Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development.

As culture continued in the absence of glucose, embryos decompacted (≈82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.

The early studies of the tn vitro energy requirements of preimplantation mouse embryos were conducted with embryos obtained from random-bred strains of mice (Whitten, 1957; Brinster, 1965a,b;,Biggers et al. 1967; Whittingham, 1969). When cultured from the 1-cell stage, the majority of these embryos arrest at the 2-cell stage; a phenomenon known as the ‘in vitro 2-cell block’. As a result, the identity and optimum concentration of the substrates required to support preimplantation development in vitro were established using the development of late 2-cell random-bred embryos to the blastocyst stage as the experimental model.

Later it was observed that the embryos of certain inbred mouse strains, and their F1 hybrids, were capable of complete preimplantation development (1cell to blastocyst) in a simple chemically defined medium containing lactate, pyruvate and glucose (Whitten and Biggers, 1968). These substrates were used in combination since they had been shown previously to support optimal development of 2-cell random-bred mouse embryos to the blastocyst stage (Biggers et al. 1967, 1971). Although the substrate requirements of random-bred mouse embryos that exhibit the ‘in vitro 2-cell block’ have been examined in considerable detail, the individual roles of the substrates lactate, pyruvate and glucose during development of early inbred and F2 hybrid embryos have not been examined.

Recently, it was shown that culture of random-bred mouse embryos from the 1-cell stage is significantly enhanced by the presence of glutamine (1 min) in the medium (Chatot et al. 1989) and that the embryos of hybrid mice are able to complete preimplantation development in the same medium (Chatot et al. 1990a). The uptake and utilization of radioactively labeled glutamine during culture of 1-cell random-bred embryos have also been described (Chatot et al. 1990b)

The present study examines the specific roles of lactate, pyruvate and glucose, and the possible role of glutamine, during culture of 1-cell F2 mouse embryos of inbred origin.

Embryo collection and culture

Fertilized 1-cell embryos were obtained from B6CBF1 (C57Bl/6×CBA/Ca) hybrid females mated to B6CBF1 males, and random-bred MF1 females mated to MF1 males. The embryos from the F2 cross are referred to as ‘F2’ embryos in the present paper. B6CBF1 mice were superovulated with intraperitoneal injections of 7.5 i.u pregnant mares’ serum gonadotrophin (PMSG, Folligon, Intervet, UK) followed 48 h later by 5 i.u human chorionic gonadotrophin (hCG, Chorulon, Intervet, UK) MF1 mice were superovulated with 5i u. PMSG and 5 i.u hCG given in a similar manner. In all experiments, except experiment 5, fertilized 1-cell embryos were recovered from the oviductal ampullae of mated females between 26 and 27 h post hCG injection. In experiment 5, 1-cell embryos were collected at three time points (19–19.5 h post hCG; 23–23 5 h post hCG; 28–28 5 h post hCG) approximating to the early, mid and late 1-cell stages, respectively The cumulus cells were dispersed with hyaluronidase (150 i u. ml-L) in Hepes-buffered mouse embryo handling medium, M2 (Fulton and Whittingham, 1978, Quinn et al. 1982), and the embryos washed in 2×2 ml changes of M2. In all experiments, embryos were cultured in bicarbonate-buffered mouse embryo culture medium, M16 (Whittingham, 1971) or modified M16 in which some or all of the energy substrates (sodium pyruvate [0.33 mM], sodium lactate [23.28mM] (Sigma, UK), glucose [5.56mM] (BDH, UK)) were omitted. These concentrations are based on those found optimal for culture of late 2-cell embryos from random-bred Swiss mice (Biggers et al 1967). Culture medium was prepared weekly from isotonic stock solutions of the various components so that changes in osmolarity, caused by the omission of an energy substrate, could be compensated for simply by the addition of extra NaCl stock. After washing thoroughly through 2×2 ml changes of the medium in which they were to be cultured, embryos were placed in small drops of medium (10 μl drops; 10 embryos/drop) under light paraffin oil (BDH, UK) in 60 mm plastic tissue culture dishes (Falcon, UK). Both medium and oil had been equilibrated previously with 5% CO2 in air at 37°C. Embryos were cultured in a humidified atmosphere of 5 % CO2 in air at 37 °C as described previously (Biggers et al. 1971), and scored daily for developmental progress. In the treatments where embryos were transferred to fresh medium, containing different combinations of substrates, the washing procedure was repeated.

Assessment of cell number

Embryonic cell number was assessed by counting the number of fluorescently labeled nuclei visualized with the vital fluorescent DNA-binding dye, Hoechst 33258 (Sigma, UK). Embryos were placed in small drops (50 μl) of M2 containing Hoechst 33258 (5 μgml-1) under oil, and maintained at 37°C for 15 min. After staining, embryos were washed three times in large drops (250 μl) of M2 and viewed using a Leitz Epifluorescence microscope.

Terminology

For the purposes of clarity and brevity, the media used in these studies are referred to according to the energy substrates present in their formulation (L=lactate; P=pyru-vate; G=glucose) Thus, unmodified M16 containing all three substrates is designated ‘LPG medium’, whilst modified formulations containing particular substrates only are designated accordingly (eg. L medium=medium containing lactate as the sole available energy substrate; LP medium=medium containing both lactate and pyruvate but lacking glucose).

Experimental design and statistical analysis

Each experiment was repeated three times on different days, except for experiments 2 and 4 which were performed once only. Since no obvious differences were observed in development between replicates, the data from each have been pooled All embryos collected on each day were pooled in the final M2 wash before being allocated to the different treatments. Differences between treatments were tested by χ2 analysis of the combined data, using Yates’ correction for continuity where d.f. = 1 (Fisher and Yates, 1963) Differences in embryonic cell number were investigated by two-tailed t- test analysis of the data.

Experiment 1: The roles of lactate, pyruvate and glucose

The in vitro development of 1-cell F2 embryos cultured in medium containing lactate and pyruvate, alone and in combination, is shown in Table 1. Lactate alone supported the first cleavage division (98%) and second cleavage (50%) in a substantial proportion of the embryos cultured, but was unable to support significant development to the morula stage (1 %). Pyruvate alone supported a high level of development to the morula stage (83%) but was unable to support the morula-to-blastocyst transition (0%). Development to the morula stage in LP medium (94 %) was significantly higher than that observed in P medium (P<0.05), but the combination was unable to support the morula-to-blastocyst transition (0%). Only those embryos cultured in medium containing glucose were able to complete preimplantation development to the blastocyst stage (95%). No differences were observed between development to the morula stage in LP or LPG medium (P>0.05).

Table 1.

The roles of pyruvate, lactate and their interaction, on development of cultured 1-cell F2 embryos

The roles of pyruvate, lactate and their interaction, on development of cultured 1-cell F2 embryos
The roles of pyruvate, lactate and their interaction, on development of cultured 1-cell F2 embryos

Experiment 2: Rate of development to the morula stage in the presence or absence of glucose

To assess whether the presence or absence of glucose affected the rate of development of 1-cell F2 embryos to the morula stage in vitro, the cell number of morulae obtained by culture in LP or LPG medium was compared after 50h of culture (76h post hCG). At this time, all of the 60 embryos cultured in each treatment had reached the compacted morula stage The mean cell number of embryos cultured in LP medium was 11.25±2.15 (n=20), and of those cultured in LPG medium was 12.1±2.10 (n=20). Comparison of the data revealed no significant difference between the two means (P>0.05). The presence or absence of glucose therefore had no effect on the rate of development to the compacted morula stage. After a further 22 h in culture (98h post hCG), all remaining embryos cultured in LP medium had decompacted and degenerated, whilst those in LPG medium had begun to cavitate. The latter subsequently proceeded to the expanded blastocyst stage after 96 h in culture (122 h post hCG). Although the mean cell number of the degenerate morulae in LP medium (12.75±2.38; n=20) was slightly higher than that observed 22 h earlier (11.25±2.15; n=20; 0.02<P<0.05), it would appear that, in the absence of glucose, F2 embryos fail to develop beyond the fourth cleavage division.

Experiment 3: Time of glucose exposure and the morula-to-blastocyst transition

A more detailed examination of the period of glucose exposure required to support the morula-to-blastocyst transition, in F2 embryos cultured from the 1-cell stage in LP medium, was undertaken. A randomized complete block design was employed, the results of which are presented m Table 2. In all treatments, the embryos developed to the compacted morula stage after 50h in culture (76h post hCG). Exposure to glucose for a 22–24 h period at any stage during the first 72 h of culture supported the morula-to-blastocyst transition (treatments 1–3), and resulted in similar levels of development compared to those observed after more prolonged (treatments 5–14) or continuous (treatment 15) exposure (P>0.05). Addition of glucose after the morulae had begun to decompact was unable to support this transition (treatment 4). Embryos cultured in LP medium throughout the 96 h culture period developed to the morula stage, but none developed into blastocysts (treatment 16).

Table 2.

Glucose exposure period required to support the morula-to-blastocyst transition in F2 embryos cultured from the 1-cell stage in LP medium

Glucose exposure period required to support the morula-to-blastocyst transition in F2 embryos cultured from the 1-cell stage in LP medium
Glucose exposure period required to support the morula-to-blastocyst transition in F2 embryos cultured from the 1-cell stage in LP medium

Experiment 4: Determination of the stage at which glucose is unable to support the morula-to-blastocyst transition

A further study was undertaken to determine at what stage exposure to glucose is unable to rescue F2embryos from the ‘morula stage block’ to development obtained by culturing from the 1-cell stage in LP medium. Starting at 70 h post hCG, and at 6 hourly intervals thereafter, groups of 50 embryos were transferred from LP to LPG medium.

Further development of the transferred embryos is shown in Fig. 1. Embryos transferred to LPG medium at 70 h and 76 h post hCG were able to develop into morphologically normal expanded blastocysts after 96 h in culture. Although the percentage response for development to blastocyst appears to tail off progressively in those groups of embryos transferred after 76 h post hCG, it should be noted that the blastocysts that did form were morphologically abnormal. They exhibited poor expansion, with many degenerate cells extruded into both the blastocoel cavity and perivitelline space. The time when transfer to medium containing glucose will not support morphologically normal development through the morula-to-blastocyst transition, therefore appears to lie between 76 to 82 h post hCG (i.e. approximately 3h after compaction of the 8-cell embryo is completed, but before decompaction becomes apparent in embryos grown in the glucose-deficient medium).

Fig. 1.

Proportion of 1-cell F2 embryos developing to the morula and blastocyst stages during 96 h of culture, after transfer from LP to LPG medium at various times between 70 and 94 h post hCG (50 embryos/treatment).

Fig. 1.

Proportion of 1-cell F2 embryos developing to the morula and blastocyst stages during 96 h of culture, after transfer from LP to LPG medium at various times between 70 and 94 h post hCG (50 embryos/treatment).

Experiment 5: Lactate support of the first cleavage division

In experiment 1, it was shown that F2 embryos undergo first cleavage (98%) in medium containing lactate as the only energy source. However, these embryos were flushed from the oviduct between 26 and 27 h post hCG, close to the first cleavage division (onset of first cleavage occurs ≈28 h post hCG; Brown, unpublished observations) and may therefore have contained sufficient endogenous energy to cleave without the need for additional exogenous substrates. The influence of ‘time of flushing’ on progression through first cleavage in L medium was therefore examined. A comparison with random-bred MF1 embryos was also made. The proportions of F2 and MF1 embryos, flushed at the early, mid and late 1-cell stages, developing to the 2-cell stage in L medium are shown in Fig. 2.

Fig. 2.

Proportion of 1-cell F2 and MF1 embryos developing to the 2-cell stage during 24 h of culture in L medium, after removal from the oviduct at various times between 19 and 28.5 h post hCG.

Fig. 2.

Proportion of 1-cell F2 and MF1 embryos developing to the 2-cell stage during 24 h of culture in L medium, after removal from the oviduct at various times between 19 and 28.5 h post hCG.

For both F2 and MF1 embryos, the ability of lactate to support first cleavage was influenced by the time of removal from the oviduct. Overall, there was a significant increase in the ability of lactate to support cleavage of both F2 and MF1 embryos between treatments (P<0.05). Partitioning of χ2 showed that significantly higher proportions of embryos flushed at the late 1-cell stage, than at the early or mid 1-cell stages, underwent first cleavage (P<0.01). No within-strain differences in development were observed between embryos flushed at the early or mid 1-cell stages (P>0.05). Within-treatment development of F2embryos was always significantly higher than that of MF1 embryos (P<0.01).

Although a high proportion of 1-cell F2 embryos cleaved in L medium, subsequent development in LPG medium was severely impaired (Table 3). Only 10 % of early-, 9% of mid-, and 29% of late-flushed 1-cell embryos developed into blastocysts (cf. 95 %, 96 %, 1-cell to blastocyst when F2 embryos were cultured in LPG medium throughout; Tables 1 and 2, respectively).

Table 3.

Subsequent development of 1-cell F2 embryos, cultured for 24 h in L medium, after transfer to LPG medium

Subsequent development of 1-cell F2 embryos, cultured for 24 h in L medium, after transfer to LPG medium
Subsequent development of 1-cell F2 embryos, cultured for 24 h in L medium, after transfer to LPG medium

Experiment 6’ The role of glutamine

The effect of glutamine on development of 1-cell F2 embryos in vitro was also examined (Table 4). LPG medium supported development to the blastocyst stage (98%). LP medium supported development to the morula stage (96%) and a few continued through the morula-to-blastocyst transition (5%). LP medium containing 1 mM glutamine supported similar development to that observed in LP medium (98 % morula, 7 % blastocyst). Thus glutamine was unable to substitute for glucose and support the morula-to-blastocyst transition. Medium containing glutamine only, supported first cleavage to a limited extent (26 %), but was unable to support further development. Arrested 1- and 2-cell embryos all degenerated within the first 48 h when cultured in glutamine-only medium.

Table 4.

Effect of glutamine on the development of cultured 1-cell F2 embryos

Effect of glutamine on the development of cultured 1-cell F2 embryos
Effect of glutamine on the development of cultured 1-cell F2 embryos

Development of 1-cell mouse embryos through the first cleavage division in medium containing lactate as the sole energy substrate, has not previously been observed. Whittingham (1969) showed that lactate alone (31.6 mM) could not support the first cleavage division of cultured embryos from random-bred Swiss mice, removed from the oviduct at the early, mid or late 1-cell stage. In the present study, the random-bred MF1 embryos were capable of limited development in L medium, even when flushed at the early 1-cell stage, but this may have been due to the somewhat lower lactate concentration used (23.28mM) and possible strain differences in the ability of the 1-cell embryo to metabolize lactate. Other investigators have reported a deleterious effect of high lactate concentrations on embryonic development at this early stage (Cross and Brinster, 1973). Although the subsequent viability of F2 embryos cultured through first cleavage in L medium was severely impaired, the results demonstrate a clear strain difference in the ability of lactate to support the first cleavage division of inbred and random-bred embryos.

Of greater interest is the observation that lactate and pyruvate appear to act synergistically in supporting normal morphological, temporal and metabolic development of 1-cell F2 embryos to the morula stage in vitro. A complex relationship exists between the type and concentration of energy sources on development of early mouse embryos in vitro (Brinster, 1965b). Optimal development of late 2-cell stage random-bred Swiss mouse embryos to the blastocyst stage was obtained in medium containing 0.25 mM pyruvate+ 30 mM lactate (Brinster, 1965b; Cross and Brinster, 1973). It is thought that the provision of both substrates in combination produces a redox equilibrium favorable to development (discussed in Whittingham, 1971; Biggers et al. 1971; Kaye, 1986) given the presence of high lactate dehydrogenase levels in the mouse embryo at all preimplantation stages (Brinster, 1965c; Auerbach and Brinster, 1967, 1968). Pyruvate carbon is the principal source of CO2 (Brinster, 1967a,b) and incorporated carbon (Wales and Whittingham, 1974) in the cultured mouse embryo up to the 8-cell stage. The complete ‘morula stage block’ to development obtained by culturing 1-cell F2 embryos in LP medium has not been observed previously however. Brinster (1965b) obtained development of 67 % of late 2-cell embryos from random-bred Swiss mice to the blastocyst stage in LP medium, but these may have been exposed to exogenous glucose during first cleavage in vivo. This could account for their ability to complete the morula-to-blastocyst transition, since F2 embryos exposed to glucose during first cleavage only in vitro were able to develop into blastocysts (experiment 3). Cross and Brinster (1973) obtained development of 53 % of 1-cell embryos of the same Swiss strain to the morula stage in LP medium, and 33% subsequently developed into blastocysts in the absence of glucose. Since none of the 1-cell F2 embryos in our studies developed into blastocysts in the absence of glucose, with the exception of a few in experiment 6, it appears that a small proportion of the Swiss embryos were able to substitute other substrates for glucose during the morula-to-blastocyst transition. Alternatively, it is possible that random-bred embryos may inherit quantitatively larger glucose stores (eg. in the form of glycogen) from the oocyte, thereby permitting a small proportion to undergo the morula-to-blastocyst transition in the absence of exogenous glucose. Since limited development of 1-cell F2 embryos to the blastocyst stage in LP medium (5%) was only observed in one experiment (experiment 6), it is probable that this resulted from contamination of the culture medium with low levels of glucose carried over from the M2 handling medium.

It is interesting that exposure of F2 embryos to glucose during the first cleavage division only, is sufficient to satisfy completely their glucose requirement during the morula-to-blastocyst transition. Previous studies have indicated very little respiratory metabolism or incorporation of glucose at this stage (Fridhandler et al. 1967; Brinster, 1967b; Leese and Barton, 1984; Gardner and Leese, 1988, 1990). Also glucose as the sole energy source is unable to support development until the late 4-to 8-cell stage (Brinster and Thomson, 1966) due to a block to glycolysis (Barbehenn et al. 1974). It may be significant that this is also the stage at which insulin and insuhn-hke growth factors and their receptors are first expressed by the preimplantation mouse embryo (Heyner et al. 1989b), and maternal insulin is taken up by receptor-mediated endocytosis (Heyner et al. 1989a), although recent evidence points to a mitogenic rather than metabolic regulatory role for insulin at this stage (Harvey and Kaye, 1990). The notable exception to this lack of glucose metabolism, however, is glycogen synthesis. Glycogen levels rise 10-fold between the 1- and 2-cell stages in mouse embryos cultured in the presence of 5.56min glucose (Ozias and Stem, 1973). Glycogen levels in vitro are lowered if glucose is omitted from the medium (Ozias and Stem, 1973), but reduction to the uterine level of 0.25 HIM glucose does not reduce incorporation to in vivo levels (Pike and Wales, 1982). Glycogen phosphorylase activity is extremely low at the 2-cell stage, but rises 8-fold by the momia stage, whilst P-glucomutase is 2000 times more active than phosphorylase in 2-cell embryos, and its activity falls by 50% in the 8-cell embryo (Hsieh et al. 1979). Incorporation of exogenous glucose into glycogen at the 1-to 2-cell stage by F2 embryos could therefore satisfy their specific requirement for glucose, and/or its metabolites, during the later morula-to-blastocyst transition.

The precise nature of the embryonic requirement for glucose remains unclear. Due to the specific timing of the requirement (between 76 and 82h post hCG), it seems unlikely that this is related to a sudden switch from a pyruvate/lactate-based metabohsm to one solely dependent on glucose. Although the blockade to glycolysis is removed at this stage (Barbehenn et al. 1974) and glucose alone becomes able to support cleavage (Brinster and Thomson, 1966), there is no evidence to suggest that TCA cycle activity is simultaneously downregulated in the momia. Continued development of the mammahan embryo relies on both temporal and stage-dependent developmental programs (Johnson et al. 1984). Glucose, and/or its metabolites, may therefore be required for the synthesis of some stage-specific, developmentally essential embryonic component(s) involved in the morula-to-blastocyst transition. Since 1-cell F2 embryos cultured in LP medium develop to the momia (8-to 16-cell) stage and compact, before decompacting and degenerating, synthesis of the calcium-dependent cell adhesion glycoprotein uvomorulin/cadherin (Hyafil et al. 1980; Damsky et al. 1983) presumably proceeds normally in the absence of glucose. However, maintenance of compaction requires the development of a calcium-independent mechanism of cell adhesion (Ogou et al. 1982). Fucosylated cell surface glycoproteins are involved in this secondary, calcium-independent stabilization of the compacted state (Kimber, 1990). Recently, it was shown that exogenous radiolabeled glucose participates significantly in the glycosylation of membrane-associated glycoproteins during culture, particularly at the momia stage, when both quantitative increases and qualitative changes in incorporation are observed (Wales and Hunter, 1990). As yet it is unknown whether synthesis of these glycoproteins is prevented by culture in the absence of glucose, providing a possible mechanism for the loss of compaction observed.

The role of glutamine in supporting mouse preimplantation development remains uncertain. Exogenous glutamine incorporation and uptake increase 10fold and 5-fold, respectively, between the 1-cell and momia stages (Brinster, 1971). Embryos cultured from the 2-cell stage in the absence of exogenous amino acids have lower levels of free amino acids than those of a similar stage in vivo (Sellens et al. 1981), whilst embryos cultured in the presence of exogenous glutamine incorporate 2–3 times more glutamine than those raised in vivo (Chatot et al. 1990b). In 1-cell F2 embryos, glutamine alone (IHIM) supported the first cleavage division to a limited extent, but was unable to substitute for glucose in supporting the morula-to-blastocyst transition. While exogenous glutamine may be helpful to promote development of 1-cell random-bred embryos to the blastocyst stage when provided in combination with lactate and pymvate (in the absence of glucose) during the first 48 h of culture (Chatot et al. 1989), no similar requirement by F2 embryos was observed in the present study. Although a small proportion of random-bred 1-cell embryos have been reported to develop into blastocysts in LP medium (with or without glutamine) in the absence of glucose (Cross and Brinster, 1973; Chatot et al. 1989), our results demonstrate that the glucose requirement of F2 embryos undergoing the morula-to-blastocyst transition is absolute. Such a system should permit the simple and rapid identification of those embryonic processes, crucial to the success of this transition, which require the provision of exogenous glucose during culture, and presumably also in vivo.

The authors would like to thank David Gilburt for excellent technical assistance, and Cindy Welch for typing the manuscnpt. J.J.G.B was the recipient of an MRC Postgraduate Studentship.

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