29, 94]. Significant uptake of these amino acids has been shown to occur during blastocyst formation and expansion in vitro, suggestive of a critical developmental role [95]. Leucine in particular has a key signaling function in early development as activator of the mammalian target of rapamycin (mTOR), a serine-threonine kinase pathway that phosphorylates regulatory targets involved in protein translation and biosynthesis [96, 97]. The mTOR signaling has been associated with several growth and patterning events and phenotypic changes during development and differentiation [98, 99]. Significantly, in the early embryo, mTOR activity has been shown to be required for inducing an invasive and migratory behavior in trophoblast cells during the implantation period [100] and, through this activity dependent on amino acid transport, could coordinate downstream signaling pathways within the embryo [101].
In another mechanistic direction, changes in amino acid environment to embryos may lead to abnormal epigenetic effects. Maternal low-protein diet fed to rodents during the periconceptional period led to elevation in maternal serum homocysteine levels [102], which may cause folate deficiency and interfere with methyl group donation required for DNA methylation [103]. The importance of maternal dietary methyl supplementation on epigenetic regulation and the extent of DNA methylation has been demonstrated in the Agouti mouse model [104, 105].
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