Metabolic Pathway Activity

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1. SIGNIFICANCE OF METABOLISM IN EMBRYO DEVELOPMENT

The early development of the mammalian embryo is directed by its inherent genetic programming and affected by its environment. Both the genetic program and the environment act as stimuli to the early embryo to induce developmental responses, initially in its intracellular biochemical activity, and ultimately in cell division, differentiation, and function. Techniques for manipulating gene expression and development, such as gene transfer, cloning, IVF, ICSI, and culture, similarly act as stimuli to induce the desired biochemical and developmental responses.

Whatever the source of the stimulus, genetic or environmental, natural or artificial, one or more intracellular processes are required to produce a developmental response. As illustrated in Figure 9.1, the pathway between the stimulus to a cell and its response can include any or all of transcription, translation, post-transitional modification, and export, activation, and function of an enzyme or other protein. Directly or indirectly, all of these processes rely on energy metabolism to provide cellular energy in the form of ATP, reducing equivalents in the form of NADH and NADPH, and precursors for the synthesis of macromolecules.

Consider, for example, the transcription of a 1200-nucleotide strand of mRNA. This would require the anaerobic metabolism of 5250 molecules of glucose for the production of approximately 10,500 ATP molecules for nucleotide synthesis and a further 1200 molecules of glucose to provide the ribose moieties. A single translation of that mRNA strand into a 400-amino-acid polypeptide would require 1200 ATP molecules, representing the anaerobic metabolism of 600 molecules of glucose. Large amounts of ATP are similarly required for DNA and lipid synthesis during cell division and for the activity of the sodium-potassium ATPase pump during formation of the blastocoele.

The energy metabolism of the early mammalian embryo can be studied indirectly by culture in varying concentrations and combinations of energy substrates. A more direct approach is to measure the disappearance of substrates from, and the release of metabolic products into, the medium. The breakdown or incorporation of radiolabeled substrates can be used to measure the activity of specific metabolic pathways, and the activity

Figure 9.1. The mechanisms involved in the response of a cell to a stimulus.

of specific enzymes can be measured in broken-cell preparations (see Rieger [1]). The technique described here is used to measure the production of 14CO2 and 3H2O from 14C- and 3H-labeled energy substrates by intact embryos, and the discussion will be limited to studies that have used that approach. More comprehensive reviews of embryo metabolism can be found elsewhere (2-7).

2. LABELED SUBSTRATES USED TO EVALUATE ENERGY METABOLISM PATHWAYS

Figure 9.2 shows the relationships among the carbon atoms of glucose, pyruvate, and lactate. Glucose can be metabolized through the pentose-phosphate pathway (PPP) to produce ribose, with a concomitant release of carbon-1 (C-1) as CO2. When metabolized through the Embden-Meyerhof pathway (EMP) of anaerobic glycolysis, each molecule of glucose produces two molecules of pyruvate. Glucose C-1 and C-6 become pyruvate C-3, glucose C-2 and C-5 become pyruvate C-2, and glucose C-3 and C-4 become pyruvate C-1. Pyruvate and lactate are interconvertible, and the carbons retain the same numbers. Pyruvate C-1 (from glucose C-3 and C-4) is released as CO2 in the conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase. The two carbons of acetyl-CoA (from glucose C-1, C-2, C-5, and C-6, or pyruvate C-2 and C-3) are released as CO2 after two or more cycles through the mitochondrial Krebs cycle of oxidative metabolism. The hydrogen on glucose C-5 is released as H2O in the conversion of 2-phosphoglycerate to phospho-enolpyruvate, the second to last step in the EMP. Gluta-mine is converted to glutamate and then to a-ketoglutarate, and its carbon and hydrogen atoms are released as CO2 and H2O in the Krebs cycle and electron transport chain, respectively.

Given these relationships, it is possible to evaluate the activity of the PPP, the EMP, and of mitochondrial oxidative metabolism by measuring the production of 14CO2 and 3H2O from 14C- and 3H-labeled energy substrates. The production of 3H2O from [5-3H]glucose is a simple measure of the activity of the EMP and can be confirmed using iodoacetate, an inhibitor of aldolase.

The production of 14CO2 or 3H2O from 14C- or 3H-labeled glutamine is a measure of the activity of the Krebs cycle and can be confirmed using 2,4-dinitrophenol, an indirect stimulator of the cycle, malonate, an inhibitor of the cycle; or inhibitors of the electron transport chain such as antimycin-A, rotenone, sodium azide, and cyanide. The activity of the Krebs cycle can also be measured using [6-14C]glucose, [2-14C]pyruvate

Intracellular Metabolism
Figure 9.2. The fate of the carbon atoms of glucose, pyruvate, and lactate metabolized through the Embden-Meyerhof pathway (EMP), the pentose-phosphate pathway (PPP), and the Krebs cycle (KC). Broken lines indicate two or more reactions.

or, [2-14C]lactate, and [1-14C]pyruvate or [1-14C]lactate can be used to evaluate the activity of pyruvate dehydrogenase.

The evaluation of the PPP is more difficult. Because glucose C-1 is released as CO2 in both the PPP and Krebs cycle, whereas C-6 is released as CO2 only in the Krebs cycle, the ratio of 14CO2 produced from [1-14C]glucose to that produced from [6-14C]glucose (the C-1/C-6 ratio) has been used as a measure of the relative activity of the PPP. Due to the possibility of recycling of the products of the PPP to fructose and rearrangement of the carbon atoms, this approach is highly questionable (8, 9). However, if the ratio is very high (10:1 or more), it is reasonable to assume that production of 14CO2 from [1-14C]glucose is a reliable measure of PPP activity. This can be confirmed using brilliant cresyl blue or phenazine ethosulfate, stimulators of the PPP.

The production of 14CO2 from [U-14C]glucose may reflect the activity of any or all of the PPP, pyruvate dehydrogenase, and the Krebs cycle and has been used as a measure of the total metabolism of glucose. The production of 14CO2 from [U-14C]lactate similarly reflects the activity of both pyruvate dehydrogenase and the Krebs cycle.

To my knowledge, the first report of the metabolism of radiolabeled substrates by the mammalian embryo was that of Fridhandler (10), who adapted the manometric Cartesian diver to measure the production of 14CO2 from [1-14C]glucose or [6-14C]glucose by rabbit embryos. The C-1/C-6 ratio was high before the blastocyst stage (as much as 20:1), suggesting that glucose metabolism was mainly by the PPP. After formation of the blastocyst, the ratio decreased to approximately 1:1, suggesting that glucose was metabolized through the Embden-Meyerhof pathway and the Krebs cycle. In the mouse embryo, the production of 14CO2 from [U-14C]glucose increased almost 5-fold between the unfertilized and fertilized ovum and 100-fold from the unfertilized ovum to the blastocyst stage (11). The C-1/C-6 ratio was significantly >1 at all stages, but the maximum was 2.33, at the two-cell stage. A similar study of the rabbit embryo (12) showed that the production of 14CO2 from [U-14C]glucose was not different between the unfertilized and fertilized ovum but increased 8000-fold by the day-6 blastocyst stage. The C-1/C-6 ratio was high until the morula stage and then dropped to approximately 1.5 thereafter. In addition to defining the basic pattern of glucose metabolism during the early development of mammalian embryos, these studies demonstrated that glucose metabolism differs significantly between embryos of different species.

Menke and McLaren (13) compared the production of 14CO2 from [U-14C]glucose by mouse blastocysts that had been cultured from the eight-cell stage or recovered from the uterus. They found that glucose metabolism by cultured blastocysts was generally greater that that by the uterine blastocysts and was increased by including fetal calf serum in the culture medium. These results demonstrated that the prior environment of the embryo can affect glucose metabolism.

The uptake of [U-14C]malate, a Krebs cycle intermediate, and its metabolism to 14CO2, was significantly greater by eight-cell mouse embryos than by two-cell embryos, suggesting that the cell membrane becomes permeable to malate between the first and third cleavage divisions (14). Conversely, the two-cell mouse embryo could produce 14CO2 from [1-14C]pyruvate, [2-14C]pyruvate, [1-14C]lactate, and [2-14C]lactate, and the amount of 14CO2 produced from C-1 was two- to threefold greater than that produced from C-2 (15). These results demonstrated that the Krebs cycle was active in the two-cell mouse embryo but that a significant portion of the amount of pyruvate that was converted to acetyl-CoA did not enter the Krebs cycle. It is important to note that the observation that little glucose carbon passes through to the Krebs cycle in the two-cell mouse embryo (11) is not inconsistent with the observation that 14CO2 is produced from [2-14C]pyruvate, because the intracellular concentration of pyruvate being taken up from the medium is likely much greater than that arising from the metabolism of glucose. Further reports of the energy metabolism of mouse and rabbit embryos continued sporadically throughout the 1970s and are covered elsewhere (2-7).

Interest in the energy metabolism of early embryos was stimulated by the advent of commercial embryo transfer in cattle and by the observation that the uptake of glucose by day-10 and day-11 cattle embryos was directly related to their development to term after transfer to recipients (16). Unfortunately, the method of measuring glucose uptake was not sensitive enough to be used for day-7 embryos, when cattle embryos are commonly transferred. However, it was possible to measure the production of 14CO2 from 14C-labeled glucose and lactate by individual in-vivo-produced, day-7 cattle embryos, and this offered a possible approach to evaluating the viability of cattle embryos before transfer (4).

O'Fallon and Wright (17) developed a technique in which individual mouse embryos were suspended in a droplet of medium above a NaOH trap to collect 14CO2 produced from 14C-labeled substrates and 3H2O from [5-3H]glucose. Rieger and Guay (18) initially developed a technique in which an individual embryo was contained in a droplet of medium in a small well within a larger well containing 25 mM NaHCO3. At the end of the incubation, NaOH was added to the bicarbonate to convert it to carbonate for counting. Rieger et al. (19) subsequently showed that the metabolism of one 14C-labeled and one 3H-labeled substrate by an embryo could be measured simultaneously. The hanging drop technique of O'Fallon and Wright (17) was then modified to use a 25 mM NaHCO3 exchange reservoir in place of the NaOH trap (20), as described below. If an NaOH trap is used, the culture environment is completely free of CO2 and bicarbonate during the incubation, which is disadvantageous because CO2/bicarbonate favors embryo development by mechanisms unrelated to pH (7). Conversely, the CO2/bicarbon ate environment is maintained by the bicarbonate exchange reservoir, which satisfies the suggestion that "meaningful measurements are made only under conditions that support embryo development" (7).

In addition to determining the general pattern of energy metabolism during early development of the cattle embryo (20), my colleagues and I have shown that glucose metabolism is related to the sex of the early cattle embryo (21) and viability following cryopreservation of cattle blastocysts (22) and is increased by early exposure to glucose in culture (23). We have also studied the patterns of energy metabolism in cattle oocytes during in vitro maturation (24, 25), in horse (26) and hamster (27) embryos, and in bird sperm (28).

3. OVERVIEW OF THE METABOLIC MEASUREMENT ASSAY

The metabolism assay apparatus is show in Figure 9.3. Individual embryos (or oocytes) are taken up in 2 ml of culture medium, with or without metabolic stimulators, inhibitors, or other test substances, and placed in the cap of a sterile 2.0-ml screw-cap microvial (Sarstedt Inc., Montreal, Canada). To this is added 2 ml of culture medium containing one 14C-labeled substrate or one 3H-labeled substrate, or a mixture of one 14C-labeled substrate and one 3H-labeled substrate. The vial contains 1.5 ml of 25 mM NaHCO3 that has been equilibrated with the gas mixture, to act as an exchange reservoir for the 14CO2 and 3H2O produced during the incubation period. The vial is flushed with the gas mixture (usually 5% CO2, 5% O2, 90% N2) just before the caps are fitted. At the end of the incubation period, the embryos are recovered and returned to culture. The NaHCO3 is mixed with NaOH and scintillation fluid, and counted in a scintillation counter to determine the content of 14CO2 and/or 3H2O.

Figure 9.3. Apparatus used for the measurement of the metabolism of individual embryos. The embryo is contained in the 4-ml suspended droplet of culture medium. (Redrawn from Rieger et al. [20], with permission.)

4. CULTURE MEDIUM

As noted above, the technique is designed to use a bicarbonate-based culture medium. The basic medium is usually that used for routine embryo culture in the laboratory, supplemented with 10-20 mM HEPES to maintain the pH of the incubation droplets while they are being prepared. The pH in the microdroplets can be monitored by including phenol red in the medium.

5. PREPARATION OF THE RADIOLABELED SUBSTRATES

The labeled substrates can be purchased from the major suppliers (e.g., Dupont, Amersham). In addition to the nuclide and the position of the label, the preparations differ in their specific activity, concentration, and solvent. In general, crystalline preparations of high specific activity are the most convenient, but they are not available for all substrates. They are often supplied in water or water:ethanol at relatively low concentrations and must be dried and then resuspended in the basic culture medium. A simple approach to drying is to insert two syringe needles into the vial and pass dry N2 into the vial through one needle, leaving the other needle open for the gas to escape. The end of the needles should be at least 1 cm above the surface of the liquid, and the gas flow rate should be only sufficient to create a dimple in the surface of the liquid. The vial can be placed into a hot water bath to hasten drying. Alternatively, the labeled substrates can be dried using a vacuum evaporator.

The concentration of the labeled substrate in the culture medium depends on the requirements of the experiment, the rate of metabolism of the substrate by the embryo, and the specific activity of the labeled substrate. Optimally, the concentration of labeled substrate should be just enough to result in the production an amount of 14CO2 or 3H2O that can be reliably measured, while the total amount of labeled and unlabeled substrate should not be significantly greater than that normally used in the culture medium. In 4 ml of medium, 0.5 mCi of [5-3H]glucose with a specific activity of 15 Ci/mmol would add only 0.01 mM to the total glucose concentration, a negligible amount. Conversely, 0.5 mCi of [1-14C-]glucose with a specific activity of 50 mCi/mmol would yield 2.5 mM. If the basic medium contained 5.55 mM glucose, then the total concentration would be 8.05 mM. This might be too great a glucose concentration to be acceptable, and less [1-14C]glucose would have to be used, or the concentration of unlabeled glucose in the basic medium would have to be reduced. At the extreme, 0.5 mCi of [2-14C]pyruvate with a specific activity of 10 mCi/mmol would yield 12.5 mM, a far greater concentration than that normally used in embryo culture media. Fortunately, the metabolism of [2-14C]pyruvate is relatively high in early embryos, and 0.05 mCi or less is usually sufficient to produced measurable amounts of 14CO2.

As an example, suppose that the metabolism of [1-14C]glucose (specific activity = 50 mCi/mmol) is to be measured in a 4-ml droplet, that the base medium contains 1.0 mM glucose, and that the final total concentration (labeled plus unlabeled) of glucose is to be 1.5 mM. A 4-ml droplet of the base medium would contain 4000 pmol of unlabeled glucose, and the total required glucose would be 6000 pmol. The difference of 2000 pmol would require 0.1 mCi (2.22 x 105 dpm) of [1-14C]glucose. The radiolabeled substrate solution would therefore have to be made to contain 2.22 x 105 dpm/2 ml.

6. PREPARATION OF THE EMBRYOS

Depending on the experimental protocol, the embryos may be exposed to test procedures or conditions before the metabolic measurement. For example, an experiment might involve comparing the metabolic activity of embryos that have been cultured under different culture media or conditions or comparing activity between fresh and frozen-thawed embryos. Aside from this, the embryos require no special treatment except that they are normally passed through several washes of culture medium and sorted into experimental groups as appropriate. Each group is then transferred into a final wash just before being placed, individually, into the caps of the metabolic measurement vials. The final wash may also contain metabolic stimulators or inhibitors, which must be at twice the desired final concentration.

7. PREPARATION OF THE METABOLIC MEASUREMENT VIALS

To avoid drying and loss of CO2 from the droplets, the vials should be prepared in batches of a maximum of 10. A single embryo is taken up in 2 ml of the final wash and placed in the cap of the incubation vial. To this is added 2 ml of the mixture of radiolabeled substrates, to produce a total volume of 4 ml. The vials are then loaded with 25 mM NaHCO3 using the apparatus shown in Figure 9.4. The delivery tubing is cut to length to have a volume of 1.5 ml. The needle at the end of the delivery tubing is placed in the incubation vial, and the 50-ml culture tube is inverted to fill the tubing with NaHCO3. When the bicarbonate solution reaches the end of the delivery tubing, the culture tube is turned upright, and the gas forces the NaHCO3 solution out of the tubing into the vial. The needle at the end of the delivery tube is left in the vial and the cap held against the needle for a few seconds to flush the vial with the gas mixture. The needle is then withdrawn and the cap screwed onto the vial.

Vial Flushing Remove

Figure 9.4. The apparatus and procedure used to load the metabolism assay vials with 25 mM NaHCO3. (a) The delivery tubing is cut to length to have a volume of 1.5 ml. The needle at the end of the delivery tubing is placed in the incubation vial. (b) The 50 ml culture tube is inverted to fill the tubing with NaHCO3. (c) When the bicarbonate solution reaches the end of the delivery tubing, the culture tube is turned upright, and the gas forces the NaHCO3 solution out of the tubing, into the vial. (d) The needle at the end of the delivery tube is left in the vial and the cap held against the needle for a few seconds to flush the vial with the gas mixture. The needle is then withdrawn and the cap screwed onto the vial.

Figure 9.4. The apparatus and procedure used to load the metabolism assay vials with 25 mM NaHCO3. (a) The delivery tubing is cut to length to have a volume of 1.5 ml. The needle at the end of the delivery tubing is placed in the incubation vial. (b) The 50 ml culture tube is inverted to fill the tubing with NaHCO3. (c) When the bicarbonate solution reaches the end of the delivery tubing, the culture tube is turned upright, and the gas forces the NaHCO3 solution out of the tubing, into the vial. (d) The needle at the end of the delivery tube is left in the vial and the cap held against the needle for a few seconds to flush the vial with the gas mixture. The needle is then withdrawn and the cap screwed onto the vial.

It is possible to use snap-cap Eppendorf or other small vials rather than the screw-top Sarstedt vials and to simply pipette the gassed bicarbonate into the vials (29), rather than using the bicarbonate delivery apparatus shown in figure 9.4. However, the Sarstedt vial caps have the advantage of having an inner well for the droplets, and securing the screw-caps is less likely to disturb the droplets than is securing a snap-cap. Moreover, Sarstedt sells a rack that holds the vials from turning so that the caps can be screwed on with one hand. The bicarbonate delivery apparatus is very cheap and easy to construct and is convenient to use.

In addition to the vials containing the embryos, each assay must include a minimum of three total count vials and a minimum of three sham vials for each treatment group. Total count vials are prepared by placing 2 ml of the solution of radiolabeled substrates into the cap of a vial containing 1.5 ml of 25 mM NaHCO3. The cap is screwed onto the vial, and the vial is inverted to mix the radiolabels with the bicarbonate. Sham vials are used to correct for counting background, chemiluminesence, spontaneous degradation of the labeled substrates, microbial contamination, and any other source of nonspecific counts. They are prepared exactly as for the vials containing the embryos, except that the droplets contain 2 ml of the final wash and 2 ml of the solution of radiolabeled substrates, but no embryo.

8. INCUBATION

Once capped, the vials should be held at the appropriate temperature, but there is no need for a humidified or gassed incubator. However, in practice, it is more convenient to culture them in the same incubator used for routine culture. We routinely culture the vials for 3 h, which is sufficient to produce measurable quantities of 14CO2 or 3H2O from labeled glucose, pyruvate, and glutamine and to ensure equilibration of the labeled products between the culture drop and the bicarbonate reservoir (see Figure 9.5).

9. TERMINATING THE ASSAY

At the end of the incubation period, the bicarbonate is transferred to scintillation vials for counting, and the embryos are recovered for return to culture or further analysis. Working in batches of a maximum of 10 vials, the caps are removed and the bicarbonate decanted into 20-ml scintillation vials containing 200 ml of 0.1 N NaOH to convert the dissolved CO2 and bicarbonate to carbonate.

To recover the embryos, 20-100 ml of fresh culture medium is added to the droplet in each cap. The embryos are picked up in 2 ml of medium and washed and returned to

Figure 9.5. The recovery of 3H2O and 14CO2 from the incubation droplet into the bicarbonate exchange reservoir of the metabolism assay vial. (Redrawn from Rieger et al. [20], with permission.)

culture, fixed, or analyzed by other procedures. To assure that the assay is not having any severe deleterious effects on the embryos, embryos that have been subject to the metabolic measurement assay should be left in culture for several days and their development compared to embryos that have not be subjected to the assay. When all the incubation vials have been decanted, 15 ml of a scintillation fluid for aqueous samples is added to each scintillation vial.

10. SCINTILLATION COUNTING

The scintillation vials are counted in a liquid scintillation beta counter for a minimum of 5 min each, and the raw counts per minute are corrected for quenching and converted to disintegrations per minute. If two labeled substrates (one 3H-labeled and one 14C-labeled) are used, then the sample must be counted in two counting windows, and the counts corrected to determine the disintegrations per minute for each label. These determinations are done automatically by most modern scintillation counters, and this requires only that the counting program be properly defined.

11. DETERMINATION OF PRODUCT RECOVERY

To determine the proportions of 14CO2 or 3H2O produced that are recovered in the bicarbonate, known amounts of NaH14CO3 and 3H2O in 4-ml droplets of culture medium are cultured in the incubation vials, exactly as for the embryos. Three to five vials are removed from culture after 0, 30, 60, 120, and 180 min and the bicarbonate counted (Figure 9.5). The recovery correction factor is calculated as the reciprocal of the integrated proportion under the curve for each of 14CO2 and 3H2O. Recovery is greater than 70% for both. Note that this determination need only be done once.

12. CALCULATIONS

The amount of each substrate (in picomoles) metabolized by an embryo is calculated by first subtracting the mean sham disintegrations per minute from the disintegrations per minute for the embryo to correct for nonspecific counts. The difference is divided by the mean total disintegrations per minute to give the proportion of labeled substrate metabolized by the embryo, which is, by definition, equal to the proportion of the total amount of substrate (labeled plus unlabeled) metabolized. This proportion is multiplied by the total amount of substrate and the recovery correction factor to give the amount of substrate metabolized. The formula is:

where Sm = the amount of substrate metabolized, De = the number of disintegrations per minute of 14CO2 or 3H2O produced by the embryo, Ds = the mean number of disintegrations per minute for the group sham preparations, Dt = the mean number of disintegrations per minute of 14C- or 3H-labeled substrate in the droplet (as counted in the total count preparations). R = the recovery correction factor, Sl = the amount of labeled substrate in the droplet, Su = the amount of unlabeled substrate in the droplet. The amount of labeled substrate in the droplet (Sl) in picomoles is determined from the mean total counts (Dt) and the specific activity (Sp. Act.) in milliCuries per millimole:

The amount of unlabeled substrate in the droplet (Su) in picomoles is determined from its concentration (C) in the medium (millimolar), and the volume of the droplet (V) in microliters:

We handle these calculations with a spreadsheet program that includes a test that the disintegrations per minute for an embryo equal or exceed the sensitivity of the assay (defined as the mean sham disintegrations per minute plus 2 standard errors), and any value below that is set to zero. The spreadsheet also calculates the embryo treatment group means and variances, which can be cut and pasted to other programs for data storage, statistical analysis, and graphing.

The variance of the measurements of metabolism of embryos of a single cleavage stage is usually sufficiently homogeneous that the data can be statistically analyzed without transformation. For experiments involving blastocysts or comparisons across stages, it is often necessary to log-transform the results to provide homogeneity of variance.

13. COMMON PROBLEMS

The procedures have been developed to make this assay as simple as possible and relatively trouble free. There are only two aspects that are of significant concern and need to be monitored closely: the nonspecific counts measured in the sham preparations and the viability of the embryos after being subjected to the procedure.

Although the sham values are included in the calculations to correct for nonspecific counts, they should be reduced to the minimum to increase the signal-to-noise ratio. Machine background can be checked by counting scintillation vials containing only scintillation fluid. If these produce more than 50 cpm, then the counter should be checked, cleaned, and adjusted by a qualified technician. Chemiluminesence can be checked by preparing counting scintillation vials containing 4 ml of culture medium, 200 ml of 0.1 N NaOH, and 1.5 ml of the bicarbonate solution together with 15 ml of scintillation fluid, but no radioactive material. Background noise can be produced by NaOH, but this is unlikely to be a significant problem because of the small amounts used. It is possible that unusual components of the culture medium could be luminescent and might have to be eliminated or reduced in concentration. Microbial contamination must be eliminated by proper sterile procedures. Chemical breakdown of the radiolabeled substrates can be a significant source of nonspecific counts. This is especially true for [5-3H]glucose, where the label is somewhat labile and can be spontaneously transferred to water. We also see relatively high sham counts with [2-14C]pyruvate, possibly due to conversion to pyropyruvate. However, this has never been a serious problem because the metabolism of [2-14C]pyruvate is also relatively high.

As noted above, the embryos must be capable of normal development after being subjected to the assay in order to be certain that the measurements reflect normal metabolic function. It is important to note that apparent morphological normality immediately after the metabolic measurement is not a reliable indicator of viability. We have seen a significant loss of viability on only two occasions. In the first case, the problem was traced to the N2 used to dry the radiolabeled substrates, and in the second, to the batch of Percoll used to prepare the sperm for in vitro fertilization. As these two examples demonstrate, it can be difficult to determine the exact cause of a loss of viability, and it may be necessary to test all parts of the culture system and the assay.

14. CONCLUSIONS

The survival and development of the early mammalian embryo depends on its ability to metabolize energy substrates, and any manipulation that affects energy metabolism may disturb or inhibit development. Evaluating the effects of molecular and mechanical manipulations on the energy metabolism of the early embryo may lead to improvements in techniques such as cryopreservation, gene transfer, cloning, and the production of stem cells. A notable example is the incidence of large lambs and calves from embryos produced by cloning (30), which may well be due to very early metabolic defects.

Acknowledgments The development and application of this technique were made possible by the efforts and kindness of my collaborators at the Universities of Montréal, Guelph, Wisconsin, Milan, Louvain-la-Neuve, Melbourne, and Dublin, the INRA research station, Nouzilly, France, and AgResearch, Hamilton, New Zealand.

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CALVIN SIMERLY RICARDO MORENO JOAO RAMALHO-SANTOS LAURA HEWITSON GERALD SCHATTEN

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Get Pregnant - Cure Infertility Naturally

Get Pregnant - Cure Infertility Naturally

Far too many people struggle to fall pregnant and conceive a child naturally. This book looks at the reasons for infertility and how using a natural, holistic approach can greatly improve your chances of conceiving a child of your own without surgery and without drugs!

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