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Objectives: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC-MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer., Methods: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts., Results: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts., Conclusions: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also increased glucose uptake and changed amino acid turnover by the cells. These changes were distinct from those induced by serum and occurred in the absence of detectable differentiation. Induction of differentiation by bone morphogenetic protein 4 (BMP4), in contrast, did not alter metabolite turnover. Deviations from metabolite turnover by ES cells in fully defined medium demonstrated that culture environment can alter metabolite use. The challenge remains to understand the impact of metabolic changes on long-term cell maintenance and the functionality of derived cell populations.

Background: The aim of this study was to determine the relationship between nutrient utilization by the human embryo and its subsequent viability after transfer. METHODS The embryos of 50 patients having single blastocyst transfer were cultured individually from Day 3 in 10 µl drops of medium G2 under Ovoil in 5%O(2), 6%CO(2), 89%N(2). Patient inclusion in the study was maternal age ≤ 38. Embryos were moved to fresh drops of medium every 24 h. Spent media samples, including controls containing no embryo, were coded, frozen and subsequently analysed blind. Analysis of glucose was performed by microfluorimetry. The sex of children born was recorded., Results: Clinical pregnancy and live birth rates were 58 and 56%, respectively. Glucose consumption by embryos which resulted in a pregnancy was significantly higher on both Day 4 and Day 5 than that by embryos which failed to develop post-transfer (P < 0.01). Furthermore, on Day 4 female embryos consumed 28% more glucose compared with males (P < 0.05). Glucose uptake was independent of embryo grade., Conclusions: The rapid screening of glucose metabolism by the human embryo on Day 4 and 5 may prove to be a useful metric in the development of algorithms for the selection of embryos for transfer in human IVF. Also, the observed sex-related metabolic difference provides preliminary data to support the hypothesis that male and female human embryos differ in their physiology due to the presence of two active X chromosomes and an altered proteome for a finite time during the preimplantation period.

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., ((c) 2005 Wiley-Liss, Inc.)

The objective was to compare effects of three metabolic regulators on development of post-compaction bovine embryos. In-vitro-produced 8- to 16-cell embryos were allocated to treatments for 72 h in G2.2 medium as follows: 0.3 microm phenazine ethosulfate (PES); 27 microm sodium azide (NaN3); 30 microm 2,4-dinitrophenol (DNP); and control, no regulator. Treatments responded similarly for blastocyst rates and embryo quality responses (P > 0.1). The PES treatment resulted in higher glucose metabolism than the NaN3 treatment (18.5 v. 14.5 pmol per embryo per h, P < 0.05), and both did not differ from DNP or the control. The PES treatment tended to result in more flux of glucose through the pentose phosphate pathway (PPP) than the control (50.5 v. 21.5%, P < 0.11). The NaN3 treatment caused more glucose uptake than the PES treatment (38.9 v. 13.1 pmol per embryo per h, P < 0.01), but neither differed from the control or DNP treatment (P > 0.1). Glycolysis for the PES treatment was 187%, which was higher than any of the other groups (88-94%; P < 0.01). There were fewer medium + large lipid granules in the cytoplasm of PES-treated embryos than any other group, including the in vitro control (P < 0.01). However, in vivo control embryos had still fewer large and medium-sized lipid granules (P < 0.01) than the PES treatment. Developmental competence to Day 14 after embryo transfer was similar among treatments. The PES treatment increased glucose metabolism, tended to increase the PPP flux of glucose and clearly reduced accumulation of lipids in embryos produced in the chemically defined media used. Use of PES in culture media may be a promising approach to improving in vitro production of embryos.

Glucose uptake was used as a measure of metabolic activity and implantation potential to compare vitrification and slow freezing in a prospective randomized trial using murine blastocysts. Frozen 2-cell embryos (n = 132) thawed and cultured for 48 h to the blastocyst stage were randomly divided into four groups: (i) control - not refrozen; (ii) slow freezing using a programmed rate (PR); (iii) vitrification by super-cooled (VSC) liquid nitrogen; and (iv) vitrification in liquid nitrogen (VLN). Upon re-thawing, embryos were cultured individually for 24 h to determine glucose uptake non-invasively. Morphological assessments included total cell counts and inner cell mass (ICM) detection following immunosurgery. Mean glucose uptake was lower for each treatment (PR and VSC, 4.3 pmol/embryo per h; VLN, 4.9 pmol/embryo per h) versus controls (6.8 pmol/embryo per h). PR and VSC embryos had fewer cells (57.4 +/- 24.2 and 64.1 +/- 31.5) versus controls (85.7 +/- 26.2), and fewer embryos containing a detectable ICM (42.9 and 61.8%) compared with controls (88.2%). The only difference between control and VLN embryos was absolute glucose uptake, although in both treatments glucose uptake was increased from embryos with an ICM compared with those without. Glucose uptake appears to be a sensitive, non-invasive method to validate cryopreservation protocols.

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus-oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 microl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0-16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8-12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8-16 h of maturation, than by oocytes that did not fertilize (8-12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12-16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

Nutrient uptakes and metabolite production by equine morula and blastocyst stage embryos were determined by non-invasive microfluorometry. Equine morula took up equal amounts of both pyruvate and glucose. However, at the early blastocyst there was a small increase in glucose uptake and, by the expanded blastocyst stage, glucose was the predominant nutrient. Expanded blastocysts took up five times more glucose than pyruvate. Expanded blastocysts exhibited an exponential increase in glucose uptake and lactate production with respect to both diameter and surface area. As less than 50% of the glucose was accounted for by lactate production, the equine blastocyst appears to have a significant capacity to oxidize glucose. Embryos with a higher morphological grade consumed more nutrients than those with a poorer morphology. However, there was a large range in nutrient consumption within the highest grade blastocysts. This suggests that nutrient uptake may be useful as a viability marker of equine blastocysts.

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., (Copyright 2001 Wiley-Liss, Inc.)

The aim of the study was to compare the energy metabolism of oocytes from pre-pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre-pubertal oocytes. The metabolism of [5-(3)H] glucose, [2-(14)C] pyruvate, and [G-(3)H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre-pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre-pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre-pubertal cows at 12 hr (P < 0.05). Oocytes from pre-pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre-pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre-pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre-pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post-aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre-pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre-pubertal and adult cows that may account for the poor developmental potential of many pre-pubertal oocytes., (Copyright 1999 Wiley-Liss, Inc.)

Metabolic reactivation of the tammar blastocyst appears to be characterized by a change in the pathway of glucose metabolism rather than an absolute increase in substrate uptake. The switch in type of metabolism used was examined to gain information on the timing and physiology of blastocyst reactivation. Fluorescent and radioisotope techniques were used sequentially to determine the activity of pathways of glucose metabolism by individual wallaby blastocysts during diapause and 3, 4, 5, 6, 7, 8, and 10 days after removal of pouch young (RPY). Maternal endometrial and luteal cell metabolism and circulating hormone levels were measured and correlated with blastocyst activity. Observed differences between rates of blastocyst reactivation could be explained by variation in the maternal response between animals. While blastocysts recovered 4 days after RPY oxidized more glucose compared with Day 0 blastocysts (p < 0.05), rates of glycolysis did not change until Day 10. Blastocysts recovered between 4 and 10 days after RPY oxidized a significantly greater percentage of the glucose taken up (p < 0.01). The reduced ATP:ADP ratio within blastocysts recovered 3 days after RPY (p < 0.05) indicates that conditions are suitable for blastocysts to undergo a metabolic switch from glycolytic to oxidative metabolism of glucose on Day 4 after RPY. The increased oxidation results in greater ATP production, which plausibly fuels the increased energy requirements of wallaby blastocysts during the early stages of reactivation.

The mouse, Mus musculus, can maintain blastocysts in embryonic diapause in the uterus while suckling young. This study used microfluorimetry to simultaneously examine glucose and pyruvate uptake by quiescent blastocysts, and at four hourly intervals after administration of the reactivating stimulus, oestradiol-17 beta. Following the non-invasive analysis of energy metabolism, blastocysts were incubated in colcemid (0.2 mg/ml), and mitotic activity determined. Mitoses and cell numbers in reactivated embryos increased significantly within 8 and 12 hours, respectively, after oestradiol-17 beta administration, compared to those of diapause (control) blastocysts (0.5 +/- 0.1 vs. 0.22 +/- 0.03 mitoses/embryo; P < 0.05, and 141.8 +/- 1.5 vs. 133.8 +/- 2.4 cells/ embryo; P < 0.05). Similarly, pyruvate uptake by reactivating blastocysts (9.3 +/- 1.1) was significantly higher than controls (5.8 +/- 0.8 pmol/embryo/hour; P < 0.05), within 4 hours of oestradiol-17 beta, but by 16 hours after oestradiol-17 beta administration, pyruvate uptake by reactivating blastocysts was no longer significantly different from the delayed controls. In contrast, significant differences in glucose uptake between the reactivated and control groups were not evident until 16 hours after oestradiol-17 beta (reactivating, 14.9 +/- 1.5; control, 10.6 +/- 1.7 pmol/embryo/hour; P < 0.05). These results demonstrate that pyruvate rather than glucose could supply the additional energy required during the first 12 hours of reactivation in the mouse, but from 16 hours after injection of the reactivating stimulus oestradiol-17 beta, glucose is the predominant energy source.

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time.

Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.

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 (approximately 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 (approximately 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.

Large White gilts, 9 to 18 months old, that had exhibited at least two natural oestrous cycles were divided into three groups (phases): unmated pre-ovulatory, unmated post-ovulatory and mated post-ovulatory (n = 16, 20 and 18). Oviductal luminal fluid samples were collected under anaesthesia by micropipette from the ampulla and ampullary-isthmic junction and analysed by an ultramicrofluorometric technique. Glucose concentrations (mmol 1(-1), means combining regions; mean +/- SEM) were significantly higher in blood plasma than in oviductal fluid (4.56 +/- 0.20 versus 0.59 +/- 0.16; P < 0.0001; n = 27), whereas lactate was higher in the oviduct (5.71 +/- 0.53 versus 2.48 +/- 0.24; P < 0.0001; n = 27). No significant differences were found between the ampulla and the ampullary-isthmic junction. However, the concentration of glucose was significantly higher (P < 0.05) in the ampulla of the pre-ovulatory group (0.97 +/- 0.20; n = 13) compared with the mated group (0.25 +/- 0.05; n = 14) and its concentration in the ampullary-isthmic junction in the pre-ovulatory group (1.65 +/- 0.63; n = 13) was significantly greater (P < 0.05) than in the post-ovulatory (0.43 +/- 0.11; n = 11) or mated groups (0.17 +/- 0.02; n = 14). Lactate in the ampulla of mated animals was higher than in the pre-ovulatory group (6.83 +/- 0.70 versus 3.86 +/- 0.38; P < 0.05; n = 15 and 13), but neither was significantly different from the post-ovulatory group. Furthermore, no change was seen at the ampullary-isthmic junction in lactate concentration with phase.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultramicrofluorometric technique was used to analyse the nutrient composition of mouse oviduct fluid. The concentrations of pyruvate, glucose and lactate in the vicinity of the cumulus mass were 0.37, 3.40 and 4.79 mM respectively. In the absence of cumulus cells, the concentration of pyruvate was significantly reduced, to 0.14 mM, while the concentration of glucose was significantly increased to 5.19 mM. Glutamine, which may help to overcome the '2-cell block' in mouse embryos in culture, was present at a concentration of 0.20 mM. A modified medium (MTF) in which the concentration of nutrients was similar to that in mouse oviduct fluid was prepared and its effects on embryo development and metabolism in vitro were compared with that of a conventional embryo culture medium (M16). The percentage of zygotes forming blastocysts in vitro by Day 5 was similar in both media (82% in M16, 79% in MTF). Rates of development, as assessed by cell number, were also comparable. However, the proportion of glucose consumed which was converted to lactate increased dramatically following culture; from 44% in fresh blastocysts, to 73% and 91% in blastocysts derived from 8-cell embryos cultured for 24 h in media MTF and M16 respectively.

The uptake of pyruvate and glucose by single, cultured mouse embryos has been measured non-invasively throughout the pre-implantation period. The embryos, derived from CBA x C57F1 females and F1 males were removed from the oviducts on day 1 or day 2 following mating, and cultured to the blastocyst stage in M16 medium. Each day they were removed from culture and incubated in 20 nl medium M2 for 3 h. Serial approximately 0.5-nl samples were analysed for pyruvate and glucose by an ultramicrofluorescence technique. Pyruvate uptake exceeded that of glucose until the 8-cell/morula stage, when glucose consumption increased dramatically. The morphological development and patterns of nutrient uptake by embryos removed on day 2 resembled more closely those of freshly collected embryos than of embryos removed on day 1. These methods will enable the biochemistry of early embryos to be monitored non-invasively prior to transferring them into the uterus of recipient females.

Preimplantation mouse embryos utilize pyruvate preferentially during the early cleavage stages before switching to glucose at around the time of compaction. This switch in substrate preference has been studied using a non-invasive ultramicrofluorometric analytical technique on single mouse embryos. On the basis of transport kinetic studies and inhibition by phloretin, cytochalasin B and sugar analogues, a component of glucose uptake by mouse blastocysts was found to be mediated by facilitated diffusion. The Jmax and Kt of this facilitated component were 3.53 pmol embryo-1 h-1 and 0.14 mM, respectively. At physiological concentrations of glucose, the facilitated component accounts for around 75% of glucose uptake. Glucose uptake by blastocysts was found to be insensitive to insulin, added at a range of concentrations. There was no evidence for glucose active transport. The carrier-mediated component of glucose entry was detectable from the 2-cell stage onwards. Pyruvate uptake was also mediated by a carrier throughout development. In the absence of glucose in the incubation medium, the characteristic decline in pyruvate uptake does not occur. The data are consistent with a role for embryonic cell transport in regulating glucose utilization prior to compaction, but do not exclude the involvement of metabolic factors, such as the allosteric regulation of the enzymes hexokinase and phosphofructokinase.

An ultramicrofluorometric technique was used to measure the uptake of glucose by single mouse blastocysts prior to transferring them into the uterus of pseudopregnant foster mothers. Of 50 blastocysts whose glucose uptake was measured prior to embryo transfer, 25 gave rise to male and 13 to female offspring. Twelve embryos failed to develop. The mean glucose uptake of these 12 embryos was significantly below that of those which developed successfully. Female embryos took up slightly more glucose than male embryos.