This review considers how our understanding of preimplantation embryo metabolism has progressed since the pioneering work on this topic in the late 1960s and early 1970s. Research has been stimulated by a desire to understand how metabolic events contribute to the development of the zygote into the blastocyst, the need for biomarkers of embryo health with which to improve the success of assisted conception technologies, and latterly by the ‘Developmental Origins of Health and Disease’ (DOHaD) concept. However, arguably, progress has not been as great as it might have been due to methodological difficulties in working with tiny amounts of tissue and the low priority assigned to fundamental research on fertility and infertility, with developments driven more by technical than scientific advances. Nevertheless, considerable progress has been made in defining the roles of the traditional nutrients: pyruvate, glucose, lactate, and amino acids; originally considered as energy sources and biosynthetic precursors, but now recognized as having multiple, overlapping functions. Other nutrients; notably lipids, are beginning to attract the attention they deserve. The pivotal role of mitochondria in early embryo development and the DOHaD concept, and in providing a cellular focus for metabolic events is now recognized. Some unifying ideas are discussed; namely ‘stress–response models’ and the ‘quiet embryo hypothesis’; the latter aiming to relate the metabolism of individual preimplantation embryos to their subsequent viability. The review concludes by updating the state of knowledge of preimplantation embryo metabolism in the early 1970s and listing some future research questions.
Henry J Leese
Nadia Gopichandran and Henry J Leese
Bovine preimplantation embryos develop more successfully when cultured in groups, proibably because of the increased production of, and exposure to, embryotrophic autocrine and paracrine factors. Using a novel embryo culture technique, this study had two aims: 1. to determine the distance over which potential paracrine interactions affect bovine embryo development in terms of blastocyst and hatching rates, cell counts and carbohydrate metabolism; 2. to investigate the effect of platelet-activating factor (PAF) supplementation on bovine embryo development and metabolism. Groups of 16 presumptive zygotes were attached to the bottom of a culture dish by the cell adhesive Cell-Tak in a 4 × 4 equidistant array. The distance between individual embryos in each group was 0–689 μm. Optimal blastocyst formation rate occurred when embryos were cultured 165 μm apart compared with control non-attached zygotes (Kruskal–Wallis followed by Mann–Whitney U test post-hoc; P < 0.05). Increasing the distance between embryos resulted in a further decline in blastocyst rate, which reached zero at 540 μm apart. Blastocyst cell number, pyruvate/glucose uptake and lactate production decreased as the interembryo distance increased from 240 to 465 μm (P < 0.05). Supplementation with PAF during conventional group culture enhanced blastocyst cell number, hatching rates and the oxidative metabolism of pyruvate and glucose. The data indicate that the distance between individual bovine embryos in culture influences preimplantation development, in particular blastocyst formation, cell number and metabolism. It is suggested that diffusible paracrine/autocrine factors, such as PAF, are in part responsible for the regulation of early embryo development.
Nicolas M Orsi and Henry J Leese
The accumulation of ammonium is a major artefact of in vitro embryo culture. This study has examined ammonium production and potential mechanisms of disposal in preimplantation bovine blastocysts. Embryos were produced by in vitro maturation and fertilisation of oocytes, and cultured in synthetic oviduct fluid containing amino acids and BSA (SOFaaBSA). Ammonium/urea concentrations were determined enzymatically. Amino acid appearance/disappearance ‘profiles’ of single blastocysts were determined at 0, 1.25 and 2.5 mM NH4Cl (with or without 0.33 mM pyruvate), and with or without 10 mM dipicolinic acid (DPCA; a glutamate dehydrogenase (GLDH) inhibitor) or 2 mM amino-oxyacetate (AOA; a transaminase inhibitor). Free ammonium was produced at a rate of 4.281 (±0.362) pmol/embryo/h, while urea production was undetectable. The presence/absence of pyruvate affected amino acid profiles, especially alanine appearance (P < 0.001), glutamate disappearance (P < 0.05) and overall turnover (the sum of appearance and disappearance) (P < 0.001). GLDH inhibition with DPCA had no effect on amino acid overall disappearance, but glutamate disappearance increased, while that of arginine decreased (P < 0.05). The transaminase inhibitor, AOA, depressed turnover (P < 0.05), aspartate and glutamate disappearance, and alanine appearance. Thus, bovine blastocysts release ammonium as free ions or fix them, not as urea, but as alanine, possibly glutamine and, less likely, arginine. An active role for GLDH and transaminases in regulating blastocyst amino acid metabolism was demonstrated.
Paul J Booth, Peter G Humpherson, Terry J Watson and Henry J Leese
Preimplantation embryos can consume and produce amino acids in a manner dependent upon the stage of development that may be predictive of subsequent viability. In order to examine these relationships in the pig, patterns of net depletion and appearance of amino acids by in vitro produced porcine preimplantation embryos were examined. Cumulus oocyte complexes derived from slaughterhouse pre-pubertal pig ovaries were matured for 40 h in defined TCM-199 medium (containing PVA) before being fertilised (Day 0) with frozen-thawed semen in Tris–based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20, in NCSU-23 medium modified to contain 0.1 mM glutamine plus a mixture of 19 amino acids (aa) at low concentrations (0.02–0.11 mM) (NCSU-23aa). Groups of 2–20 embryos were removed (dependent on stage) on Day 0 (1 cell), Day 1 (two- and four-cells), Day 4 (compact morulae) and Day 6 (blastocysts) and placed in 4 μl NCSU-23aa for 24 h. After incubation, the embryos were removed and the spent media was analysed by HPLC. The net rate of amino acid depletion or appearance varied according to amino acid (P < 0.001) and, apart from serine and histidine, stage of development (P < 0.014). Glycine, isoleucine, valine, phenylalanine, tryptophan, methionine, asparagine, lysine, glutamate and aspartate consistently appeared, whereas threonine, glutamine and arginine were consistently depleted. Five types of stage-dependent trends could be observed: Type I: amino acids having high rates of net appearance on Day 0 that reached a nadir on Day 1 or 4 but subsequently increased by Day 6 (glycine, glutamate); Type II: those that exhibited lower rates of net appearance on Days 0 and 6 compared with the intermediate Days 1 and 4 (isoleucine, valine, phenylalanine, methionine, arginine); Type III: amino acids which showed a continuous fall in net appearance (asparagine, aspartate); Type IV: those that exhibited a steady fall in net depletion from Day 0 to Day 6 (glutamine, threonine); Type V: those following no discernable trend. Analysis of further embryo types indicated that presumptive polyspermic embryos on Day 0 had increased (P < 0.05) net rates of leucine, isoleucine, valine and glutamate appearance, and reduced (P < 0.05) net rates of threonine and glutamine depletion compared with normally inseminated oocytes. These data suggest that the net rates of depletion and uptake of amino acids by pig embryos vary between a) amino acids, b) the day of embryo development and, c) the type of embryos present at a given stage of development. The results also suggested that the net depletion and appearance rates of amino acids by early pig embryos might be more similar to those of the human than those of the mouse and cow.
Constantine A Simintiras, Thomas Fröhlich, Thozhukat Sathyapalan, Georg J Arnold, Susanne E Ulbrich, Henry J Leese and Roger G Sturmey
Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage and genome activation. However, the composition and regulation of this critical environment remain rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium to investigate the formation and composition of in vitro-derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct-specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation was evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial–fibroblast interactions, probing the molecular aetiologies of subfertility and optimising embryo culture media.
Sarah E Harris, Iris Adriaens, Henry J Leese, Roger G Gosden and Helen M Picton
Metabolic markers are potentially valuable for assessment of follicle development in vitro. Carbohydrate metabolism of murine preantral follicles grown to maturityover 13 days in vitro has been measured, and metabolism of resulting oocyte–cumulus complexes (OCCs) and denuded oocytes has been compared with in vivo ovulated control counterparts. Spent follicle culture media were analysed for glucose, lactate and pyruvate concentrations. During follicle in vitro growth, glycolysis accounted for a rise from ∼24 to 60% of all glucose consumed. Ovulation induction caused a significant increase in glucose uptake and lactate production by in vitro-grown follicles to 71.7±1.2 and 96.6±4.8 nmoles/day respectively. OCCs grown in vitro had significantly higher rates of glucose consumption and lactate and pyruvate production (110.1± 3.5, 191.8± 8.9 and 31.7± 1.7 pmoles/h respectively) than in vivo ovulated controls (67.4± 8.1, 113.9± 17.1 and 20.2± 4.0 pmoles/h respectively), but a reduced capacity for pyruvate consumption (1.13± 0.06 vs 1.49± 0.06 pmoles/h by in vivo ovulated oocytes). Metabolism of OCCs was affected by the quality of the original follicle. In vitro-grown oocytes had a reduced cytoplasmic volume when compared with controls (168.3± 2.0 vs 199.0± 3.2 proportionately respectively) but a similar rate of metabolism per unit volume. Meiotic status influenced metabolism of both OCCs and denuded oocytes. In conclusion, glucose consumption and lactate production by cultured follicles increased in tandem with developmental progression and were stimulated prior to ovulation. Additionally, the metabolic profiles of in vitro produced OCCs and the oocytes within them are affected by long-term exposure to the culture environment.
Niamh Lewis, Katrin Hinrichs, Henry J Leese, Caroline McGregor Argo, Daniel R Brison and Roger G Sturmey
The use of in vitro embryo production in the horse is increasing in clinical and research settings; however, protocols are yet to be optimised. Notably, the two most commonly used base media for in vitro maturation (IVM) supply glucose at markedly different concentrations: physiological (5.6 mM, M199) or supraphysiological (17 mM, DMEM/F-12). Exposure to high glucose has detrimental effects on oocytes and early embryos in many mammalian species, but the impact has not yet been examined in the horse. To address this, we compared the energy metabolism of equine COCs matured in M199-based maturation medium containing either 5.6 or 17 mM glucose, as well as expression of key genes in oocytes and cumulus cells. Oocytes were fertilised by ICSI and cultured. Analysis of spent medium revealed that COC glucose consumption and production of lactate and pyruvate were similar between treatments. However, the glycolytic index was decreased at 17 mM and analysis of mitochondrial function of COCs revealed that IVM in 17 mM glucose was associated with decreased ATP-coupled respiration and increased non-mitochondrial respiration compared to that for 5.6 mM glucose. We also found that the metabolic enzyme lactate dehydrogenase-A (LDHA) was downregulated in cumulus cells of oocytes that completed IVM in 17 mM glucose. There was no difference in maturation or blastocyst rates. These data indicate that COC mitochondrial function and gene expression are altered by high glucose concentration during IVM. Further work is needed to determine if these changes are associated with developmental changes in the resulting offspring.
Nicolas M Orsi, Nadia Gopichandran, Henry J Leese, Helen M Picton and Sarah E Harris
Bovine oocyte maturation in vitro frequently results in abnormal cytoplasmic maturation and failure to acquire developmental competence. This is, in part, likely to be due to the non-physiological nutritional milieu to which oocytes are exposed. Improvements in oocyte developmental potential may be achieved by modelling nutrient profiles on those of preovulatory follicular fluid (FF). However, little is known about fluctuations in FF nutrient levels according to follicle dominance and oestrous cyclicity. This study therefore characterised the carbohydrate and amino acid profile of FF according to these parameters, and compared preovulatory FF composition with that of maturation medium. Carbohydrate concentrations (n = 121) were determined enzymatically whilst amino acid profiles (n = 40) were determined by reverse-phase HPLC. Pyruvate and glucose concentrations were unaffected by follicle dominance, whereas Stage III–IV lactate profiles were higher in non-dominant FF (P < 0.01). While most dominant FF amino acid concentrations were affected by oestrous stage, only glutamate, alanine, leucine and lysine levels fluctuated in non-dominant FF. Glucose and lactate concentrations were significantly negatively correlated, whereas most amino acids were significantly positively correlated with each other. Maturation medium had higher pyruvate and lower lactate concentrations than preovulatory FF (P < 0.001), whereas glucose level was similar. All amino acid levels (except histidine, taurine, alanine and tryptophan) differed significantly between maturation medium and preovulatory FF. These data indicated that FF composition varies throughout the oestrous cycle. Preovulatory FF nutrient profile differed from that of maturation medium, perhaps accounting for the poor developmental competence of in vitro matured oocytes. These data may contribute to the formulation of a nutritionally more physiological maturation medium.