Studies on the development of mouse embyros <i>in vitro</i>. II. The effect of energy source

Brinster, Ralph L.

doi:10.1002/jez.1401580106

Cited by 272 publications

(109 citation statements)

References 11 publications

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“…The basic medium used was a modified Krebs-Ringer bicarbonate solution containing 5·56 mM glucose, 1 mg/ml bovine serum albumin, 60 Ilg/ml penicillin, and 50llg/ml streptomycin (Brinster 1965).…”

Section: (A) Collection Culture and Recovery Of Embryosmentioning

confidence: 99%

“…Another major difference between the early mouse and rabbit embryo is the energy requirements for in vitro culture. Pyruvate or lactate is required by two-cell mouse embryos for development and glucose cannot support development before the eight-cell stage (Brinster 1965;Brinster and Thomson 1966). The two-cell rabbit embryo, however, can develop in vitro for 48 hr without an exogenous energy source but with low concentrations of amino nitrogen in the culture medium, the addition of pyruvate or lactate increases the number of embryos developing (Brinster 1970).…”

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confidence: 99%

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The in Vitro Metabolism of [U-14C]Glucose by the Preimplantation Rabbit Embryo

Quinn¹,

Wales²

1973

Aust. Jnl. Of Bio. Sci.

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The incorporation of glucose carbon into various components of embryos and culture medium was studied throughout the preimplantation period of development in the rabbit embryo.During the first 3 days after ovulation, embryos were able to develop for 24 hr in a simple chemically defined medium containing glucose and serum albumin. After blastocyst formation, this medium could maintain normal morphological appearance of day 4, day 5, and day 6 blastocysts for at least 3-4 hr.The uptake of glucose carbon by the embryo increased as development progressed, with substantial increases in the incorporation of glucose occurring after blastocyst formation. A considerable amount of the label incorporated was present in the embryos as the parent substrate, indicating that the uptake of glucose was greater than its conversion to other metabolites. Nevertheless, throughout development glucose carbon was incorporated into a wide variety of the carbon pools of the embryo, such as acidic and basic acid-soluble compounds, proteins, and lipids. Lactate, pyruvate, and acetate also accumulated in the culture medium. The rate of protein synthesis declined in relation to the increase in cell number during development and may reflect the requirements of the rabbit embryo for exogenous protein and amino acids for blastocyst expansion.A considerable amount of the incorporation of substrate carbon into day 6 blastocysts was due to the accumulation of lactate, pyruvate, acetate, amino acids, and the parent substrate within the blastocoel fluid. The higher concentration of metabolites, such as carboxylic acids and amino acids, in the blastocoel fluid than in the incubation medium indicated that there was some active mechanism for retaining these compounds within the blastocyst. The blastocyst tissue also contained labelled metabolites such as lactate, citrate, and malate as well as the amino acids glutamate, alanine, and aspartate. The accumulation of glucose carbon in these metabolites indicates that glucose is metabolized in the rabbit embryo by the operation of the Embden-Meyerhof pathway and tricarboxylic acid cycle.

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Section: (A) Collection Culture and Recovery Of Embryosmentioning

confidence: 99%

mentioning

confidence: 99%

The in Vitro Metabolism of [U-14C]Glucose by the Preimplantation Rabbit Embryo

Quinn¹,

Wales²

1973

Aust. Jnl. Of Bio. Sci.

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“…Significantly, lactate is only able to support development from the two-cell stage (4), and glucose could support development from the eight-cell stage (5). Interestingly, mouse zygotes can metabolize both pyruvate and lactate (6,7) even though cleavage only occurs in the presence of pyruvate.…”

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confidence: 99%

Mitochondrial Malate-Aspartate Shuttle Regulates Mouse Embryo Nutrient Consumption

Lane

Gardner

2005

Journal of Biological Chemistry

107

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Pyruvate has been considered the sole substrate that can support development of the mouse zygote to the two-cell stage, with lactate able to support development from the two-cell stage. This study has determined for the first time that mitochondrial reducing equivalent shuttles regulate metabolism in the early embryo. Activity of the malate-aspartate shuttle was found to be essential for the metabolism of lactate in the two-cell embryo. Furthermore, the inability of the mouse zygote to use lactate as an energy source was a result of a lack of malate-aspartate shuttle activity. The mRNA for the four enzymes for shuttle activity were detected at all stages of development. It was determined that aspartate was a rate-limiting factor in the activity of the malate-aspartate shuttle in mouse zygotes probably due to the high K m of the cytoplasmic aspartate aminotransferase. Addition of high concentrations of exogenous aspartate to the culture medium enabled mouse zygotes to utilize lactate in the absence of pyruvate and develop normally to the blastocyst stage as well as produce normal viable offspring. This study determined that the malateaspartate shuttle is a key regulator of embryo metabolism and therefore viability and is the first report that mouse zygotes can develop normally to term in the absence of pyruvate.Studies on the preimplantation mouse embryo have indicated that the zygote has an absolute requirement for pyruvate to complete the first cleavage division (1-3). Significantly, lactate is only able to support development from the two-cell stage (4), and glucose could support development from the eight-cell stage (5). Interestingly, mouse zygotes can metabolize both pyruvate and lactate (6, 7) even though cleavage only occurs in the presence of pyruvate. While this has been documented for more than 30 years, there is no explanation for this unusual pattern of metabolism at the zygote nor is it known what changes occur in the embryo that enable the two-cell embryo to use lactate to support development.Energy metabolism in cells is compartmentalized to either the cytoplasm or the mitochondria. Pyruvate is transported into the mitochondria by specific transport proteins and is metabolized within the mitochondria. In contrast, lactate is converted to pyruvate within the cytosol with the concomitant conversion of NAD ϩ to NADH. Cells must maintain a balance of metabolic intermediates between the cytosol and the mitochondria to enable lactate metabolism to continue. An inability to maintain this balance results in impairment of either mitochondrial and/or cytoplasmic metabolism. In addition to many enzymes existing in either the cytoplasm or mitochondria, the pyridine nucleotides (NAD ϩ , NADH, NADP ϩ , and NADPH) are also compartmentalized between the cytoplasm and the mitochondria. Intact mitochondria are impermeable to NADH (8). Therefore to transfer NADH across the mitochondrial membrane for oxidation and ATP production and for the regeneration of cytoplasmic NAD ϩ , there is an indirect pathway involving th...

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“…Although acetate, like citrate, will not support development of the two-cell mouse embryo (Brinster 1965b), its uptake by the embryos at this stage of development differs considerably from that of citrate. Determinations by method 1 showed that significant amounts of substrate carbon accumulated in embryos from acetate and the extent of this accumulation depended on the concentration in the medium.…”

Section: Discussionmentioning

confidence: 89%

Further Studies on the Accumulation of Energy Substrates by Two-cell Mouse Embryos

Wales¹,

Whittingham²

1974

Aust. Jnl. Of Bio. Sci.

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The accumulation of substrate carbon by two-cell mouse embryos after incubation for 30 min in isotopically labelled substrates was compared by two different methods. The embryos were centrifuged either through an isotonic sucrose solution to free them from radioactive medium before counting or in radioactive medium in a capillary tube when substrate uptake was calculated from the relative counts in packed embryos and incubation medium.Measurements of the accumulation of substrate carbon from [1_ 14 C]pyruvate gave the same values by both methods, but the accumulation of C-1 of lactate was much greater after centrifuging the embryos in a capillary tube. Fractionation of the intracellular isotope accumulated from lactate showed that the increased radioactivity accumulated was associated with the parent substrate. A similar difference was found in the accumulation of C-1 of acetate with the two methods of recovery, indicating that both substrates diffuse freely between the medium and embryo. Incorporation of acetate into two-and eight-cell mouse embryos was similar. No intracellular substrate carbon was accumulated from [1,[5][6][7][8][9][10][11][12][13][14] C]citrate by two-cell mouse embryos during 30 min incubation.

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Studies on the development of mouse embyros in vitro. II. The effect of energy source

Cited by 272 publications

References 11 publications

The in Vitro Metabolism of [U-14C]Glucose by the Preimplantation Rabbit Embryo

The in Vitro Metabolism of [U-14C]Glucose by the Preimplantation Rabbit Embryo

Mitochondrial Malate-Aspartate Shuttle Regulates Mouse Embryo Nutrient Consumption

Further Studies on the Accumulation of Energy Substrates by Two-cell Mouse Embryos

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