Ž .
had shown that early development 1- to 8-cell stage in the mouse was supported by the oxidation of carboxylic acids pyruvate and lactate, especially pyruvate. Glucose was
poorly utilised, which was attributed to a block to a regulatory glycolytic enzyme, Ž
. phosphofructokinase Barbehenn et al., 1974 . During progression through compaction
and blastulation, this block was removed and glucose contributed increasingly to ATP production via oxidation, so that mouse morula development to the blastocyst stage
Ž .
could be supported by glucose alone. Formulations, such as M16 Whittingham, 1971 , which included glucose, pyruvate and lactate, were found to be effective in the
Ž development of 2-cell embryos of most mouse strains and 1-cell embryos from hybrid
. strains .
Against this background, the pioneers of ruminant embryo culture struggled to Ž
. develop comparably successful systems e.g. Wright and Bondioli, 1981 . Tervit et al.
Ž .
Ž .
1972, 1974 described ‘‘Synthetic Oviduct Fluid’’
SOF , a culture system that produced viable blastocysts from early cleavage stage embryos produced in vivo from
both cattle and sheep. However, little uptake of this system was initially attempted, with Ž
. the exception of Walker et al.
1989 . Indeed, for ruminant embryos, there were conflicting reports as to the appropriate in vitro requirements. For example, Betterbed
Ž .
and Wright 1985 advocated the use of concentrations of glucose of 5.6 mM and an atmospheric oxygen concentration for the in vitro development of sheep embryos,
Ž .
whereas Tervit et al. 1972 had advocated a low O tension and energy substrate levels
2
based on oviduct concentrations. Underpinning these conflicting results was the inability to reliably culture ruminant embryos through the so-called ‘‘8- to 16-cell developmental
block’’, a culture-induced phenomenon which was irreversible but did not result in Ž
. immediate embryonic death Wright and Bondioli, 1981; Eyestone and First, 1991 . Due
to a general lack of understanding as to what caused the block to development and the few but highly variable results obtained with SOF and other simple media, an alternative
Ž strategy was sought which led to the advent of co-culture systems Gandolfi and Moor,
. 1987; Eyestone and First, 1989 . Co-culture significantly advanced the application of in
vitro embryo production, in as much as viable blastocysts were obtained. Nevertheless, in terms of our understanding of factors influencing embryo development, much less
information has been generated using co-culture systems compared to defined systems Ž
. Bavister, 1995 . Furthermore, positive factors influencing embryo development, such as
a low O environment and low glucose levels, which have been attributed to the success
2
Ž .
of co-culture conditions Watson et al., 1994b; Edwards et al., 1997 , were first
identified using defined systems.
2. Oxygen, carboxylic acids, carbohydrates and lipids
Ž .
Utilising both radioisotope-labelled substrates e.g. Rieger and Guay, 1988 and Ž
non-invasive quantitative fluorescence-linked substrate uptakerproduction assays e.g. .
Gardner et al., 1993 , the rates of oxygen uptake, uptakerproduction and utilisation of carboxylic acids and glucose have been well documented over the past 10 years. As a
brief synopsis, Fig. 1 represents the relative contributions of glycolysis and oxidative phosphorylation to ATP production in early and post-compaction stage cattle and sheep
Fig. 1. Simple representation of the subjective differences in metabolic pathway preferences of carboxylic Ž .
Ž . acids and glucose between the early cleavage stages a and compaction and blastulation stages b in cattle
Ž .
and sheep embryos. During early stages, uptake of glucose is low dotted line , whereas uptake of pyruvate Ž
. and lactate provide the fuel for the TCA-cycle solid line , which predominates. In general, rate of ATP
production is low during the early cleavage stages, as demand is not high. In comparison, ATP demand Ž
. increases as compaction and blastulation proceeds
heavy lines . In addition, the contribution to ATP production from glycolysis also increases with development, causing a significant increase in glucose
consumption. However, little of the glucose carbon is oxidised through the TCA cycle. Rather, it appears to preferentially be directed to lactate production.
embryos. In general, embryos throughout pre-elongation development are reliant on Ž
. oxidative phosphorylation via oxidation of pyruvate and amino acids for the generation
Ž of ATP for embryo development Javed and Wright, 1991; Rieger et al., 1992a,b;
. Gardner et al., 1993; Thompson et al., 1996, 2000 . However, there is a switch to an
Ž increased contribution of glycolysis during compaction and blastulation Gardner et al.,
. 1993; Thompson et al., 1996, 2000 . Failure to depress glycolysis during pre-compaction
Ž is one factor associated with the ‘‘8- to 16-cell’’ developmental block Gardner et al.,
. 1997; Thompson et al., 1992a . Therefore, culture media should be designed to suppress
glycolysis during pre-compaction development followed by removal of the suppression during post-compaction development. The former is often confused with complete
removal of glucose from culture medium throughout pre-elongation development. However, this is unlikely to benefit the embryo, as glucose plays other roles including
ribose and NADPH production through the pentose–phosphate pathway. In particular, ribose synthesis is important for the embryo, as this molecule is a precursor for DNA
and RNA synthesis, which is essential for embryonic development. Such metabolic w
14
x intermediates have been detected in sheep embryos following incubation with U- C -
Ž glucose, as well as incorporation into non-glycogen macromolecules Thompson et al.,
. 1995a .
There remains one major question regarding the metabolism of these substrates. Why is glucose poorly oxidized through the tricarboxylic acid cycle, when exogenous
pyruvate appears to be readily oxidised via this pathway? The current and yet inade- quately tested hypothesis is that there is a block to the NAD
q
rNADH shuttle mecha- nism across the mitochondrial membrane. Thus, to maintain an appropriate redox
equilibrium, cytoplasmic NADH must be oxidised in order to maintain glycolytic flux Ž
. Thompson et al.,1993; Edwards et al., 1997 . Thus, glucose is nearly 100 converted
Ž to
L
-lactate, especially at the blastocyst stage Gardner et al., 1993; Thompson et al., .
1996 . A possible explanation for this phenomenon is that there is substrate compart- mentalisation within the cytoplasm of the developing embryo. Glucose-derived pyruvate
is almost entirely converted to lactate, possibly through a mechanism such as substrate channelling.
Another major question still unresolved within ruminant embryos is the role of intracellular lipid in ATP production and other cellular functions. There is ample
Ž .
evidence that lipid is present in reproductive tract secretions e.g. Grippo et al., 1994 , as Ž
there is evidence that lipid can accumulate within embryos cultured in vitro e.g. .
Thompson et al., 1995b; Ferguson and Leese, 1999 . More recently, attempts have been Ž
initiated to characterise the lipid content of cattle and sheep embryos e.g. Ferguson and .
Leese, 1999 . It is hoped that further analysis and improved understanding will answer the question of lipid metabolism.
3. Amino acids
