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Embryo; |
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, 4072 Australia
ABSTRACT
Although mouse oocytes and cleavage-stage embryos are unable to utilize glucose as a metabolic fuel, they have a specific requirement for a short exposure to glucose prior to compaction. The reason for this requirement has been unclear. In this study we confirm that cleavage-stage exposure to glucose is required for blastocyst formation and show that the absence of glucose between 18–64 h after hCG causes an irreversible decrease in cellular proliferation and an increase in apoptosis. More importantly, this glucose signals to activate expression of Slc2a3 transcript and SLC2A3 protein, a facilitative glucose transporter (previously known as GLUT3) associated with developmental competence and increased glucose uptake used to fuel blastocyst formation. Glucosamine could substitute for glucose in these roles, suggesting that hexosamine biosynthesis may be a nutrient-sensing mechanism involved in metabolic differentiation. Inhibition of the rate-limiting enzyme in this pathway, glutamine-fructose-6-phosphate amidotransferase (GFPT), inhibited expression of the SLC2A3 transporter protein and blastocyst formation. Glucosamine, a substrate that enters this pathway downstream of GFPT, was able to overcome this inhibition and support SLC2A3 expression. These data suggest that early embryos rely on hexosamine biosynthesis as a glucose-sensing pathway to initiate metabolic differentiation.
blastocyst formation, early development, gene regulation, glucose signaling, hexosamine biosynthesis
Adequate nutrient supply to the embryo prior to implantation is not only essential to early embryonic growth and development but has also been implicated in metabolic programming that influences later growth and the onset of adult disease [1]. However, the molecular mechanisms involved in early embryonic nutrient sensitivity and subsequent programming have not yet been determined. In this study we examined the basis of the long established cleavage-stage requirement for glucose during early development and blastocyst formation.
Preimplantation embryos from a number of species are unable to utilize glucose as an energy substrate prior to compaction. In the mouse, the first cleavage division requires the presence of pyruvate [2], and the second is supported by both pyruvate and lactate [3, 4]. Glucose becomes the preferred fuel following compaction at the 8-cell stage [5] when it is required to fuel the high energy requiring Na+/K+ ATPases that facilitate cavitation. Acquisition of the metabolic competence to utilize glucose for energy correlates with the apical expression of the high-affinity, high-capacity glucose transporter SLC2A3 at compaction [6]. Ablation of this transporter results in failure to form a blastocyst, suggesting a link between expression of this transporter and metabolic differentiation to a state amenable to burning glucose and blastocyst formation [6].
Although the requirement for glucose after compaction and the high ATP turnover required for cavitation are well established, the role of glucose in fertilized oocytes and cleaving embryos prior to compaction remains elusive. Embryos isolated from the maternal tract at the 2-cell stage can be cultured in the absence of glucose and can form blastocysts. These 2-cell embryos therefore have the adaptive capacity to accommodate the increased energy demands of cavitation by increasing their pyruvate consumption to cope with the absence of glucose. Moreover, these embryos have also developed the capacity to transport glucose at high rates should it become available [7]. However, if zygotes are collected and cultured without glucose, they do not progress beyond the morula stage. These embryos do not develop the capacity to transport glucose should it become available, nor the adaptive capacity to cope with its absence [7]. Remarkably, brief exposure to glucose prior to the morula stage is sufficient to "prime" these naïve embryos and permit subsequent blastocyst formation [8–10]. This inability to transport bulk glucose, coupled with failure to form a blastocyst, suggests that SLC2A3 expression is compromised in these embryos. To test this hypothesis, expression of Slc2a3 mRNA and SLC2A3 protein was assessed in embryos that had been cultured in the absence of glucose. Moreover, using the previously established model of a glucose pulse [10], we examined the hypothesis that early glucose exposure acts as a signal that is required to activate Slc2a3 transcription and translation and thus facilitates metabolic differentiation and blastocyst formation. The possibility that this "glucose sensing" was mediated via the hexosamine biosynthetic pathway (HBP) was also explored.
All experiments on mice were approved by the Animal Ethics and Experimentation Committee of the University of Queensland, a committee approved by the National Health and Medical Research Council of Australia.
Zygotes were collected from 6- to 10-wk-old superovulated and mated outbred Quackenbush mice (bred and housed in our own colony within the School of Biomedical Sciences Animal Facility) at 18 h after hCG in M2 medium [11] without glucose and modified to contain 0.33 mM Na pyruvate and 4 g BSA per liter [12]. Cumulus cells were removed with 1500 IU hyaluronidase per liter, and zygotes were cultured under mineral oil at 37°C in a humidified atmosphere of 5%CO2/5%O2/90%N2 in KSOM medium [13] in the presence and absence of glucose for up to 120 h after hCG or as per experimental design below. Amino acids were not included so as to limit gluconeogenic activity. It should be noted, however, that glutamine, a standard component of KSOM medium, was included because it is required for the conversion of fructose-6-phosphate to glucosamine-6-phosphate by glutamine-fructose-6-phosphate amidotransferase (GFPT).
The permissive effect of short exposure to glucose or glucosamine "pulse" on blastocyst formation and Slc2a3 transcription and translation was explored using a model of glucose activation based on that of Chatot et al. (1994) [10]. This model used a short exposure to 27 mM glucose to restore blastocyst formation. Pulse duration was minimized to 1–3 h to facilitate use of pharmacologic inhibition without the added complications of longer-term nonspecific effects. Thus, zygotes collected and cultured in the absence of glucose were exposed to a 1–3 h pulse of either 27 mM glucose or glucosamine prior to 67 h after hCG [10]. Assessment of cell number and apoptosis was carried out at 90–94 h after hCG as embryos cultured in the absence of glucose degenerate shortly thereafter. The effect on blastocyst formation of continuous culture in the presence of 1 mM glucosamine in the absence of glucose was also explored. Though in our experimental model we assessed blastocyst formation at approximately 96–112 h after hCG to mirror the in vivo model (see Fig. 1), we have also used extended culture to 120 h after hCG (see Figs. 4 and 5) to more closely reflect the more commonly used model in the published literature [10]. To test for participation of the HBP, the effect on blastocyst formation of GFPT inhibition with 4 µM azaserine was assessed in embryos exposed to a 3-h pulse of 27 mM glucose (control treatment) or a 3-h pulse of 27 mM glucose or glucosamine with a 4-h pulse (30 min before, during, and after addition of glucose/glucosamine) of 4 µM azaserine. The effect of these treatments on expression of the SLC2A3 protein was assessed.
Apoptosis and Cell Number Determination
Embryos were fixed and permeabilized [14], and apoptosis was determined by TUNEL assay using the In Situ Death Detection Kit (Boehringer Mannheim, Mannheim, Germany). Following permeabilization, embryos were washed twice in PBS containing 5 mg/ml polyvinyl-pyrrolidone (PBS/PVP; Sigma Chemical Co., St. Louis, MO) and incubated in 15-µl drops containing 10 U/µl terminal deoxynucleotidyl transferase (DNTT), 20 µM fluorescein-conjugated dUTP, and reaction buffer for 1 h at 37°C in the dark. Positive controls were incubated with DNase I (100 U/ml; Sigma), which cleaves all DNA, for 20 min at 37°C before TUNEL. Negative controls were incubated in fluorescein-dUTP in the absence of DNTT. After TUNEL, embryos were washed three times with PBS/PVP, counterstained with 0.5 µg/ml propidium iodide (Sigma) after RNase A treatment (50 µg/ml, 60 min, 37°C), and mounted in hanging drops of glycerol prior to examination.
Expression and Immunolocalization of SLC2A3
The anti-SLC2A3 antibody was raised in rabbits against a synthetic carboxyl-terminal dodecapeptide of mouse SLC2A3 coupled to thyroglobulin. This antiserum was monospecific for the SLC2A3 transporter and found to immunolocalize on the apical trophectoderm of morulae and blastocysts [6].
Embryos were fixed, permeabilized, immunoprobed for SLC2A3 protein as previously described [6], and examined using a Bio-Rad MRC-600 confocal laser scanning microscope mounted on a Zeiss Axioskop equipped with a Zeiss Plan-APOCHROMAT x 63 oil immersion objective. An embryo was counted as expressing SLC2A3 if SLC2A3 immunoreactivity was above immunological control levels and distinctly concentrated apically as previously described [6].
Reverse Transcription Polymerase Chain Reaction
Total embryo RNA obtained using phenol/chloroform extraction was reverse transcribed and amplified by PCR using Slc2a3-specific primers as previously described [6]. The 284-bp PCR product was resolved on 2% agarose gels and its identity confirmed by sequencing on an Applied Biosystems 373A DNA sequencer. Contamination of cDNA samples with genomic DNA was tested by PCR for mouse actin with a primer pair that generates a predicted 243-bp fragment for the cDNA and a 330-bp fragment if contaminating genomic DNA is present due to presence of an intron [15].
Prism (version 3; GraphPad) was used for the Fisher exact test, chi-square analysis, ANOVA, and multiple comparison tests as noted in Results and the figure legends.
Effect of Glucose Deprivation on Blastocyst Formation, Cell Number, Apoptosis, and SLC2A3 Expression
Zygotes cultured in the complete absence of glucose degenerated as morulae and failed to form blastocysts, in contrast to those cultured with glucose (Fig. 1), which, albeit displaying the typical developmental delay of cultured oocytes, nonetheless formed blastocysts by 112 h after hCG. Not only did embryos cultured in the absence of glucose fail to form blastocysts, they displayed fewer cells as late morulae (90 h after hCG; P < 0.01; Fig. 2A) and increased levels of apoptosis as assessed by counting the proportion of TUNEL-positive nuclei in each embryo (P < 0.01; Fig. 2B). Though exposure to a pulse of glucose at 64 h after hCG reversed this effect on apoptosis to a level not different from control (Fig. 2B), the effect on cell number was not fully reversed (Fig. 2A). Expression of Slc2a3/SLC2A3 was inhibited by absence of glucose as assessed both by RT-PCR and immunofluorescence, respectively (Figs. 3 and 4), but restored by exposure to glucose before the 8-cell stage (Figs. 3 and 4).
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Glucosamine could substitute for glucose in supporting both SLC2A3 protein expression (Fig. 4) and blastocyst formation (Fig. 5). Zygotes cultured in KSOM without glucose and supplemented with 1 mM glucosamine overcame the block to blastocyst formation and developed to blastocysts. Embryos exposed to a pulse of glucose or glucosamine also developed to the blastocyst stage at the same rate as those cultured continuously in the presence of glucose or glucosamine (Fig. 5). Immunoreactive SLC2A3 was characteristically present in the outer apical trophectoderm in blastocysts cultured in the presence of glucose (Fig. 4) and in embryos pulsed with either glucosamine or glucose (Fig. 4). In the absence of glucose, the embryos had largely degenerated by 120 h after hCG, and the characteristic pattern of SLC2A3 immunoreactivity was not apparent. Given that the majority of embryos were degenerated, corresponding mRNA expression was sought, and Slc2a3 mRNA was found absent at 94 h after hCG (Fig. 3). Slc2a3 transcription was restored by a glucose pulse at 50 h after hCG (Fig. 3).
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GFPT Inhibition and Blastocyst Formation
Azaserine, a glutamine analogue and competitive inhibitor of GFPT, the rate-limiting enzyme of the HBP (Fig. 6), was used to assess the role of this pathway in the glucose activation of blastocyst formation and SLC2A3 expression. To limit potential effects due to interference with other glutamine utilization pathways such as purine biosynthesis, the duration of azaserine exposure was minimized by bracketing the azaserine treatment around the glucose pulse. Glucosamine, which can be converted to glucosamine-6-phosphate and thus enter the HBP downstream of GFPT (thus bypassing GFPT inhibition; Fig. 6), was to act as a positive control to demonstrate the effect of GFPT inhibition on glucose-mediated SLC2A3 expression.
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Azaserine blocked the effect of the glucose pulse, reducing blastocyst formation and the distinctly apical expression of SLC2A3, whereas glucosamine bypassed the azaserine inhibition and allowed SLC2A3 expression (Fig. 7).
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Despite their inability to utilize glucose for energy prior to compaction, mouse embryos have an absolute requirement for at least a brief glucose exposure to permit normal proliferative, differentiative, and metabolic development [7, 8, 10]. In the absence of this glucose, which may be as brief as 1 min [10], postcompaction development is inhibited. Our data confirm that embryos deprived of glucose cleave to form morulae, but fail to form blastocysts and subsequently degenerate. Although two previous studies report that some blastocyst formation does occur in the absence of glucose (15% or less [16]; 36% [17]), it should be noted that slight differences in methodology, including time of collection (and hence prior exposure to oviductal fluid relative to this study), culture medium, and conditions used, may account for these differences. Specifically, in the latter study the culture medium used (DM2 –glucose) contained amino acids and thus a mechanism for gluconeogenesis to occur. In this study, we have omitted amino acids from the culture medium to specifically eliminate the confounding impact that potential gluconeogenesis would provide. Moreover, in the study by Leppens-Luisier [16] the use of M16 as the base medium provides a higher level of glucose (5.5 vs. 0.2 mM) than the universally preferred KSOM, making the findings more difficult to interpret given the negative impact that higher levels of glucose are reported to have on development.
Nonetheless, our study clearly shows that the complete absence of glucose during the early cleavage stages as well as not supporting blastocyst formation also causes a significant decrease in cell number. Although previous studies had similarly shown that the absence of glucose leads to a significant reduction in the number of cells present in late blastocysts 122–124 h after hCG [7, 10], it should be noted that embryos in our studies cultured in the absence of glucose were largely degenerate by this time. A glucose pulse applied at 64 h after hCG significantly improves cell number, but not to the level of embryos continuously exposed to glucose. This decrease in cell number in the absence of glucose may arise from inhibited mitosis and/or increased apoptosis. Consistent with this proposal, the proportion of apoptotic cells in embryos cultured in the complete absence of glucose was also significantly increased.
The ability of a glucose pulse to reduce the rate of apoptosis to a level not different from control, in contrast to its partial restoration of cell number, suggests that the absence of glucose during the first two or three cleavage divisions (18–64 h after hCG) irreversibly reduces the rate of proliferation and thus potentially affects later stage development. More importantly, however, embryos cultured in the absence of glucose display a morphologic and metabolic phenotype that matches that of embryos in which SLC2A3 expression is blocked, in the sense that blastocyst formation is inhibited and glucose transport capacity is significantly reduced [6, 7], suggesting that early glucose exposure may be involved in the activation of SLC2A3 expression.
That a pulse of glucose is able to restore both expression of Slc2a3 mRNA and protein indicates that glucose can act as a signal, with impact on embryonic gene expression. Moreover, embryos cultured in the absence of glucose fail to maintain their capacity to transport pyruvate and thus to accommodate for the absence of glucose [7]. We have previously shown that this reduced pyruvate transport capacity correlates with a decline in the expression of the monocaroxylate transporter protein SLC16A1 in the absence of glucose, but this expression is also responsive to a glucose pulse [18], suggesting that glucose is indeed essential for metabolic differentiation in the mouse embryo. How does this cleavage stage exposure operate to control apoptosis, cell proliferation, SLC2A3 and SLC16A1 expression, and blastocyst formation?
That nonmetabolizable glucose analogues such as methyl glucose and 2-deoxy glucose are unable to restore blastocyst formation [10] suggests that a transcriptionally active downstream metabolite of glucose is responsible for this phenomenon. The reported efficacy of fructose in improving cell number [10] suggests that conversion to fructose-6-phosphate is involved. Moreover, though glutamine alone is unable to substitute for glucose [8], its incorporation increases 10-fold between the zygote and morula stage [19], and there is a 3-fold increase in Vmax of glutamine uptake, and thus uptake capacity, between the 2-cell and blastocyst stages [20]. Fructose-6-phosphate formation is the first metabolite in hexosamine biosynthesis, and glutamine acts as the amide donor, suggesting that this may be the pathway by which these effects of glucose are mediated.
The HBP is implicated as a glucose sensing and effector pathway in many somatic cell types [21]. The ability of glucosamine, an alternate substrate to glucose for hexosamine biosynthesis, to overcome the block to blastocyst formation and SLC2A3 expression supports and further implicates hexosamine biosynthesis in embryonic glucose sensing. Inhibition of the rate-limiting enzyme GFPT with azaserine was able to block the signaling of glucose to activate SLC2A3 transcription. Azaserine, a widely used inhibitor of GFPT, was non-embryo toxic and specific to GFPT in mesangial cells at this concentration [22]. That glucosamine can overcome this inhibition is further evidence that as used azaserine has no toxic effects, but more importantly that hexosamine biosynthesis is involved in the glucose-facilitated expression of SLC2A3 and consequent metabolic differentiation to reliance on glucose.
As its end product, hexosamine biosynthesis produces the acetylated amino-sugar nucleotide uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), which acts as the donor substrate for both N- and O-linked glycosylation of lipids and proteins and the synthesis of other sugar nucleotides. The formation of 
-1–6-N-acetylglucosaminyl-phosphatidyl inositol from UDP-GlcNAc and phosphatidyl inositol is the first step in the biosynthesis of the GPI lipid anchor [23]. UDP-GlcNAc may also undergo a series of epimerase reactions to form N-acetyl-D-galactosamine or acetylmannosamine and ultimately CMP-N-acetyl-neuraminic acid, an important carbohydrate moiety for many glycosides, glycoproteins, and glycolipids that are likely to be important in embryo development and maturation. Indeed experiments exploring [U-14C] glucose incorporation into embryonic glycoproteins demonstrate both quantitative and qualitative increases in glucose incorporation into the glycoprotein fraction with development [24]. While tunicamycin, a specific inhibitor of N-linked glycosylation, suppressed glucose incorporation into glycoproteins and inhibited compaction [24, 25], it had no effects on other parameters of metabolism including glycolysis [24], suggesting that metabolic differentiation proceeds normally in these embryos in direct contrast to the effects of glucose deprivation. Interestingly, [14C] amino acid incorporation into the glycoprotein fraction was found to be insensitive to tunicamycin treatment [24], whereas an earlier study using 3H-glucosamine showed that 40% of the incorporated glycoprotein label in blastocysts is tunicamycin insensitive [25].
The most likely downstream effector pathway in activating metabolic differentiation is O-linked modification of cellular proteins by N-acetylglucosamine. This modification alters the activity and/or stability of key regulatory proteins and factors, thus regulating cellular homeostasis in response to glucose availability. Unlike most other forms of glycosylation it is found primarily in the nucleus and cytoplasm [26], where it modifies numerous regulatory proteins including transcription factors such as Sp1 and RNA polymerase II at serine and threonine residues with a single molecule of O-linked β-N-acetylglucosamine (O-GlcNAc) [27]. Sp1 bears multiple O-GlcNAc residues and when hypoglycosylated is subject to rapid proteasomal degradation. The transcription factors Sp1 and Sp3 regulate transcription of Slc2a3 in murine neuroblasts and trophoblasts, suggesting that this may be the conduit for glucose regulation of SLC2A3 expression in preimplantation embryos. Experiments using D-[U-14C] glucosamine to pulse blastocysts revealed that though there was labeling in the blastocoelic cavity, the Golgi complex, and other membranous regions, labeling was also detected at the nuclear envelopes and in association with chromatin [28], consistent with nuclear protein O-linked glycosylation. Though the role of O-linked glycosylation has not specifically been examined in the mouse, recent studies in bovine oocytes implicate this mechanism in oocyte developmental competence [29]. The role of this dynamic modification on metabolic differentiation and blastocyst formation requires further study.
In conclusion, we have identified that glucose acts as a signal via hexosamine biosynthesis to activate embryonic gene expression, differentiation, and development. As in somatic cells, this glucose-sensing pathway provides the early embryo with a mechanism with which to tightly couple key cellular processes with nutrient availability. This mechanism may explain the molecular basis of the impact of disturbed glucose supply on embryonic development.
ACKNOWLEDGMENTS
We thank Sheree Hughes-Stamm and Emmy Hung for expert technical assistance.
FOOTNOTES
1Supported by grants to M.P. and P.L.K. from the National Health and Medical Research Council of Australia Project Grant 210194 and NICHD National Cooperative Program on Female Health and Egg Quality under cooperative agreement U01 HD044644. 
Correspondence: 2FAX: 617 3365 1766; e-mail: m.pantaleon{at}uq.edu.au
Received: 15 May 2007.
First decision: 16 June 2007.
Accepted: 19 November 2007.
REFERENCES
This article has been cited by other articles:
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  S. Jansen, M. Pantaleon, and P. L Kaye Characterization and Regulation of Monocarboxylate Cotransporters Slc16a7 and Slc16a3 in Preimplantation Mouse Embryos Biol Reprod, July 1, 2008; 79(1): 84 - 92. [Abstract] [Full Text] [PDF]
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2 = 271.6, df = 3, P < 0.0001, n = 360 [+GLU] or 322 [–GLU]).
x200.
