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GLYCOLYTIC PATHWAY IN PREIMPLANTATION MAMMALIAN EMBRYOS John D. Biggers - INTRODUCTION A highly integrated network of chemical reactions Cal leaf metabolism enables cells to extract energy and reducing power from their environments and to synthesize the building blocks of their macromolecules. The role of . . . metabolism in the generative process is therefore fundamental. It provides the energy whereby a new organism can develop following the blueprint determined by its genetic endowment. In aerobic organisms metabolism is subserved ubiquitously by three linked systems - the glycolytic pathway, the citric acid cycle and the oxidative respiratory chain. Intuitively it seems trivia] to study these highly conserved systems in detail in preimpJantation mammalian embryos, since they are more readily studied in other systems. Observations that began to accumulate over 30 years ago (see Biggers, 1987, for a review), however, show that such a conservative view cannot be sustained. Pioneering studies by Whitten (1956, 1957) on media for the culture of preimplantation mouse embryos found that 2-cell stages that would not develop if glucose alone was the only energy source would develop if lactate was present. Subsequent work by several investigators led to the conclusion that during oogenesis in the mouse the metabolic process is restricted and that it is restored at about the 8-cell stage of development (Brinster, 1965a,b; Brinster and Thompson, 1966; Biggers et al., 1967). A key finding was that metabolism is restricted in the maturing oocyte stage and in the zygote to the extent that only pyruvate or oxaloacetate could support development (Biggers et al., 1967). These early observations are the origin of the widely assumed view that pyruvate is an important source of energy in early mammalian development and should be - 282 -

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incorporated in all embryo culture media. These observations al-so led-to an extensive li.terature on the metabolism of preimplantation embryos (see Biggers and Stern, 1973; Biggers, 1976; Biggers: and Borland, 1976; Wales, l9?5; Pike, 1981; Weitlauf and Nieder, 1984; Kaye, 1986; Biggers et-al., 1989, for reviews). In this paper I wish to revisit particularly the glycolytic pathway, whose function seems to account for the original observations on the nutritional requirements.of the preimplantation mouse embryo. I suggest that three areas can be usefully discussed at the present time to provide a basis for future research. These areas are: (l).What is the evidence that metabolic pathways are restricted in preimplantation development? (2) What overal1 metabolic fluxes are functional during preimplantation development?^t3) How is.the glycolytic pathway controlled during early mammalian development? . EVIDENCE THAT METABOLIC PATHWAYS ARE RESTRICTED IN OOGENESIS AND EARLY MAMMALIAN DEVELOPMENT ~ . Four types of evidence suggest that metabolic pathways are restricted during oogenesis and early mammalian development. The evidence comes from changes in the morphology of the mitochondria, which can be correlated with the metabolic state of the embryo, nutritional studies with chemically defined media, the uptake of metabolic substrates, and the metabolic fate of substrates. The morphological evidence is important since it is the only evidence that does not involve the manipulation of embryos in vitro. Morphology of the Mitochondria during Oogenesis and . preimplantation Development . A pioneer study with the electron microscope on oocyte matuation described the pleiomorphic stucture of.cristae in.. the mitochondria of the

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mouse (Yamada et al., 1957). Some of the cristee were oriented transversely across the organelle, while others were arranged concentrically inside the plasma membrane. Later Mazanec and Dvorak (1964) described mitochondria with concentric cristee in the early cleavage stages of the rat, and the presence of these were soon confirmed in the mouse (Sackler, 1968; Calarco and Brown, 1969; Hillman and Tasca, 1969; Reinius, 1969). This type of mitochondrion has been reported also in oocytes of the guinea-pig, hamster, rabbit, cow, monkey and human, and in the cleavage stages of the rabbit [see Stern et al.(l971), for a review]. Carefully timed studies showed that after a critical time of development almost all mitochondria with concentric cristee disappeared, after which time all mitochondria have transverse cristae. In the mouse this maturation in mitochondrion morphology is complete by the B-cell stage (Stern et al., 1971) and in the rabbit by the morula stage (Anderson et al.,l970). Of importance is the fact that this maturation is complete by the time the oxygen consumption of the mouse embryo (Mills and Brinster, 1967) and the rabbit embryo (Fridhandler et al., 1957) increases suddenly. Moreover, the mitochondrial maturation is associated in the mouse with the restoration of the ability of the embryos to utilize malate, citrate and 2-oxoglutarate (Kramer and Diggers, 1971). Although the genesis of these morphological changes in mitochondrial morphology is unknown, one possible explanation is that they reflect the energy state of the blastomeres, as has been described in other cell types (Hackenbrock, 1968~. Nutritional studies with chemicaliv defined media The early results obtained on the nutritional requirements of the preimplantation mouse embryo are summarized in Fig. 1. Meiotic maturation of the oocyte and cleavage through the first division after fertilization require 284

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the presence of pyruvate or oxaloacetate. Development of the 2-cell stage can only be supported by pyruvate, oxaloacetate, phosphoenolpyruvate and lactate. Glucose alone is unable to support development of the preimplantation mouse embryo until after the 8-cell stage. Comparable studies on the nutritional requirements of the rabbit have led to conflicting results. The design of definitive experiments is complicated for two reasons [see Kane (1987a) for a review]. First, the large rabbit ovum, with a volume about five times that of the mouse, has considerable energy reserves which allow it to undergo several cleavage divisions without the incorporation of any energy source in the medium (Kane, 1972). Second, rabbit preimplantation embryos are able to use as energy sources a variety of long- and short-chain fatty acids that contaminate many samples of bovine serum albumin (Kane, 1979). Using a complex modification of Ham's F10 without a macromolecule, Daniel (1967) reported that pyruvate, phosphoenolpyruvate and lactate improved the development of rabbit embryos. These results conflict with those obtained by Kane (1987b), who used a basic protein-free medium, containing polyvinylalcohol as a macromolecule. He found that pyruvate and glucose can separately increase the number of cell divisions of one-cell rabbit embryos over a 48h period (Fig. 2), while phosphoenolphosphate, oxaloacetate and lactate had no significant effects. Thus there may be an advantage to including pyruvate in the medium, though the requirement is not absolute, as in the mouse. The need for pyruvate and lactate in the culture of pig preimplantation embryos has been studied [see Davis (1985), for a review]. Davis and Day (1978) and Stone et al (1984) have reported that the incorporation of pyruvate and lactate is detrimental to development. Petters (personal communication) 285

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has not found these substrates detrimental, but has failed to show they have any beneficial effects on the development of 1-cell and 4-cell pig embryos. In vitro studies using chemically defined media with other species are far less complete. Pyruvate is often incorporated into media for the culture of non-human primate (Bavister et al., 1983) and human embryos (Edwards et al., 1970), but the practice does not seem to be based on any critical observations. To summarize, the in vitro studies on the development of the preimplantation stages of three mammalian species (mouse, rabbit, pig) suggest the requirements for energy substrates may differ widely. Uptake of metabolic substrates Recently, by using very sensitive u~tramicrofluorometric methods of biochemical analysis (Mroz amd Lechene, 1980), it has become possible to ~ analyze the uptake of pyruvate and glucose by single preimp~antation embryos (Leese et al., 1984; Butler et al., 1988). The results obtained by Leese and Barton (1984), using embryos flushed directly from the genital tract,!are shown in Fig. 3. Prior to the 8-cell stage the embryos preferentially take up pyruvate, while older embryos take up glucose. The change-over in relative uptakes occurs roughly at the time of compaction. Similar results have been obtained using embryos produced in vitro (Gardner and Leese, 1986~. Both of these sets of results are consistent with the earlier nutritional studies on the mouse which suggested that metabolic fluxes of preimplantation mouse embryos change with development. Recently it has been possible to measure the uptake of pyruvate by single human oocytes and spare human embryos obtained from an IVFET Program (Leese et al., 1986~. The results are shown in Fig. 4. The fresh oocytes took - 286

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up an average of 36 pmol/oocyte/h, while normal, cleaving embryos took up less. The uptake was diminished still further by the blastocyst stage. These very preliminary observations suggest that the utilization of pyruvate by the human preimplantation embryo follows a developmental pattern similar to that observed in the mouse. i Metabolic Fate of Substrates A physiological test of the utilization of glucose by the glycolytic pathway is to compare the amount of CO2 produced from exogenous glucose and pyruvate respectively. Such comparisons were done some years ago on the mouse preimplantation embryo by Brinster (1967). The results of Brinster are summarized in Fig. 5. At the one- and two-cell stages relatively little glucose is metabolized to CO2-, whereas considerably more pyruvate gives rise to CO2. After the 8-cell stage, however, relatively more glucose is metabolized to CO2. In another study Brinster and Harstad (1977) showed that the preferential utilization of pyruvate over glucose occurs in primordial germ cells isolated from the germinal ridge of 15-day-old mouse fetuses. These results suggest that in the mouse relatively little glucose is metabolized through the glycolytic cycle during oogenesis, and that glycolysis is not restored until after the 8-cell stage. Similar results on the production of CO2 from glucose have recently been reported by Wales (1986) using single mouse preimplantation embryos. - Comparable studies on the rabbit (Brinster, 1968, 1969) also show that the ability of the mature oocyte and one-cell rabbit embryo to oxidize glucose to CO2 is very restricted, whereas pyruvate can be readily oxidized. Further studies on the oocyte of the monkey (Brinster, 1971) and cow (Rushmer and Brinster, 1973) have also shown that these cells oxidize pyruvate to carbon 287 -

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dioxide much more readily than glucose. Recently Rieger and Guay (1988) have examined the metabolism of glucose and pyruvate in the seven-day-old cow blastocyst. They found that pyruvate was readily oxidized to CO2 and that the rate of this process could be significantly stimulated by dinitrophenol, a decoupled of oxidative phosphorylation. Glucose, on the other hand, seemed to be converted in quantity to phosphoenolpyruvate. The inhibitor, however, had no stipulatory effect. These results suggest that in the cow full glycolysis is not restored until after the seventh day of development. A Caveat - Be Alert for Artifacts There is always the possibility that the metabolic effects that have been observed in preimplantation embryos are due to the in vitro conditions under which the embryos are observed. An example has recently come to light in studies on the breakdown of glycogen by mouse preimplantation embryos. The concentrations of glycogen in the mouse preimplantation embryo analyzed immediately after flushing from the female genital tract are shown in Fig. 6. There is little glycogen in the one-cell stage. At the two-cell stage considerable quantities of glycogen are synthesized, reaching a peak concentration at the 8-cell stage (Stern and Biggers, 1968; Ozias; and Stern, 1973). Synthesis of glycogen also occurs in early mouse preimplantation embryos developing in vitro (Ozias and Stern, 1973). At the blastocyst stage the amount of glycogen diminishes in utero. In contrast the glycogen content remains elevated in blastocysts raised in vitro. Recent work has shown that the amount of glycogen present in mouse blastocysts that developed in the uterus is regulated by the rate of degradation, which is low in blastocysts produced under in vitro conditions (Edirisinghe and Wales, 1984). Very

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recently Khurana (1987) has obtained evidence that the po2 controls the rate of degradation of glycogen in mouse blastocysts. The results shown in Fig. 7 demonstrate that 20% oxygen almost completely inhibits glycogen breakdown and that it is still 50% inhibited at an oxygen concentration of 2.5%. It is well established that an oxygen concentration of 20% is not optimal for the culture of mouse preimplantation embryos (Whitten, 1971; Quinn and Harlow, 1978; Harlow and Quinn, 1979). There is also evidence that a low oxygen tension is beneficial for the culture of sheep and cattle preimplantation embryos (Tervit et al., 1972; Wright et al., 1976). Measurements of the oxygen tension in the uterus of the rabbit (Bishop, 1956) and the rhesus monkey (Mass et al., 1976) suggest that the oxygen concentration may be less than 5 percent, particularly if the organ is under the influence of progesterone. Thus the recent work of Khurana (1987), showing that oxygen tension affects the rate of degradation of glycogen, is possibly the first demonstration of a biochemical artefact in metabolism produced by conditions commonly used in embryo culture. METABOLIC FATE OF GLUCOSE An analysis of the fate of exogenously supplied glucose to an organism In vitro is complex. The possible pathways are shown in Fig. 8. The glucose is first transported through the plasma membrane of the cells into the glucose pool. Then it can be used through either the glycolytic pathway, the pentose phosphate pathway or the glycogen synthesis/degradation shuttle. A simplistic interpretation of the experiments on the fate of glucose and pyruvate at different stages of preimplantation development in the mouse is that the glycolytic pathway is shut down during oogenesis while the citric acid cycle and the respiratory pathways are left intact. As development proceeds the glycolytic pathway becomes functional. Such an interpretation 2eg -

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could be explained in terms of"the absence of an enzyme in the g~ycolytic pathway in the early stages,'which is remedied by the activation of the appropriate gene at a critical time in preimp~antation development. In a critical review of earlier studies (Biggers, 197'6) it was argued that the tote] shutdown of glycolytic pathways in the mouse does not occur at any stage. The evidence, in fact, suggested that the g~ycolytic pathway in the mouse oocyte and preimplantation embryo operates at a low'leve] throughout development, and that the citric acid cycle is also not operating to full capacity. Under such circumstances the metabolic pathways should be considered intact in the sense that all enzymes and their substrates are present. -~ Appropriate analyses of the metabolic networks are in terms of the fluxes passing at any given time through the component pathways [see Reich and Sel'Kov (1981), for a thorough theoretical review]. Such studies have barely begun in the investigation of preimplantation development. So far two approaches have been used. One is an examination ofithe relative metabolic fluxes originating with glucose through the g~ycolytic pathway and the pentose-phosphate pathway in the rabbit. The other i'nvo~ves the identification of specific rate-limiting steps in a metabolic pathway u'sing starvation and replacement feeding experimental strategies. The first attempts to estimate the relative amounts of glucose ~ metabolized through the glycolytic pathway and the pentose-~phosphate pathway were done on the rabbit (Fridhandler, 1961; Brinster,-1968) and mouse (Brinster, 1967) by measuring the amounts of ,4CO2 produced from glucose labelled in the C-1 and C-6 positions repectively. The results suggested that during the early cleavage stages flux through" the pentose-phosphate pathway is high in the rabbit and low in the mouse. The interpretation of these - 990

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experiments, however, is equivocal (Biggers and Stern, 1973). A more reliable assessment of the relative fluxes through~the two pathways can be obtained only if the total glucose utilization is also known (Katz et al., 1966). Recently O'Fallon and Wright (1986) have re-examined the relative fluxes of the glycolytic pathway and the pentose-phosphate pathway in the preimplantation mouse embryo using the methods described by Katz et al. in their studies of rat adipose tissue. They showed that glucose is metabolized to CO2 through both the glycolytic and pentose pathways from the 2-cell to the late blastocyst. Evidence was presented which suggests that the relative activities of these two pathways fluctuate during the preimplantation period, with peak relative fluxes through the pentose pathway at-the 2-cell and compacted morula stages and a low relative flux at the late blastocyst. A very recent study on the relative activities of the glycolytic pathway and the pentose-phosphate pathway in the pig suggests that the rate of glucose utilization is low up to the time of compaction, after which it rapidly increases. Prior to compaction the glucose is used predominantly through the pentose-phosphate pathway, while after compaction it is used almost exclusively by glycolysis. The technique of starvation and refeeding to locate- rate-limiting steps in the metabolic pathways in pre-implantation mouse embryos was introduced by Barbehenn et al. (1974). The principle of this method is to compare the levels of certain metabolites in the glycolytic and citric acid pathways in single embryos at different stages of development in the~presence and absence of glucose or pyruvate. Fig. 9 compares the effect of starvation and refeeding glucose on the concentrations of glucose-6-phosphate and fructose-1,6- bisphosphate in the 2-cell, 8-cell, morula and blastocyst stages of mouse 29i . .

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preimplantation development. Following starvation the concentrations of glucose-6-phosphate fell in all developmental stages. On refeeding glucose the concentration of the metaboli.te was restored to basal levels or above within five minutes. In contrast starvation had little effect on the concentrations of fructose-l,6-bisphosphate at all developmental stages. Refeeding glucose had no effect on the concentration of metabolite at the 2- and B-ce11 stages. However, at the moru~a and blastocyst stages the concentration of the metabolite increased significantly. The prompt rise in the concentration of glucose-6-phosphate on refeeding glucose at the 2-cell stages suggests that neither the transport of glucose into the b~astomeres nor the activity of hexokinase are rate-limiting steps that prevent glucose from supporting development of this early stage of development. The observations that the concentration of fructose-l,6-bisphosphate is unaffected by starvation or - refeeding of glucose suggest that the activity of the enzyme phosphofructokinase is minimal. The fact that the concentration of the metabolite increases on refeeding glucose in the 8-cell, morula and blastocyst stages suggests that the activity of the enzyme increases after the two-cell stage of development. The fact that the response to refeeding increases in amount with development provides evidence that the activity of the enzyme is increasing. These results provide strong evidence that phosphofructokinase is a rate-limiting enzyme that controls the utilization of glucose by the i preimplantation mouse embryo. In a further study, Barbehenn et al. (1978) compared the effect of starvation and refeeding glucose and glucose plus pyruvate on seven metabo~ites in single 2-cell, 8-cell, moru~a and b~astocyst stages of mouse development. The metabolites were glucose-6-phosphate, fructose-6-phosphate, - 292

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fructose-1,6-bisphosphate, citrate, isocitrate, alpha oxoglutarate and palate. The results are shown in Fig. 10. Two major effects of pyruvate were observed. First, glucose caused an increase in fructose-1,6-bisphosphate above resting levels; particularly from the 8-cell stage onwards the addition of pyruvate strongly inhibited the accumulation of the metabolize. Clearly the presence of pyruvate strongly inhibits the conversion of fructose-6-phosphate to fructose- 1,6-bisphosphate. Secondly, glucose had no effect on the concentrations of citrate, isocitrate, alpha-oxoglutarate and palate. When pyruvate was also present, the concentrations of citrate, isocitrate and alpha-oxoglutarate were raised at all four stages of development. The concentration of malate was not raised by the presence of pyruvate. Barbehenn et al. (1978) suggest that there is a rate-limiting step between alpha-oxoglutarate and palate which regulates fluxes through the citric acid cycle. CONTROL OF GLYCOLYSIS From an evolutionary point of view the glycolytic pathway is one of the ancient, conserved metabolic pathways (Boiteux and Hess, 1981; Fothergill- Gilmore, 1986), being used by both aerobic and anaerobic organisms. The pathway consists of a chain of three classes of enzyme that serve regulatory functions (Fig. 11). These classes are interconvertible enzymes, cooperative enzymes and Michaelis-Menten enzymes [see Boiteux and Hess (1981) for a review]. The two interconvertible enzymes, glycogen phosphorylase and pyruvate dehydrogenase, are placed at the beginning and end of the chain. The activities of these enzymes are controlled by chemical interconversion to different molecular species by processes which occur at relatively slow rates. The enzymes control the movement of metabolites into and out of the pathway. 2g3

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Glycogen phosphorylase controls the entry of glucose-6-phosphate from glycogen stores. Pyruvate dehydrogenase controls the channeling of acetycoenzyme A to the citric acid cycle or the fatty acid cycle. Two cooperative enzymes - phosphofructokinase and pyruvate kinase - are also located at either end of the glycolytic pathway. The activites of these cooperative enzymes, in contrast to the interconvertible enzymes, are modified at fast rates through allosteric control mechanisms.' They also regulate the exit of metabolites from the glycolytic pathway. Phosphofructokinase controls the transfer of metabolites from the glycolytic pathway into lipid synthesis pathways, through the production of dihydroxyacetone phosphate. Pyruvate kinase regulates the exit of substrates from the~glycolytic pathway into pathways that result in the synthesis of amino acids and related compounds. The Michaelis-Menten enzymes, if they are in a simple chain, control each othe'r'sequential'ly through substrate concentrations (Crabtree and Newsholme, 1987~. However, if they are present at an intersection with 'another metabolic pathway, they may also participate in a control point. As an example Boiteux and Hess (1981) use the enzyme giceraldehyde phosphate dehydrogeniase, which is located at the intersection of the glycolytic and pentose-phosphate pathways. Fluxes through this control point and their direction will depend on the concentrations of two substrates and the redox potential of the cofactor nicotinamide adenine dinucleotide. Our knowledge about th'e control points of the glycolytic pathway ' in preimplantation mammalian embryos is still s-parse. Among the interconvertible enzymes there is evidence that the amount of glycogen phosphorylase is low unti] the morula stage in the mouse (Hsieh et al., 1979). These investigators, however, assumed that their'assay method for the enzyme measures both the 2g4

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active a and inactive b forms of the enzyme. If by any chance the b form in the embryo resembles the b form found in 'the liver,'the total amount of enzyme Would be grossly underestimated. We do not know, therefore, whether this enzyme is controlled by being switched-from an inactive'to active form at the module stage.'By this stage-there is~also a marked droptin'the concentration of glycogen synthetase:(Stern, 1970), and glycogen concntrations in the embryo fall (Stern and Biggers, 1968) (Fig. 12). At present we have no information on the regulation of the other interconvertible enzyme, pyruvate dehydrogenase, in preimplantation embryos. ~ Most of our information on the regulation of the glycolytic pathway in the preimplantation embryo concerns the cooperative enzyme phosphofructokinase. This enzyme is controlled allosterically by several factors - concentrations of adenylates, citrate, protons and phosphate (Wu and Davis, 1981). The role of the adenylates (ATP, ADP, AMP) in the regulation of the glycolytic pathway in mouse preimplantation embryos-has been discussed in terms of Atkinson's energy charge (Atkinson, i968; Biggers, 1976; Biggers and Borland, 1976). The energy charge of the adenylate pool is defined as half of the number of anhydride-bound phosphate groups per adenine moiety, which is given by; ' ' ' - Energy charge = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]) If all of the adenylate pool is in the form of ATP, tine' energy charge equals one, and if all of the pool is in the form of-AMP, the energy charge is~zero. A high energy charge favors the biosynthetic pathways, while a low energy charge favors energy-generating pathways that maintain the levels of ATP. Further details on Atkinson's energy charge are given by Reich and Sel'kov (1981). It has been known since the work of Quinn and Wales (1971) that the 295

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mouse zygote has a high ATP/ADP ratio and that this ratio falls as cleavage proceeds. As a result of developing a method to estimate ATP, ADP and:AMP simultaneously on single embryos, Leese et al. (1984) have been able to estimate both the ATP/ADP ratio and the energy charge at different stages of mouse preimplantation development (Table I). The results show both a high ATP/ADP ratio and a high energy charge in the 1-cell mouse embryo and that both parameters fall with development. The high energy charge at the beginning of development may allosterical~y inhibit phosphofructokinase and cause a restriction in the use of glucose by early mouse embryos. It is also known that citrate is present in high concentrations in preimplantation mouse embryos (Barbehenn et al., 1974). These high concentrations of citrate could also reinforce the inhibition of phosphofructokinase. However, the effects of citrate on phosphofructokinase depend on the pH and Pj concentration. Until the interactions between the effects of these factors is studied, the physiological role of phosphofructokinase in the regulation of glyco~ysis in preimplantation mouse embryos will be obscure. Little is known about the other cooperative enzyme in the g~yco~ytic pathway - pyruvate kinase. However it has been suggested recently that this enzyme is rate limiting in the use of glucose by the cow blastocyst (Rieger and Guay, 1988). i CONCLUSION The evidence assembled in this review establishes that the metabolism of the mature oocyte differs from that of adult cells. The modifications, which are presumably produced during oogenesis, are later restored by the blastocyst stage. In particular the glycolytic pathway seems affected, so that glucose utilization is restricted over a period spanning fertilization. As yet our understanding of the rate-limiting processes which are operative in the oocyte 29o

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and early cleavage stages is limited and explanations are sought in terms of the well known control points. These explanations, however, may not be the final answer, since recent work has emphasized other mechanisms which may control glycolysis. These involve the role of the cellular structure of the cytoplasm, such as the cytoskeleton (Swezey and Epel, 1986; Masters et al., 1987), and metabolite-modulated dynamic enzyme associations (Ovadi, 1988). Future research should keep these alternative possibilities in mind. 297 -

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REFERENCES Anderson, E., Condon, W. and Sharp, D. 1970. A study of oogenesis and early embryogenesis in the rabbit, OrYctolaqus cunicu~us, with special reference to the structural changes of mitochondria. d. Morph. 130: 67- 92. Atkinson, D.E. 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochem. 7:4030-4034. Barbehenn, E.K., Wales, R.G. and Lowry, O.H. 1974. The explanation for the blockade of g~ycolysis in early mouse embryos. Proc. Nat. Acad. Sci. U.S.A. 71: 1056-1060. Barbehenn, E.K., WaJ es, R.G. and Lowry, O.H. 1978. Measurement of metabolizes in single preimplantation embryos: a new means to study metabolic control in early embryos. J. Embryol. exp. Morph. 43: 29-46. Bavister, B.D., Boatman, D.E., Leibfried, By., Loose, M. and Vernon, M.W. 1983. Fertilization and cleavage of rhesus Bio1. Reprod. 28: 983-999. monkey oocytes in vitro. Diggers, d.D. 1976. Bioenergetic aspects of fertilization and embryonic development of the mouse. In: Ebling, F.~.G. and Henderson, I.W. eds. Biological and Clinical Aspects of Reproduction. Excerpta-Medica, Amsterdam-Oxford, pp 128-133. Diggers, d.D. 1987. Pioneering mammalian embryo culture. In: The Mammalian PreimpJantation Embryo. ed. B.D. Bavister, pp.-22. Plenum Press, New York. Biggers, d.D. and Borland, R.M. 1976. Physiological spects of growth and development of the preimplantation mammalian embryo. Ann. Rev. Physio1. 38: 95-~19. Biggers, d.D. and Stern, S. 1973. Metabolism of the preimpJantation embryo. Adv. Reproductive Physiology 6: I-59. Biggers, d.D., Gardner, D.K., and Leese, H.~. 1989. Control of carbohydrate metabolism in preimpJantation mammalian embryos. In: Regulation of Growth in Development, eds. T.Y.Rosenb~um and S. Heyner, CRC Press, Boca Raton, FL (in press). Biggers, d.D., Whittingham, D.G. and Donahue, R.P. 1967. The pattern of energy metabolism in the mouse oocyte and zygote. Proc. Nat. Aced. Sci., USA, 58: 560-567. Bishop, D.W. 1956. Oxygen concentrations in the rabbit genital tract. Proc. 3rd. lot. Congress Animal Reprod. Cambridge. Section I, 53-55. 2ge

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