Glucose inhibits development of hamster 8-cell embryos in vitro

Relative preferences of energy substrates (glucose, pyruvate, and lactate) for in vitro development of hamster 8-cell embryos were investigated. Using protein-free modified Tyrode's medium (TLP-PVA) containing 10 mM lactate (L), 0.1 mM pyruvate (P), and amino acids (Phe, Ile, Met and Gln), we found that development of hamster 8-cell embryos to blastocysts was supported better in the absence of glucose than in medium containing (standard) 5 mM glucose (88.1% and 50%, respectively). Addition of even 0.25 mM glucose to the medium significantly inhibited blastocyst formation (54.1%). Medium T-PVA, containing 5 mM glucose as sole energy substrate (without pyruvate, lactate, and amino acids), very poorly supported embryo development (less than or equal to 7.9% blastocysts), but addition of 0.1 mM pyruvate enhanced blastocyst formation (52%). Elimination of pyruvate in TL-PVA medium containing 5 mM glucose and amino acids markedly reduced blastocyst formation by 4-fold (13.5%); the optimal pyruvate concentration was 0.2 mM. However, if the same medium was devoid of glucose, blastocyst formation was high both in the absence (71.1%) and presence (83.3%) of 0.1 mM pyruvate. Similarly, in glucose-free T-PVA medium, addition of either 10 mM lactate or amino acids supported 8-cell embryo development to blastocysts (61.7% and 60.5%, respectively) as opposed to 18.8% and 30.6%, respectively, in the presence of 5 mM glucose. This augmented development in the absence of glucose is suggested to the due to the efficient conversion of lactate to pyruvate and of amino acids to amphibolic intermediates and hence their utilization via the Krebs cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.     

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.     

Energy substrate requirements for in vitro development of early cleavage-stage bovine embryos

Energy substrate preferences of bovine cleavage-stage embryos produced by in vitro maturation and in vitro fertilization were examined in a chemically-defined (protein-free) culture medium modified hamster embryo culture medium-3, (mHECM3). Few inseminated ova cleaved without energy substrates. Glucose and/or glutamine could not support embryo development, but lactate alone was effective (37% 5–8-cells), equivalent to complex medium TCM-199 (44%). Addition of 11 selected amino acids to lactate increased embryo cleavages, although this treatment was not significantly different from pyruvate alone. Addition of glucose to lactate or to pyruvate depressed development. Lactate + amino acids was significantly better than TCM-199 (54% and 26% ≤8-cells, respectively). Blastocyst development was evaluated after transferring ≤8-cell embryos into a complex medium (TCM-199) containing serum. Cleavage-stage embryos produced with pyruvate alone or with lactate + amino acids yielded the highest proportions of blastocysts (36% and 41%, respectively, of inseminated ova). Between 33–63% of blastocysts derived from embryos that were initially developed in mHECM-3 supplemented with various substrates escaped from their zonae (hatched) depending on the treatment, but none of the embryos from the pyruvate + glucose combination hatched. This study shows that optimal energy substrates for bovine cleavage-stage embryo development can be determined using a chemically-defined culture medium, that a simple medium with selected substrates can support early development as well as or better than a complex medium, that a two-step culture system can be used to evaluate blastocyst development from these cleavage-stage embryos, and that timing and hatching of embryos may provide additional information about discriminating between the suitabilities of different substrates for early embryo development. © 1996 Wiley-Liss, Inc.     

The effect of energy substrate concentration and amino acids on the in vitro development of preimplantation porcine embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.     

Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo

The uptake of pyruvate and glucose by individual sheep oocytes and preattachment sheep embryos at each state of development up to the hatching blastocyst was determined using a microfluorescence technique. After an initial increase at fertilization, pyruvate uptake was relatively constant (?15 pmol/embryo/h) from the zygote through to the morula. Upon blastocyst formation and hatching, there were significant increases in uptake (39 pmol/embryo/h, P < 0.001; and 53 pmol/embryo/h, P < 0.001, respectively). In contrast to that of pyruvate, glucose uptake was very low (?1 pmol/embryo/h) up to the time of genome activation (eight- to 16 cell stage), after which there were significant increases in uptake at each successive stage of development. By the hatching blastocyst stage, glucose uptake had reached 54 pmol/embryo/h. The ability of day-7 hatching blastocysts to oxidize pyruvate and glucose was determined indirectly by measuring the production of lactate when either substrate was present as the sole energy source. Unlike the mouse blastocyst, which has a considerable oxidative capacity for both pyruvate and glucose, the day-7 sheep blastocyst showed limited ability to oxidise either substrate. Rather, in the sheep blastocyst, 65% of pyruvate and 98% of glucose taken up could be accounted for as lactate. Such low levels of substrate oxidation appear to be inconsistent with the energy requirements of the proliferating preattachment ruminant blastocyst. The utilization of alternative substrates at the blastocyst, such as amino acids, is proposed. © 1993 Wiley-Liss, Inc.     

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).     

Pyruvate and oxygen consumption throughout the growth and development of murine oocytes

Growing oocytes in vitro from the most immature stages until they are developmentally competent is a major goal of reproductive technology, requiring fundamental knowledge of metabolic processes. Carbohydrate metabolism and oxygen consumption have been analysed in a series of experiments designed to investigate important energy substrates for mouse oocytes and to reveal any qualitative or quantitative changes between the primordial and ovulatory follicle stages. Primordial follicles were incubated in groups in modified-KSOM medium, whereas growing or ovulated oocytes were studied singly and, in both cases, the depletion or accumulation of metabolites in spent medium were analysed using ultramicrofluorometric assays. The rates of glucose (0.014 +/- 0.006 pmol/hr) and pyruvate (0.028 +/- 0.009 pmol/hr) consumption and l-lactate (0.058 +/- 0.023 pmol/hr) production by primordial follicles suggested that energy production was supported by a combination of metabolic pathways, including glycolysis. Pyruvate and oxygen consumption per oocyte increased two- and ninefold, respectively, between the primary and pre-ovulatory stages (0.82 +/- 0.1 and 1.67 +/- 0.1 pmol pyruvate/hr, respectively and 1.4 +/- 0.3 and 7 +/- 0.6 pmol oxygen/hr) after which oxygen (12.7 +/- 1.1 pmol/hr) utilisation nearly doubled. Oxygen consumption by fully grown oocytes was in excess of oxidation requirements for pyruvate. When pyruvate and oxygen consumption rates were normalised for oocyte cellular volume, which increased over 130-fold during growth, oocyte metabolism was higher in primary follicles than at any subsequent stage, indicating that energy needs are greater during a developmental transition. To conclude, pyruvate and oxygen were consumed throughout oocyte development at increasing rates. When oocyte cellular volume was accounted for, oocytes from primary follicles displayed greatest metabolic rates.     

Effect of exogenous carbohydrates in a serum-free culture medium on the development of in vitro matured and fertilized porcine embryos

This study was conducted to examine the effect of energy substrates in a serum-free culture medium on in vitro development of porcine embryos. Presumptive zygotes derived from in vitro fertilization were cultured in glucose-free North Carolina State University (NCSU)-23 medium with glucose, pyruvate, fructose and lactate added to the culture medium singly or in various combinations. In experiment 1, a higher percentage of embryos cleaved (53-63% vs 10-13%) and developed to the blastocyst stage (18-27% vs 0) after the single addition of glucose (5.6 mM), pyruvate (0.5 mM) or lactate (10 mM) than with no energy substrate addition or the addition only of fructose (5.6 mM). In experiment 2, the addition of pyruvate and lactate resulted in higher blastocyst formation (25%) than other combinations (6-22%), while the addition of glucose and pyruvate significantly inhibited blastocyst formation. Increasing lactate concentration, as a single energy supplement, from 5 to 20 mM significantly improved blastocyst formation (7% vs 14-18%), while no benefit was achieved from increasing pyruvate concentration up to 2 mM (experiment 3). Glucose-free NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate significantly improved blastocyst formation (28% vs 17%) compared with NCSU-23 medium supplemented with 5.6 mM glucose (experiment 4). In conclusion, pyruvate and lactate are preferable energy substrates to support in vitro development of porcine embryos cultured in a serum-free NCSU-23 medium.     

The involvement of mitochondria in carbon metabolism in cleavingXenopus embryos

Summary The major carbon sources inXenopus oocytes and cleavage-stage embryos appear to be amino acids, which are oxidized to form pyruvate (to support the Krebs cycle) and phosphoenolpyruvate (for anabolic processes). Metabolism of various metabolites in vitro into aspartate or glutamate, and then partially into phosphoenolpyruvate, requires the presence of mitochondria, suggesting that metabolism in vivo utilizes mitochondrial enzymes. The rate limiting step in metabolism in the stage VI oocyte appears to be uptake and/or metabolism of compounds by the mitochondria; the rate of metabolism increases during maturation. During early cleavage no qualitative differences in metabolism were observed either as a function of development, or spatially along the animal/vegetal or prospective dorsal/ventral axes.     

In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins

To elucidate the effect of nutrient substrates on embryo development, in vitro fertilized bovine one-cell embryos were cultured in a medium similar to synthetic oviduct fluid (SOF) but without glucose and containing 3.3 mM lactate, 0.3 mM pyruvate and 3 mg/ml bovine serum albumin (BSA) at 39 degrees C in 5% CO(2) in air. Results indicated that addition of glucose was not only unnecessary, but it also had a deleterious effect on embryo development to the morula stage. Lactate supported embryo development up to the morula stage as well as pyruvate. Supplementation with 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the morula stage dramatically and increased the cell number compared with that of the controls. Addition of the vitamins from MEM to SOF had no beneficial effect. The SOF with amino acids did not increase the frequency of blastocysts 7 days after in-vitro fertilization but did increase the total number of cells compared with that of the controls. Frequency of blastocysts at Day 7 in SOF with amino acids was equivalent to that of co-culture although the total cell number was lower. These results demonstrate that a semi-chemically defined medium can successfully support the development of bovine embryos to the morula stage to a limited extent, but the medium lacks some nutrients or growth factors to fully support development through the blastocyst stage.     

Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells

The objective was to determine ovarian follicular fluid concentrations of glucose, lactate, and pyruvate in relation to follicle size in buffalo and sheep. The effect of varying concentrations of these substances on in vitro oocyte maturation, oocyte protein content, and granulosa and cumulus cell growth was also investigated. Follicular fluid was aspirated from various sizes of follicles (from ovaries without a dominant follicle) collected from adult, cycling nonpregnant buffalo (Bubalus bubalis) and sheep (Ovis aries) during the breeding season. Overall, mean (+/-S.E.M.) concentrations (mM) were glucose 2.42+/-0.31 and 1.40+/-0.22, lactate 7.56+/-2.61 and 10.42+/-1.64, and pyruvate 0.02+/-0.01 and 0.002+/-0.00, in buffalo and sheep, respectively. In both species, as follicles became larger, concentrations of glucose significantly increased, lactate significantly decreased, but pyruvate was not affected. Oocyte maturation was higher (P<0.05) in medium containing supra-physiological concentrations of either glucose (5 mM), or pyruvate (10 mM) alone, or physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub- or supra-physiological concentrations of glucose, lactate and pyruvate in combination (both species). The protein content of oocytes was not significantly affected by the concentration of glucose, lactate, and pyruvate in the maturation medium. However, growth of granulosa and cumulus cells was higher (P<0.05) in medium containing supra-physiological concentrations of glucose (5 mM) alone, or pyruvate (10 mM) alone, or physiological, or supra-physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub-physiological concentrations of glucose, lactate and pyruvate in combination (both species). In conclusion, concentrations of glucose, pyruvate and lactate in the medium had cell type-specific effects on oocyte maturation, and on growth of granulosa and cumulus cells. Furthermore, glucose and pyruvate were the principal energy sources for oocytes and follicular somatic cells in buffalo and sheep.     

Nutrient concentrations in murine follicular fluid and the female reproductive tract

The culture of murine oocytes and preimplantation embryos in vitro has been used successfully for many years. However, this practice can result in cellular stress and reduced viability. Since this phenomenon is partly attributable to differences in nutrient composition between culture media and maternal tract fluids, we determined the concentrations of glucose, pyruvate, lactate and 19 amino acids in murine preovulatory follicles and oestrous oviductal and uterine fluids. Follicular fluids were aspirated from hyperstimulated ovaries, whereas oviductal fluids (with/without oocyte-cumulus complexes) and uterine fluids were collected from naturally cycling animals. Glucose, pyruvate and lactate concentrations were analysed using ultramicrofluorometric methods, whilst amino acid profiles were determined by reverse-phase high performance liquid chromatography. Mean glucose concentrations in follicular, oviduct (with/without cumulus cells) and uterine fluids were 0.46, 1.09/1.65 and 0.61 mmol l(-1), respectively. Pyruvate concentrations were 0.38, 0.37/0.17 and 0.25 mmol l(-1), respectively, and lactate concentrations were 17.34, 10.92/11.68 and 9.41 mmol l(-1), respectively. Oviductal pyruvate concentration was significantly higher, and glucose significantly lower, in the presence of cumulus cells. Taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected. Concentrations of amino acids differed among fluids, with highest levels being found in the oviduct. The follicular fluid and tract nutrient profiles differed from those of murine maturation, fertilisation and embryo culture media. These data extend our understanding of cellular metabolism and of nutritional environments of the oocyte and early embryo as they progress along the reproductive tract in vivo. These results may also contribute to the formulation of nutritionally more physiological media for mouse oocyte maturation and embryo culture.     

Phosphate is required for inhibition by glucose of development of hamster 8-cell embryos in vitro

The influence of sodium dihydrogen phosphate (Pi) and glucose on the development of hamster 8-cell embryos mediated by pyruvate (P) or amino acids (A) or lactate (L) was investigated using modified Tyrode's medium, TLP-PVA. When pyruvate was tested as the only energy substrate in medium TP-PVA for embryo development, blastocyst formation ranged from 81.3 to 90.9% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these two compounds were present together, blastocyst formation fell to 51.8%. Similarly, in TA-PVA medium containing four amino acids: Phe, Ile, Met, and Gln), embryo development to blastocyst ranged from 74.1% to 90.4% whether or not the medium contained 0.35 mM Pi or 5 mM glucose; but, when these compounds were present together, blastocyst formation fell to 16.0%. In TL-PVA medium, 10 mM sodium lactate supported embryo development (84.4% blastocysts); the addition of 0.35 mM Pi decreased blastocyst development to 65.6%. However, addition of glucose to Pi-free TL-PVA medium did not decrease blastocyst formation (81.3%); when the medium contained 0.35 mM Pi, glucose curtailed blastocyst development to 7.5%. When glucose and Pi interactions were studied at different concentrations, glucose up to 1 mM was not inhibitory in Pi-free TL-PVA medium (74.3% blastocysts), but 0.25 mM glucose in the presence of 0.35 mM Pi markedly inhibited embryo development (7.7% blastocysts). Phosphate at a relatively high concentration (1 mM) was inhibitory (37.9% blastocysts), even in the absence of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the phosphatidylinositol 3-kinase pathway and is associated with oocyte maturation

Successful in vitro maturation (IVM) of oocytes obtained from medium-sized antral follicles could avoid the need for superovulation for in vitro fertilization. The wide range of doses of FSH used in IVM prompted us to study the effect of varying concentrations of FSH on the dynamics of nutrient uptake and production by individual maturing mouse cumulus-oocyte complexes (COCs). COCs isolated from the antral follicles of unprimed, prepubertal B6CBF(1) mice were cultured individually in increasing concentrations of FSH (0-2000 ng/ml). Following culture, pyruvate, glucose, and lactate uptake or production by individual complexes were noninvasively assessed and compared with the stage of nuclear maturation of the enclosed oocyte. FSH significantly increased oocyte maturation and produced a two- to threefold increase in glucose uptake and lactate production by COCs in which the enclosed oocyte completed maturation. In these COCs, pyruvate was taken up under control conditions but was produced in progressively higher quantities in increasing concentrations of FSH. In COCs where the oocyte failed to complete maturation, pyruvate was taken up (rather than produced) and glucose uptake and lactate production were lower and unaffected by the presence or absence of FSH. This suggests that there is dialogue between cumulus cells and the maturing oocyte that influences FSH responsiveness and substrate metabolism of the whole COC. Finally, inhibition of FSH-stimulated glucose uptake by the PI3-kinase inhibitor LY294002 and the finding of GLUT4 protein in granulosa cells suggest that FSH increases glucose uptake by PI3-kinase-mediated translocation of GLUT4 to the granulosa cell membrane.     

Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.