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a Department of Biotechnology, Institute for Animal Husbandry and Behaviour, Mariensee, 31535 Neustadt, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the possible causes for the various differences observed between embryos generated in vitro and in vivo could be suboptimal in vitro culture conditions. Energy substrates are among the important ingredients of any culture medium. A substantial body of information indicates the changing energy substrate requirements during early development of mammalian embryos with important differences among species [11–14]. An increased knowledge of the metabolic fate of various energy substrates and the nutrient preferences of preimplantation embryos is crucial for improvements of culture media. Although several reports have dealt with the metabolism of bovine embryos produced in vitro or in vivo [15–20], a direct comparison of the metabolic activity of embryos derived either in vitro or in vivo at similar stages of development within the same experiment has not yet been carried out. The purpose of this study was to determine the metabolic needs of the bovine oocyte as it undergoes in vitro maturation (IVM), fertilization (IVF), and development to the hatched blastocyst stage, as well as to compare these requirements with those of their in vivo counterparts to evaluate the biochemical normality of in vitro-produced embryos. The feasibility of using metabolic activity as an index of developmental potential was also examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals, reagents, hormones, media, and antibiotics were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Ovaries, primarily from Holstein cows and heifers, were collected from a local abattoir and transported to the laboratory within 2–3 h of collection in PBS at ambient temperature (~25°C). Upon arrival in the laboratory, the ovaries were washed twice with fresh PBS. Cumulus-oocyte complexes (COC) aspirated from follicles 2–8 mm in diameter were allowed to settle for 8–10 min. COC were collected into fresh PBS and classified into one of four morphological categories mainly based upon the characteristics of their cellular investments [21]. Oocytes having a homogenous, evenly granulated cytoplasm surrounded by a compact cumulus oophorus with more than three layers were classified as category I, and oocytes with fewer than 3 layers of cumulus cells or those partially denuded but having a homogenous evenly granulated cytoplasm were classified as category II. Oocytes surrounded by corona radiata cells only were put in category III, and denuded oocytes were grouped under category IV. Only oocytes belonging to categories I and II yielded morulae/blastocysts in vitro and were used in these experiments. However, in some metabolic studies, oocytes of category III were also included for comparison.
The basic medium for in vitro maturation and culture was TCM-199 (no. M2520) containing glutamine and 25 mM Hepes and supplemented with 22 µg/ml pyruvate, 2.2 mg/ml sodium bicarbonate, and either penicillin G sodium and streptomycin sulfate (0.5 mg/ml each) or gentamycin sulfate (50 µg/ml). The medium was prepared fresh every week and stored at 4°C. For IVM, this medium was supplemented with 1 µg/ml estradiol-17ß (Serva, Heidelberg, Germany), 0.5 µg/ml FSH (Beckers, Faculte de Medicine Veterinaire, Universite de l'Etat a Liegé, Belgium), 0.06 IU hCG (Ekluton; Vemie, Kempen, Germany), and 20% heat-inactivated (30 min at 56°C) estrous cow serum (ECS) (i.e., maturation medium) [22].
COC were washed twice in maturation medium without estradiol, FSH, and hCG and transferred in groups of 10 into 100-µl drops of maturation medium under silicone oil (Serva). They were incubated for 24 h at 39°C in an atmosphere of high humidity and 5% CO2 in air. The IVF medium consisted of Fert-TALP (TALP: Tyrode's, albumin, lactate, pyruvate) [23] containing 1 µM epinephrine, 10 µM hypotaurine, 20 µM penicillamine, 0.56 µg/ml heparin, and 6 mg/ml BSA. After maturation, COC were inseminated for 18 h in 100-µl droplets of fertilization medium under silicone oil with frozen/thawed semen from one Holstein bull with a history of proven fertility in our IVF program. After semen was thawed in a water bath at 37°C for 1 min, motile spermatozoa were separated by the swim-up procedure [24]. Sperm concentration was determined using a Coulter counter (Coulter Electronics Ltd., Krefeld, Germany) or a hemocytometer, and the final concentration in fertilization droplets was adjusted to one million sperm per milliliter. After fertilization, presumptive zygotes with cumulus cells were washed twice and then transferred into 0.5 ml of development medium (TCM-199+20%ECS) placed under silicone oil in a 4-well Nunclon dish (Nunc, Roskilde, Denmark). Cumulus cells attached to the bottom of the dish and formed a monolayer within 2–3 days of culture. When early cleavage-stage embryos were required, cumulus cell remnants were removed by slight pipetting without disturbing the cells attached to the bottom of the dish. According to the requirements of the individual experiments, culture was either maintained undisturbed for 8–10 days when the embryos should have reached the blastocyst stage or was terminated at appropriate intervals to obtain embryos of predefined developmental stages for use in the metabolic studies. The development medium was not replaced during the 8- to 10-day culture period. All other culture conditions were the same as for IVM/IVF. The viability of IVP embryos was examined by transferring blastocysts to 14 synchronized recipients, of which 6 become pregnant. A total of 2281 embryos generated in 60 IVP runs over a period of 18 mo were used for the present study.
Collection of In Vivo Embryos
Holstein cows and heifers were superovulated using either eCG or pFSH followed by prostaglandin F2
injection. Uterine embryos were collected nonsurgically according to standardized procedures. Oviductal stages were obtained after slaughter in the institute's own slaughterhouse, using PBS supplemented with 1% newborn calf serum as the flushing medium. In vivo-matured oocytes were obtained by aspirating follicles > 12 mm either from abattoir ovaries or from the ovaries of superovulated animals slaughtered 72–74 h after the prostaglandin injection. Follicles measuring > 20 mm in diameter were considered cystic and were not included in the study. In total, 373 in vivo-produced embryos recovered from 42 animals in 15 sessions over a period of 18 months were used for these experiments.
Metabolic Studies
Energy substrate-free medium Studies on the metabolic competence of bovine embryos generated in vitro or in vivo were undertaken using medium based on Dulbecco's PBS without any additional energy substrates. It consisted of NaCl, 130 mM; KCl, 2.38 mM; KH2PO4, 1.47 mM; Na2PO4, 8.10 mM; CaCl2, 0.71 mM; MgSO4, 0.92 mM; penicillin G sodium, 60 µg/ml; streptomycin sulfate, 50 µg/ml; and BSA (fraction V), 1.0 mg/ml. The medium was prepared fresh every week and stored at 4°C. This medium was used for initial washing and after being enriched with the respective radiolabeled substrate was employed for the final washing and incubation of the embryos for estimation of metabolism.
Isotopes
All radioactive substrates including [U-14C]glucose, [1-14C]pyruvate, and [1-14C]lactate were obtained from Amersham Buchler GmbH & Co. KG (Braunschweig, Germany) and stored at -20°C upon arrival. In preliminary experiments, an aliquot of each supply was tested for potential toxic or inhibitory effects on embryonic development in vitro. The stock isotopic solutions were dried under nitrogen and reconstituted by dilution with the respective nonradioactive substrate to give the required specific activity and concentration. Calculated aliquots were dried in sterile tubes and stored at -20°C until use. Prior to use, the dried radioactive substrate was reconstituted by addition of a known quantity of sterile culture medium. To increase the accuracy of the measurements, particularly in studies with single embryos, it was necessary to use the isotopic marker at a high specific activity. In the present experiments, [U-14C]glucose was used at a concentration of 0.28 mM (0.56 mM in a few preliminary experiments for comparison) at a specific activity of 30 µCi/µmol (1.11 Mbq/µmol). Similarly, pyruvate was used at a concentration of 0.5 mM and a specific activity of 15 µCi/µmol (0.55 MBq/µmol), while lactate had a concentration of 2.5 mM at a specific activity similar to that of pyruvate.
Estimation of CO2 Production
Basically, the methodology used for measuring oxidative metabolism of embryos was similar to that used previously [16, 25] with slight modifications. Embryos at a particular stage of development, either single or in groups of 2–5, were incubated for 4 h in a closed chamber in the presence of a radioactive substrate, and the CO2 released was continuously collected in a NaOH trap. A washed (3 times) and heat-sterilized 20-ml scintillation vial served as the incubation apparatus. Embryos were washed twice in energy substrate-free medium and placed in 50-µl drops of radioactive medium for a final wash before being transferred in a minimum volume (< 1 µl) into a sterile 600-µl microcentrifuge tube (Eppendorf-Netheler-Hinz, GmbH, Hamburg, Germany) containing 50 µl of radioactive incubation medium. This tube was then placed in the scintillation vial with the lid of the tube acting as a support. Simultaneously, 0.5 ml of 2.5 M sodium hydroxide was added to the bottom of the vial, which was then closed and incubated at 39°C for 4 h. At the end of the incubation period, the tube with embryos was removed, and the radioactivity in the trapped CO2 was determined after addition of Hionic Fluor (Packard Instruments, Dreieich, Germany), a pseudocumene-based scintillation cocktail. In all experiments, at least two sham incubations were run in parallel to correct for background counts. The rate of CO2 production was estimated from the disintegrations per minute detected in the sample and the specific activity of the parent substrate. It was converted into picomoles per embryo per hour. This technique was preferred in the present study as it required no transfer of NaOH to the scintillation vial and provided sufficient postincubation medium for the analysis of lactate production. Similarly, the viability of the embryos subjected to metabolic measurements was examined by returning a total of 25 embryos at the 2-cell stage to coculture with cumulus cells after measuring their CO2 production. While 16 of these embryos underwent two or more further divisions, 6 advanced to the morula/blastocyst stage. The rate of development was similar to that of control embryos.
Incorporation of Substrate Carbon
After the 4-h incubation period for measuring CO2 production, the embryos were recovered in a minimum volume of incubation medium, washed three times in medium containing nonradioactive substrate at the same concentration, and transferred to a scintillation vial containing 0.5 ml of distilled water and processed for assay of radioactivity. A sample of the last wash before radioassay was always included to provide background levels. The incorporation was expressed as picograms atoms per embryo per hour as described in relation to CO2 production.
Estimation of Lactate Production
Lactate production was estimated by separating lactate from glucose in the postincubation media with descending paper chromatography [26]. A total of 20 µl medium was spotted on each strip of Whatman chromatography paper (no. 3030917; Whatman Scientific Ltd., Kent, England) and run for 16 h using butanol:water:acetic acid (4:5:1, v/v) as solvent. Dried chromatograms were serially cut into 1-cm lengths and processed for assay of radioactivity. The lactate peak was identified by the Rf value and its comigration with authentic lactate. Disintegrations per minute were converted to picomoles per embryo per hour as described for CO2 production.
Experimental Design
The metabolism of three radiolabeled energy substrates including glucose, pyruvate, and lactate during in vitro development from the immature oocyte to the hatched blastocyst was investigated during a 4-h incubation period. The indicators of metabolic activity included production of carbon dioxide and incorporation of substrate carbon for all three energy substrates, and accumulation of lactate from glucose only. The cumulus cells surrounding early-stage embryos were carefully removed prior to use for the metabolic studies. Complete absence of cumulus was verified by microscopic examination (x100). In the present study, only very good quality embryos were used for metabolic investigations [27].
Metabolism of Glucose
Four experiments were conducted to study metabolism of glucose by bovine embryos.
Experiment 1 The first experiment addressed utilization of radiolabeled glucose as the sole energy substrate at a concentration of 0.28 mM from the bovine immature oocyte up to the hatched blastocyst stage derived in vitro or in vivo. In preliminary trials, a 2-fold higher concentration (0.56 mM) was also included to ensure a sufficient availability of the substrate.
Experiment 2 Based on the finding of the first experiment indicating large variations in repetitions from sizes and individual blastocysts, the relationship between blastocyst size and its metabolic activity was evaluated. The blastocysts were divided into three groups according to their diameter (150–175, 176–200, and > 200 µm).
Experiment 3 The data from experiment 1 suggested that preexposure of in vivo embryos to in vitro culture (IVC) conditions affected their lactate production. To confirm this suggestion, this experiment addressed the metabolism of glucose by mid- and expanded blastocyst stage embryos obtained after IVC of fresh in vivo-collected early blastocysts. Utilizing data from experiment 1, a comparative study of glucose metabolism in embryos from three different sources (in vitro produced, collected in vivo and used immediately [fresh], and fresh embryos preexposed to culture conditions) was accomplished.
Experiment 4 The fourth experiment examined glucose metabolism in the presence of additional substrates (as would be normally expected in vivo or under IVC conditions) in the form of pyruvate (0.5 mM) and lactate (2.5 mM) by embryos produced in vitro or in vivo at different stages of preimplantation development.
Metabolism of Pyruvate and Lactate
Three experiments were conducted to evaluate the metabolism of radiolabeled pyruvate and lactate by various stages of preimplantation bovine embryos derived in vitro or in vivo.
Experiment 5 This experiment evaluated the relationship between the morphological quality of oocytes (categories I, II, III) and their ability to metabolize pyruvate and lactate immediately after aspiration (immature) or after IVM for 24 h. In vivo-matured (follicular) oocytes were also included. Nude oocytes (category IV) were not used, since preliminary trials showed that these oocytes had very low and inconsistent metabolic activity.
Experiment 6 The sixth experiment was an investigation of the utilization of [1-14C]pyruvate and [1-14C]lactate present as sole energy substrates by bovine embryos produced in vitro or in vivo from the immature oocyte to the hatched blastocyst stage.
Experiment 7 As pyruvate and lactate are known to have interactions in embryos of various species [28], the effects of both substrates on each other's metabolism from the immature oocyte to the 16-cell stage using IVP embryos were tested. In the first part of this experiment, the metabolism of radiolabeled pyruvate (0.5 mM) was assessed in the presence of unlabeled lactate at increasing concentrations of 0, 0.5, 2.5, 5.0, and 25 mM; in the second part, the metabolism of radiolabeled lactate (2.5 mM) was evaluated in the presence of 0 or 0.5 mM unlabeled pyruvate.
Statistical Analysis
Statistical significance of differences was tested by Student's t-test, chi-square test, and protected least significant difference (LSD) test where appropriate [29]. Differences in the frequencies of maturation, fertilization, and cleavage, as well as in the proportion of zygotes reaching the morula/blastocyst stage in the two groups, were compared by chi-square test. The statistical significance of the differences of the various indicators of metabolism between the two sources of embryos and stages of development was calculated by Student's t-test. Multiple means were compared by protected LSD test. The data are presented as mean ± SEM. Values at P < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of experiment 1 are presented in Table 1. In initial trials, a 2-fold higher concentration (0.56 mM) of glucose was also used. Since the pattern of glucose metabolism was not altered by this higher concentration of substrate, the data pertaining to the 0.56 mM concentration are not presented. While immature oocytes did not oxidize any glucose, there was a 38-fold increase in the rate of CO2 production as the matured oocyte progressed to the hatched blastocyst stage in vitro. The first significant increase in oxidation of glucose was noticed at the 16-cell stage when the rate of CO2 production exceeded the level of 1 pmol/embryo per hour. The in vitro-matured oocyte exhibited aerobic glycolysis by accumulating small quantities of lactate in the incubation medium. Lactate production by IVP embryos fell to undetectable levels until the 12-cell stage, when lactate was again detected. In contrast, in vivo-generated embryos did not produce any lactate up to the 16-cell stage. Thereafter, a gradual increase in the production of lactate was recorded as the embryo progressed to the blastocyst stage and underwent hatching. The bulk of glucose was utilized through aerobic glycosis by later preimplantation stages. In general, the in vivo-grown embryos produced only half the quantity of lactate that their in vitro counterparts did. However, no true comparisons could be made at the hatching and hatched blastocyst stages between embryos of differing origin, as these could not be collected in vivo and were obtained by IVC of freshly collected early blastocysts for 48–72 h. Interestingly, lactate production of these in vivo-grown and in vitro-cultured blastocysts reached levels almost equal to that of their IVP counterparts. While only a small quantity of glucose was incorporated by the immature oocyte, it increased steadily thereafter, showing a 15-fold increase between the 1-cell and the blastocyst stages. In vivo-generated embryos followed a similar pattern with a few minor differences.
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The results of experiment 2 are summarized in Table 2. There were no differences among the three groups of blastocysts based on their diameters in their metabolic activity as measured by CO2 production, lactate accumulation, and carbon uptake from glucose. CO2 production of 22 blastocysts employed in this experiment ranged from 3 to 12.7 pmol/embryo per hour. Lactate production and substrate carbon uptake ranged from 27.3 to 133.8 pmol and 5.2 to 79.3 pg atoms per embryo per hour, respectively.
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The results of experiment 3 are shown in Table 3. The preexposure of in vivo-generated embryos to culture conditions significantly increased the rate of aerobic glycosis, and the differences disappeared at the hatched blastocyst stage obtained after 72 h of IVC.
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The data on the metabolism of radiolabeled glucose in the presence of unlabeled pyruvate and lactate (experiment 4) are presented in Table 4. The overall pattern of glucose metabolism and the production of lactate by the IVM oocyte remained unaltered in the presence of alternate energy substrates. However, the total consumption of glucose was reduced by 60–70% as compared to values obtained in experiment 1 when glucose was used as the sole energy substrate. Approximately 70–90% of it was metabolized through aerobic glycolysis. The IVP embryos continued to produce twice as much lactate as did their in vivo counterparts but on a much lower scale in absolute terms. The incorporation of glucose carbon at the blastocyst stage was not affected when compared with the data from experiment 1.
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Metabolism of Pyruvate and Lactate
The results of experiment 5 indicate that the morphological quality of the immature oocyte significantly affected oxidation and uptake of pyruvate but not that of lactate (Table 5). In general, oocytes from categories I and II had a similar pattern of metabolic activity. In vivo-matured oocytes had significantly lower rates of oxidation of pyruvate and lactate as compared to IVM oocytes in categories I and II.
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The results of experiment 6 are presented in Table 6. Irrespective of the origin of the embryos, pyruvate was the preferred substrate until the 16-cell stage. This was evident from its rate of oxidation as compared to that of lactate or glucose (Fig. 1). The rate of utilization of pyruvate was relatively constant as the oocyte advanced to the morula stage in comparison to few ups and downs noted in the utilization of lactate. Only a small but consistent amount of substrate carbon was incorporated throughout preimplantation development with no effects of source of embryos or the employed substrate.
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The data pertaining to both parts of experiment 7 are summarized in Table 7. In general, the oxidation of pyruvate was significantly reduced as the concentration of lactate was increased. At 25 mM lactate, the highest level tested, oxidative metabolism of pyruvate was drastically reduced to 4–15% of the control values in all stages of development. Oxidation of C-1 of lactate in the presence of 0.5 mM pyruvate was significantly decreased in all developmental stages except at the 1- and 2-cell stage.
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General Considerations
An overview of oxidative metabolism of IVP embryos is depicted in Figure 1. The metabolic activity was more or less constant until the 12-cell stage, with pyruvate being the preferred energy substrate. The immature oocyte was not able to utilize any glucose, but glucose utilization gradually increased from the mature oocyte to the hatched blastocyst stage with the first marked increase occurring between the 12- and 16-cell stages. In contrast, the oxidation of pyruvate and lactate remained within a narrow range of 1–3 pmol until the morula stage, when there was a 2- to 4-fold increase. Only a small amount of substrate carbon from pyruvate and lactate was incorporated throughout preimplantation development. In comparison, incorporation of glucose increased more than 150-fold from the immature oocyte to the blastocyst stage. The bulk of glucose was metabolized through aerobic glycolysis at the expanded and hatched blastocyst stages. The addition of one or more alternate energy substrates significantly affected the utilization of glucose, pyruvate, or lactate.
In general, the pattern of metabolism was similar between the embryos from the two sources, with some important differences such as the higher metabolic activity of IVM oocytes with characteristic aerobic glycolysis. Similarly, embryos at later stages of preimplantation development, either generated in vitro or preexposed to culture conditions for 1–3 days after being collected in vivo, produced double the amounts of lactate from glucose as compared to their in vivo counterparts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In general, the pattern of utilization of glucose, pyruvate, and lactate was similar between the embryos derived in vitro and in vivo. The overall pattern of glucose metabolism from the 1-cell to the blastocyst stages was similar to that reported for murine [34], human [35], ovine [36], porcine [37], and bovine [15–17] embryos. The first marked increase in the metabolism of glucose was observed between the 8- and 16-cell stage, the time of activation of the embryonic genome [38]. However, in bovine embryos collected from superovulated animals, the first marked increase in utilization of glucose was detected between the 16-cell and morula stage [16]. This significant increase in glucose metabolism in early embryos may either be related to the synthesis of one or more key glycolytic enzymes as it coincides with the activation of the embryonic genome or be due to changes in the allosteric regulation of enzyme activity [17]. However, the present observations on the production of lactate by in vitro-matured oocytes would suggest that bovine embryos possess the full machinery of glycolytic enzymes from very early stages onward. This suggestion is supported by a study in which lactate production by all stages of in vitro-derived bovine embryos between the 1-cell and the blastocyst stage was detected [15]. In contrast, lactate production was not detected from the 1-cell to the 12-cell stage in the present study. However, these two studies used different methodologies and may not be directly comparable. It is possible that the enzymes were present throughout the early period of development, but were inhibited by intracellular regulations until the 12-cell stage when the growth rate and energy demands of the embryo increased significantly.
The present data on incorporation of glucose carbon suggest that glucose metabolism plays a crucial role in the synthetic activity, as incorporation increased more than six times between the 12-cell and compact morula stages. This suggestion is further supported by the observations made in experiment 4, in which glucose carbon incorporation was substantially reduced in the presence of alternate energy substrates at all stages of development but not at the blastocyst stage when a great deal of synthetic activity is expected. Several studies using mouse morulae/early blastocysts have shown that a significant fraction of glucose is incorporated into macromolecules [26, 39, 40]. In general, the developmental pattern of changes in the incorporation resembled that of human [35] and murine [41] embryos.
One of the striking findings of the present metabolic studies was the production of lactate and the considerably higher rates of oxidation of all three energy substrates by IVM oocytes as compared to their in vivo counterparts. Differences at the ultrastructural level between oocytes matured in vitro and in vivo [42, 43] are likely reflected at the biochemical level. Alternately, follicular oocytes used in the present study might not yet have acquired their full metabolic competence, which occurs prior to the time of ovulation. A comparison of the metabolism of oocytes collected from preovulatory follicles at different intervals prior to ovulation and oocytes collected from the oviduct immediately after ovulation would be useful.
The most apparent characteristic of glucose metabolism in the present study was the 2-fold higher production of lactate by the in vitro-produced blastocysts as compared to their in vivo counterparts. The rate of lactate production of hatched blastocysts obtained after 72-h IVC of freshly collected early blastocysts matched that of their IVP counterparts. Others have also reported findings similar to ours, that is, that the bulk of glucose is metabolized through aerobic glycolysis by mammalian morulae and blastocysts [15–17, 36, 41, 44–46]. In contrast, earlier it was suggested that glycolysis was blocked in bovine blastocysts because of a lack or inhibition of pyruvate kinase [47]. The high rate of aerobic glycolysis does not seem to be incompatible with an active tricarboxylic acid (TCA) cycle. This is shown by the 3- to 4-fold increase in the rates of oxidation of pyruvate and lactate during development from the morula to the blastocyst stage in the present experiments as well as in other studies [15, 48]. The results of experiment 4 also suggest the operation of an active TCA cycle in embryos, as the total consumption of glucose was reduced by more than 50% when pyruvate and lactate were added to the incubation medium.
Several explanations for the high rate of lactate production by IVP embryos can be put forward. Glycolysis was functional at the blastocyst stage (except in IVM oocytes), when there is a high demand for energy to meet the needs of compaction, expansion, and blastocoele formation [49]. Presumably, bovine embryos are able to enhance glycolysis to a maximum capacity to gain additional energy when the other pathways are already operating at maximum. This is supported by the finding that the rate of oxidation of glucose did not increase significantly beyond the morula stage although the energy demand is increased during blastulation. Why, in spite of the high demands of energy, the embryo chose this less efficient pathway of energy production is not clear, particularly in light of the fact that the TCA cycle was active. It is most likely a manifestation of metabolic stress [16] attributable either to separation from the natural environment or to the extended period of suboptimal IVC conditions. A stress response could also explain the lactate production shown by in vitro-matured oocytes in the first stage of separation from their natural environment (follicle) and the high rates of lactate production at the blastocyst stage after additional exposure to IVC conditions. Similarly, the low rate of lactate production by in vivo-collected embryos could also be explained on this basis. An enhanced aerobic glycolysis is a characteristic feature of adult cells when they are isolated from their natural environment and cultured in vitro. The same findings have been obtained for tumor cells, which resemble early embryos in that they have pluri- or totipotent properties and show rapid cell division [13].
Recently, it has been reported that culture medium used for production of embryos can affect metabolism of the resulting embryos. Embryos generated in a completely defined medium had lower rates of glycolysis than embryos grown in medium supplemented with serum [50]. This could be similarly true for the present study, since the IVP embryos were produced in medium containing 20% ECS. Whether this high rate of lactate accumulation that can cross cell membranes and affect intracellular pH [51] has any physiological significance or any effect on viability of the bovine embryos raises questions yet to be resolved. The production of lactate was suggested to be a physiological requirement for the maintenance of cytoplasmic redox equilibrium of (NADH):(NAD+) [52]. Embryos that are likely to have reduced viability such as those exhibiting a slow rate of development and having fewer blastomeres displayed higher rates of glycolysis [32]. Similarly, increased rates of glycolysis have been associated with reduced embryo viability in mice [53].
All three measures of glucose metabolism displayed wide individual variations at the blastocyst stage. The data presented in Table 2 suggest that these variations are not related to size or morphological quality of the embryo, as only very good quality embryos were employed for these metabolic studies. However, glucose uptake and lactate production have been employed to select viable bovine blastocysts upon freezing and thawing [54]. Perhaps cryopreservation is particularly detrimental to embryos with a metabolism operating at its maximum capacity and only those embryos can survive that have a metabolism within the physiological range.
The data on the metabolism of pyruvate by immature oocytes belonging to different morphological categories indicated that the morphological differences were reflected at the biochemical level. However, these metabolic indicators cannot be used as selection criteria for the developmental potential of oocytes in vitro, as the overall metabolic activity of immature oocytes was relatively low and the differences in metabolic activity between category I and II oocytes disappeared after maturation. The pattern of utilization of pyruvate and lactate suggested that pyruvate was the preferred substrate up to the 12-cell stage, and its oxidation further increased during compaction and blastulation, similarly to that of glucose. A similar increased uptake and oxidation of pyruvate has been reported for bovine [13,47, 48] and ovine [36, 52] embryos. This is in contrast to murine embryos, which show a dramatic decrease in pyruvate uptake at the blastocyst stage [55, 56]. Our results show that only a small fraction of pyruvate and lactate carbon was incorporated by embryos at all stages of development, which is in agreement with the findings for murine embryos [57]. We observed that early embryos up the to 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than their in vivo counterparts. These observations indicate differences in ATP/ADP ratio and oxidation/reduction status of the embryos grown in vitro versus in vivo. A proper lactate-to-pyruvate ratio is essential for balancing the oxidation/reduction potential in embryos [58]. However, it has been reported that lactate alone can support development of early bovine [59] and ovine [60] embryos.
An interesting finding of our study was that lactate and pyruvate each affected metabolism of the other. In the presence of 25 mM lactate, utilization of pyruvate was drastically reduced, raising doubts about the need for high amounts of lactate in culture media. Most culture media have a higher pyruvate-to-lactate ratio than the maximum (1:50) used in the present study. It is possible that the presence of somatic cells and glucose during IVC of bovine embryos may change this ratio, as in the mouse, cumulus cells are capable of producing pyruvate [61]. Additionally, pyruvate was required for meiotic maturation in denuded but not in cumulus-enclosed bovine oocytes [62]. The present results suggest that a low pyruvate-to-lactate ratio should be beneficial for culture of bovine oocytes/embryos in chemically defined medium. A low pyruvate-to-lactate ratio for culture of bovine embryos in semidefined media was compatible with rates of development similar to those obtained with complex culture conditions and a high lactate-to-pyruvate ratio [59].
In conclusion, the results of the present experiments demonstrate that in vivo- and in vitro-derived bovine embryos possess to a large extent similar metabolic activities. However, several distinct metabolic differences were identified for in vitro-derived oocytes and embryos as compared with their in vivo counterparts. The most prominent features were the production of lactate and high rates of oxidation of energy substrates by in vitro-matured oocytes. Similarly, IVP blastocysts produced twice as much lactate as did embryos generated in vivo. A stress response to suboptimal culture conditions is the most probable explanation for this high rate of lactate production. The overall low metabolic activity of immature oocytes and the high individual variability of the metabolic rates of blastocysts may not allow employment of metabolic indicators as an index of development potential.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 N.K.K. was financially supported by the Alexander von Humboldt Foundation, Bonn, Germany, in the form of a postdoctoral fellowship. 
2 Correspondence. FAX: 49 05034 871 101; niemann{at}tzv.fal.de
3 Current address: Sector-I, Govt. Livestock Farm, Hisar-125001, Haryana, India.
Accepted: November 8, 1999.
Received: September 9, 1999.
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D. O. Brandao, P. Maddox-Hyttel, P. Lovendahl, R. Rumpf, D. Stringfellow, and H. Callesen Post Hatching Development: a Novel System for Extended in Vitro Culture of Bovine Embryos Biol Reprod, December 1, 2004; 71(6): 2048 - 2055. [Abstract] [Full Text] [PDF]
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M. L Sutton-McDowall, R. B Gilchrist, and J. G Thompson Cumulus expansion and glucose utilisation by bovine cumulus-oocyte complexes during in vitro maturation: the influence of glucosamine and follicle-stimulating hormone Reproduction, September 1, 2004; 128(3): 313 - 319. [Abstract] [Full Text] [PDF]
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M. Tamassia, F. Nuttinck, P. May-Panloup, P. Reynier, Y. Heyman, G. Charpigny, M. Stojkovic, S. Hiendleder, J.-P. Renard, and S. Chastant-Maillard In Vitro Embryo Production Efficiency in Cattle and Its Association with Oocyte Adenosine Triphosphate Content, Quantity of Mitochondrial DNA, and Mitochondrial DNA Haplogroup Biol Reprod, August 1, 2004; 71(2): 697 - 704. [Abstract] [Full Text] [PDF]
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R. M. Rivera, G. M Dahlgren, L. A. de Castro e Paula, R. T Kennedy, and P. J Hansen Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development Reproduction, July 1, 2004; 128(1): 33 - 42. [Abstract] [Full Text] [PDF]
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A. Oropeza, C. Wrenzycki, D. Herrmann, K.-G. Hadeler, and H. Niemann Improvement of the Developmental Capacity of Oocytes from Prepubertal Cattleby Intraovarian Insulin-Like Growth Factor-I Application Biol Reprod, June 1, 2004; 70(6): 1634 - 1643. [Abstract] [Full Text] [PDF]
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