While much is known about the metabolism of exogenous nutrients such as glucose, lactate, pyruvate, amino acids by oocytes and pre-implantation mammalian embryos, the role of endogenous stores, particularly lipid, has been largely overlooked. The presence of lipid within oocytes and early embryos has been long known, and comparisons between species indicate that the amounts and types of lipid present vary considerably. Large amounts of intracellular lipid can compromise the success of cryopreservation and the removal of such lipid has been the subject of considerable effort. In this review, we present evidence that strongly suggests a metabolic role for lipid, specifically with regard to energy provision, in the late-stage oocyte and the pre-implantation embryo. We focus initially on oxygen consumption as a global indicator of metabolic activity, before reviewing different approaches that either have been designed to investigate directly, or have revealed indirectly the role of endogenous lipid in energy generation. These fall under five headings: (i) fatty acid oxidation; (ii) inhibition of triglyceride oxidation; (iii) culture in the absence of exogenous substrates; (iv) cytoplasmic organization; and (v) delipidation. On the basis of the data derived from these studies, we conclude that there is strong evidence for the utilization of endogenous lipid as an energy substrate by oocytes and early embryos.

The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0\p=n-\6of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l \ m=-\ 1, 10 mmol d,l-lactate l \m=-\1 and 1.5 mmol glucose l \m=-\1and also 3 mg BSA ml \m=-\1and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0\p=n-\4of culture (0.24\p=n-\0.27 nl per embryo h \ m=-\ 1) , but increased with the initiation of compaction (0.39 nl per embryo h\m=-\1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h \m=-\1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake at day 0 of culture (putative zygotes), increasing to 70% by day 2 (eight-cell stage) and 100% of glucose uptake from day 4 of culture onwards. ATP production followed a similar pattern to that of oxygen consumption (60\p=n-\85 pmol per embryo h \ m=-\ 1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h \ m=-\ 1) and blastulation (221 pmol per embryo h\m=-\1). For precompaction stages, 93\p=n-\96%of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.

Pig oocytes and embryos differ from those of other species in having a large quantity of endogenous lipid, a potential role for which has yet to be identified. In the present study, the hypothesis that endogenous triglyceride acts as a metabolic substrate during in vitro maturation and early embryo development was tested. Embryos were produced by in vitro fertilization (IVF) of in vitro-matured, abattoirderived immature oocytes, cultured in medium NCSU23 up to the blastocyst stage. The triglyceride content of single oocytes and embryos was measured throughout development. Oxygen and glucose consumption and the formation of lactate were measured non-invasively over the same period, enabling total ATP production to be calculated. The triglyceride content of oocytes before maturation (135 ± 4.9 ng) decreased by 13 ng (P < 0.05) during in vitro maturation, but there was no apparent change in triglyceride content during embryo development (117.68 ng). Oxygen consumption was low throughout embryo cleavage before reaching a peak at the blastocyst stage (P < 0.01), a pattern similar to that seen in other mammals studied. Glucose consumption and lactate production were also at a maximum at the blastocyst stage (P < 0.05). These data indicate that pig oocytes may use endogenous triglyceride as an energy source during in vitro maturation and that most (91-97%) of the ATP produced during embryo development comes from oxidative phosphorylation. The high exogenous glucose concentration in NCSU23 (5.5 mmol l −1 ) may be needed to form pyruvate, which in turn, produces oxaloacetate, which is required to prime the tricarboxylic acid cycle. However, the reason for the high lipid content in early pig embryos remains to be elucidated.

The role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes has been studied. Fluorescence resonance energy transfer (FRET) acceptor bleaching methods have been used to examine mitochondrial:lipid droplet co-localisation in live oocytes. FRET experiments demonstrate whether organelles are within the FRET-distance (i.e. 6-10 nm), thus showing true association on a molecular scale. Immature and in vitro-matured porcine oocytes were stained with Mitotracker Green (MTG; mitochondria) and Nile Red (NR; lipid droplets). The data indicated sufficient overlap between MTG emission and NR excitation to support a FRET reaction and that mitochondria and lipid droplets were sufficiently co-localised for a FRET reaction to occur. When NR-stained lipid droplets were specifically bleached, a significant increase in the MTG signal in stained mitochondria was observed (FRET efficiency, EZ22.2G3.18%). These results strongly suggest a metabolic role for lipid metabolism during oocyte maturation. This conclusion was reinforced by the use of inhibitors of fatty acid b-oxidation, methyl palmoxirate or mercaptoacetate, exposure to which during oocyte maturation led to developmental failure post-fertilisation. These data provide strong evidence that MTG and NR can act as a FRET pair and that in porcine oocytes, mitochondria and lipid droplets lie within 6-10 nm of each other, indicating association on a molecular scale. The findings also suggest that endogenous triglycerides play an important role in energy metabolism during porcine in vitro maturation. Reproduction (2006) 132 829-837

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Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid b-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid b-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

A variety of embryo-based technologies used in farm animal reproduction, including embryo culture, nuclear transfer, embryo-somatic cell co-culture and asynchronous embryo transfer can lead to the production of large offspring; the so-called large calf/lamb syndrome. In some cases, abnormalities in the fetus and newborn are apparent. The nature of these associations is explored with emphasis on the biological differences between in-vivo- and in-vitro-produced embryos. A unifying framework and research programme aimed at explaining anomalies in early embryo development is then proposed in terms of the response of somatic cells and embryos to cellular stress. The review concludes with a caution against developments in assisted conception technologies, in man and domestic animals, being determined too much by the needs of commerce at the expense of research on the molecular, biochemical and physiological basis of early mammalian development.

Vascularly perfused Fallopian tubes have been used to study the formation and composition of human tubal fluid and the response to adrenergic agents. An artery serving the tube was cannulated and perfused with Medium 199 supplemented with bovine serum albumin (BSA) and antibiotics. A second cannula was attached to the fimbriated end for native tubal fluid collection. The preparation was viable for up to 2 h. Tubal fluid was only obtained in tubes removed in the proliferative and early secretory phases of the ovarian cycle. Isoproterenol (1 mM) added to the perfusate stimulated fluid production, whereas dibutyryl cyclic AMP (1 mM) reduced fluid formation by 66%. Glucose, pyruvate and lactate concentrations in tubal fluid, measured by microfluorescence assays, were 1.11, 0.14 and 5.4 mM respectively. The concentrations of 17 amino acids in tubal fluid were measured by high performance liquid chromatography following fluorescence derivatization. Arginine (0.19 mM) > alanine (0.11 mM) > glutamate (0.09 mM) were present in highest concentration in all phases of the cycle. All 17 amino acid concentrations in tubal fluid were below those in the vascular perfusate. These data provides the basis for a culture medium whose composition mimics the physiological environment to which early human embryos are exposed.