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Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Mary O. Carayannopoulos or Kelle H. Moley, Department of Obstetrics and Gynecology, Washington University School of Medicine, 4911 Barnes Hospital Plaza, St. Louis, Missouri 63110. E-mail: carayannopm{at}msnotes.wustl.edu or moleyk{at}msnotes.wustl.edu.
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Abstract |
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Introduction |
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Recently new members of the GLUT family of transporters have been identified based on sequence similarity to GLUTs 1–5 (11). Because of the critical role glucose homeostasis plays in early embryogenesis, we seek to identify members of this family that are expressed at this stage of development and to determine their role in progression to the blastocyst stage. Human GLUT9 was initially identified as a gene of unknown function transcribed in adult kidney, liver with lower expression in placenta, lung, blood leukocytes, and heart (12). Its expression in embryonic tissue was not examined. Here we report that the mouse homolog is expressed as early as a one-cell embryo or zygote. Interestingly, we have also identified two additional isoforms of GLUT9, which have not been identified in the human. These two isoforms, GLUT9a(
209–316) and GLUT9b(NH2b/209–316), have each deleted transmembrane domains 6 and 7 and appear to be splice variants of the same transporter gene. There appears to be differential expression of the GLUT9 isoforms in early embryonic development. In addition, we show that GLUT9 is a functional glucose transporter and that down-regulation of this transporter increases embryo demise. This suggests that GLUT9 may be another glucose transporter critical for embryonic development.
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Materials and Methods |
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Northern analysis
A mouse multiple tissue blot purchased from Clontech was probed following the manufacturer’s instructions. Briefly, the blot was prehybridized at 68 C for 30 min in ExpressHyb hybridization solution (Clontech). A 360-nucleotide cDNA probe corresponding to amino acids 34–154 of mouse GLUT9 was generated by PCR and designated as the 5' probe. This probe will recognize all isoforms of GLUT9. Additionally, a second probe corresponding to the deletion in the shorter isoforms, from amino acid 209–316 was used to determine the differential expression of these isoforms. Hybridization of the probes was performed at 68 C for 1 h. The blot was washed at room temperature for 40 min in 2x saline sodium citrate/0.01% SDS and 50 C for 40 min in 0.1x saline sodium citrate/0.1% SDS. A ß-actin probe, provided by the company, was used as a positive control. The expected transcript size is 2.0 kb.
Embryo recovery and culture
All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Embryos were recovered as previously described (8). Briefly, female mice (B6 x SJL F1, Jackson Laboratories, Bar Harbor, ME) 4–6 wk of age were superovulated with an ip injection of 10 IU/animal of pregnant mare serum gonadotropin (PMSG) (Sigma Chemical Co., St. Louis, MO) followed 48 h later by 10 IU/animal of human chorionic gonadotropin (Sigma). Mating was confirmed by identification of a vaginal plug. Animals were killed on embryonic day (E)3.5, 96 h after human chorionic gonadotropin administration and mating. Embryos were obtained by flushing dissected uterine horns and ostia as described previously (8). Embryos were immediately placed in HTF media (Irvine Scientific, Irvine, CA). Culture conditions for specific experiments are described below.
RT-PCR of mouse embryos
Total RNA was extracted from 80–150 embryos (two-cell or blastocyst) using the RNeasy mini kit (Qiagen, Santa Clarita, CA). RT-PCR was performed in a single tube reaction using the Titan one-tube RT-PCR system (Roche, Basel, Switzerland). The primers used in the PCR flank the deletion found in the shorter forms and will give different sized products, depending on the isoform amplified. The primers used were: forward primer, 5'-CCCAAGGAGATCCGGGGCTCTCTG; reverse primer, 5'-GGAGAGGTCCCCTTCCTAAGCGC. To amplify the alternative amino termini, the following forward primers were used: mG9a, 5'-ATGGATTCCAGGGAGCTTGCTTTAGC and mG9b, 5'-ATGAAGCTCAGTGAAAAGAACTCCG.
GLUT9 localization within the mouse blastocyst
A polyclonal sheep antibody was raised against a keyhole limpet hemocyanin conjugate to the last 20 amino acids of the C-terminal tail (SQTEPDSSSTLDSYGQNKIV) of mouse GLUT9 (Strategic Biosolutions, Ramona, CA). To confirm the specificity of our antibody, GLUT9a was cloned into pcDNA3.1 (Invitrogen) and engineered with an amino terminal FLAG tag (DYKDDDDK) and overexpressed in COS cells. Whole-cell lysates were generated from COS cells transfected with either GLUT9FLAG or empty vector and were immunoprecipitated with 10 µg of the mouse anti-FLAG M2 antibody (Sigma) and probed with our GLUT9-specific antisera (see Fig. 4A
) Attempts to identify endogenous GLUT9 in adult tissue (adipose tissue, brain, heart, kidney, liver, muscle, and testes), whole-cell lysates were unsuccessful. For localization of GLUT9 in embryos, embryos were fixed for 20 min in 3% paraformaldehyde and permeabilized for 30 min with 0.1% Tween 20 and placed on glass slides. Embryos were immunostained with GLUT9 peptide-purified antisera (1 µg/ml) and a secondary antisheep Alexa 488 at a 1:50 dilution (Molecular Probes, Eugene, OR). Embryos were counterstained with propidium iodide, which stains nuclei red. Fluorescence was detected with laser-scanning confocal immunofluorescent microscopy (MRC-600, magnification, x63, Bio-Rad Laboratories, Hercules, CA) as described previously (8). As controls, fixed embryos were stained with preimmune sera.
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2-Deoxyglucose uptake in Xenopus laevis oocyte
Stage V-VI stage oocytes were injected with 50 ng RNA prepared from GLUT9a cDNA, GLUT9a (206–316), or GLUT4 cDNA as previously described (13, 14). Three days after injection, uptake of 2-deoxy-D-[3H] glucose (2-DG) was measured as previously described (13, 14). Groups of 10–20 oocytes were assayed per experiment. A total of three separate experiments were performed
Blastocyst transfer after exposure in vitro to GLUT9AS or GLUT9S oligonucleotides
Two-cell embryos were obtained as described above and cultured for 72 h in either GLUT9 sense or antisense oligonucleotide probes as described previously (6). Briefly, 5 µM GLUT9 sense (5'-ATG GAT TCC AGG GAG CTT GCT-3') or GLUT9 antisense (5'-AGC AAG CTC CCT GGA ATC CAT-3') phosphorothioate oligonucleotides (Oligos Etc. Inc., Wilsonville, OR) were added to HTF media (Irvine Scientific) and the embryos cultured to a blastocyst stage in 5% O2, 5% CO2, 90% N2 at 37 C. After the incubation, a total of eight blastocysts were transferred into recipient pseudopregnant Institute for Cancer Research (ICR) female mice as described by Hogan (15). The mice were killed on d 14.5, and the numbers of normal implantation vs. resorption sites were recorded as previously described (16). Implantation rate represents normal gestational sacs divided by total number of sacs including resorptions. Distinctions were not made between abnormalities in decidualization, placentation, or fetal development because most resorptions appeared to have occurred much earlier and the contents of the sacs were necrotic. Three experiments were performed for each group, both sense-treated and antisense-treated embryos. Only foster mice that had at least one successful implantation in either the sense or the antisense embryos group were used in the data collected.
In concurrent experiments, sense and antisense-treated embryos were also stained for GLUT9 expression as described above, to confirm decreased protein expression. In addition, antisense-treated embryos were subjected to terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay to assess apoptotic nuclei. In these experiments, blastocysts were fixed in 3% paraformaldehyde, permeabilized with 0.1% Tween 20, and then incubated in fluorescein-labeled deoxyuridine 5-triphosphate and terminal transferase in the dark for 1 h at 37 C to label fragmented 3' DNA (TUNEL, cell death in situ kit, Roche Molecular Biochemicals, Indianapolis, IN) as previously described (7, 16). Counterstaining of nuclear DNA was achieved by incubating the embryos in propidium iodide (0.01 mg/ml, red channel). Embryos were visualized using confocal immunofluorescent microscopy (Bio-Rad MRC-600) at x63 magnification. A Z-series was performed on each blastocyst to determine the total number of nuclei and the number of apoptotic or TUNEL-positive nuclei. Apoptosis was expressed as percent TUNEL-positive nuclei per total nuclei per embryo.
To determine whether the antisense oligoprobe blocked GLUT9 function as well as expression, glucose uptake was measured using a nonradioactive microanalytic procedure described previously (8, 17). In brief, blastocysts after 72 h of culture were incubated at 25 C in 200 µM 2-DG for 15 min, washed in DG-free, BSA-free buffer for 1 min, and then quick frozen on a glass slide. After freeze drying overnight, the embryos were extracted in microliter volumes under oil and assayed for DG and DG6P as described previously. The final measurements are expressed as picomoles per embryo per 15 min.
Statistical analysis
Differences between control values and experimental values were compared by Student t test or one-way ANOVA coupled with Fisher test (Statview 4.5) when comparisons were made between more than two experimental groups. All data are expressed as mean ± SEM. All experiments were performed in duplicate or triplicate. For confocal immunofluorescent microscopy studies, at least 12 embryos were examined for each experimental group on three different days. For the oocyte glucose uptake studies, at least 12 oocytes were assayed in three different experiments. Significance was defined as P < 0.01.
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). Upon cloning and sequencing of these products, we discovered that these clones were identical except for a 107-amino acid deletion. We designated these clones mouse GLUT9a (accession number AF469480) and mouse GLUT9a(209–316) (accession number AF490463) (Fig. 2A). GLUT9a has an open reading frame of 539 amino acids, whereas GLUT9a(209–316) is 432 amino acids in length. In addition, a more recent search of the NCBI database resulted in the discovery of another GLUT9 isoform (accession number BC006076). This clone is identical to GLUT9a(209–316) except that it has a much shorter amino terminus. This isoform is 417 amino acids and has been designated GLUT9b(NH2b/209–316) (Fig. 2B). These clones share 85% sequence identity with the human homolog. Mouse GLUT9 diverges from human GLUT9 at both the amino and carboxy termini (Fig. 2A). The sequences of all three isoforms were analyzed using the program HMMTOP that is accessible at http://www.enzim.hu/hmmtop/. This analysis suggests the presence of 12 putative transmembrane spanning domains for the full-length form of GLUT9 and 10 putative transmembrane spanning domains for the deleted forms (Fig. 2C). Transmembrane 6 and 7 and the large intracellular loop between them have been deleted in the shorter isoforms.
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Expression analysis of GLUT9
Northern blot analysis using a probe that would identify all isoforms of GLUT9 (Fig. 3A) revealed a 4.0-kb transcript with highest expression observed in adult liver and kidney and to a lesser extent in heart. To determine whether there was differential expression of these two forms of GLUT9, a probe specific for the full-length isoform was used. There was no difference in expression pattern for this probe (data not shown), suggesting that both isoforms are expressed in these tissues. Next, we performed RT-PCR on embryos to look for expression of GLUT9 and determine whether there was differential expression of the full-length and deleted isoforms. We performed RT-PCR on two-cell and blastocyst stage embryos. At both stages, only the shorter isoform of GLUT9 is present (Fig. 3B). In addition, using primers that differentiated between the a and b isoforms, both forms were detected at the blastocyst stage (Fig. 3, C and D). We know that the longer form is present as early as E7 because both isoforms of GLUT9 were originally cloned from d 7 embryo cDNA (Fig. 1
). These data suggest that the shorter isoform of GLUT9 is expressed in vivo and is the only GLUT9 isoform expressed during preimplantation development, whereas both isoforms are expressed at later embryonic stages.
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Next, we examined the expression of GLUT9 in embryonic tissue. Western blot analysis of blastocysts revealed a diffuse band having a molecular mass of about 46 kDa, corresponding to the shorter isoform, GLUT9a(209–316), which has a predicted molecular mass of 46 kDa, compared with 66 kDa for the full-length isoform (Fig. 4B). Immunofluorescence microscopy of embryos stained with a GLUT9-specific antibody show staining as early as the one-cell and two-cell stages. (Fig. 4C). Although the antibody detects the short and long forms of GLUT9, the RT-PCR data suggest that it is the short forms that are detected by this immunofluorescent staining. At the one- and two-cell zygote stages, GLUT9 appears to localize to the plasma membrane. In contrast, in the blastocyst, GLUT9 appears to be localized perinuclearly (Fig. 4, D–F) and exclusively in the trophectoderm cells, not the inner cell mass (ICM) cells.
Functional analysis of mouse GLUT9
To determine whether GLUT9 functions as a glucose transporter, a Xenopus laevis oocyte expression system was used as described previously (14). 2-DG uptake was significantly higher in oocytes injected with either GLUT9a or GLUT9a(209–316) when compared with sham -injected oocytes (4.94 ± 0.47 pmol/embryo·30 min vs. 1.46 ± 0.16 pmol/embryo·30 min; P < 0.001; 7.55 ± 0.40 pmol/embryo·30 min vs. 1.46 ± 0.16 pmol/embryo·30 min; P < 0.001) (Fig. 5). Immunohistologic staining of sectioned oocytes demonstrated increased expression of both GLUT9 isoforms at the plasma membrane of injected oocytes over sham-injected oocytes (data not shown). In addition, GLUT4 was used as a positive control and showed similar increased staining and glucose uptake (14).
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, A and B). Decreasing GLUT9 expression by antisense oligonucleotide treatment, however, did not alter basal glucose uptake at a blastocyst stage when compared with sense-treated embryos or untreated controls (1.65 ± 0.052 pmol/embryo·15 min vs. 1.66 ± 0.041 pmol/embryo·15 min vs. 1.64 ± 0.046 pmol/embryo·15 min) (Fig. 6C). The intracellular location of GLUT9 at this stage may explain this finding. In addition, TUNEL staining of antisense-treated embryos, used as a marker for apoptosis, showed no significant difference in the number of apoptotic nuclei, compared with that seen in sense-treated controls. These findings are in contrast to what is seen in blastocysts exposed to GLUT1, GLUT3, and GLUT8 antisense oligonucleotides. Embryos treated with antisense oligonucleotides to these transporters exhibit significant apoptosis caused by a decrease in glucose uptake and resultant decrease in intraembryonic-free glucose. Therefore, because no change in basal glucose uptake was seen in the GLUT9 antisense-treated embryos, it is not surprising that there was no effect on apoptosis induction in similarly treated blastocysts.
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). This suggests that expression of GLUT9 is important for embryo development and survival. |
Discussion |
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209–316) and GLUT9b(NH2b/209–316)] (Fig. 2C). Splice variants resulting in alternative amino termini have recently been described for GLUT11 (19, 20). Two of the GLUT11 isoforms display functional glucose transporter activity and are localized to the plasma membrane. Although all of the known mammalian glucose transporters have 12 transmembrane-spanning domains and not 10, several of the nucleoside hexose transporters and monocarboxylate transporters contain only 10 transmembrane domains (21, 22). It is possible that the shorter isoform, expressed during the preimplantation period may serve a dual transport function, depending on its location and isoform.
Based on RT-PCR and Western analysis, only the shorter isoforms are expressed during the preimplantation stage of development (Figs. 3B and 4B). As early as a two-cell stage, GLUT9a(209–316) appears to be expressed exclusively and persists through the blastocyst stage (Fig. 4, C–F). Because we originally cloned both long and deleted isoforms from d 7 embryo cDNA, we know that expression of the longer isoform, GLUT9a, must be initiated between E4 and E7. Localization of GLUT9a(209–316) in one- and two-cell embryos appears to be at the cell surface. This is in contrast to localization at the blastocyst stage in which GLUT9a(209–316) shows intracellular perinuclear staining. This change in localization may be due to a different amino terminus and/or related to functional differences. Intracellular localization of a glucose transporter is atypical but not unprecedented. GLUT4 and GLUT8 are the only other glucose transporters that reside intracellularly under basal conditions, and of these, only GLUT8 is expressed in the blastocyst. GLUT8, however, redistributes to the apical plasma membrane in response to insulin (6). Insulin has no effect on GLUT9 localization within the blastocyst (data not shown).
Similar differential patterns of expression have been described for GLUT1 in mouse preimplantation embryos (23). In mouse oocytes and cleavage-stage embryos, GLUT1 is predominantly expressed in the nucleus and pronucleus. Not until embryo compaction is GLUT1 expressed predominantly at the plasma membrane. The differential localization of GLUT1 during preimplantation development correlates with the metabolic requirements of the developing embryo. Although necessary for blastocyst formation, glucose alone cannot support embryonic development before compaction (24, 25, 26).
In contrast to GLUT1, GLUT9 has the opposite differential pattern of expression. This transporter is expressed primarily at the plasma membrane in cleavage stage embryos and moves to an intracellular location during the blastocyst stage of development. This change in localization suggests that GLUT9 may play a role in the delivery of glucose or perhaps an alternate substrate (lactate or pyruvate) to the developing embryo before compaction. Pyruvate and lactate are preferred substrates until the early blastocyst stage in murine embryos, and therefore this truncated transporter may serve as a monocarboxylate transporter (MCT). MCT 1, MCT 2, and MCT 4 have been detected in murine preimplantation embryos by RT-PCR, but protein expression and function have not been confirmed (27). GLUT9a(209–316) at the plasma membrane may serve as a functional transporter of pyruvate to optimize monocarboxylate uptake early in development. Upon progression to a blastocyst stage when glucose is the predominant substrate, this transporter may translocate to an intracellular location and in its place, GLUT3, a high-affinity glucose transporter, is expressed at the apical plasma membrane and serves as the predominant substrate transporter.
Uptake and use of glucose during the early cleavage stages are predominantly for glycogen synthesis and storage in preparation for implantation. In flux studies, it has been shown that the largest amount of glucose taken up by two-cell embryos is incorporated in glycogen (28, 29, 30, 31). It is possible that the shorter form of GLUT9 present at the plasma membrane in one- and two-cell embryos may be complexed or coexpressed with the enzymatic and protein machinery necessary for glycogen synthesis. Recent studies in human placental tissue have shown an association between expression of glycogenin, the protein primer necessary for synthesis of glycogen and GLUT3 (32). Glycogenin is a self-glycosylating protein, critical for the initiation of glycogen biosynthesis and associated with membrane structures (33).
This difference in GLUT9 localization and the role this transporter may play in early embryo metabolism may possibly explain why antisense treatment did not result in immediate effects at the blastocyst stage, as seen with the other GLUTs. Down-regulation of GLUT1, GLUT3, and GLUT8 all result in decreased basal or insulin-stimulated glucose transport, which manifests as decreased intraembryonic glucose and triggers a programed cell death pathway (6, 16, 18). These blastocyst changes translate into increased pregnancy loss and embryonic malformations when these embryos are transferred into foster mothers (16). Decreased expression of GLUT9 from a two-cell to a blastocyst stage embryo may affect glucose transport at a two-cell stage and thus affect cleavage stage metabolism. The consequence of this abnormality may not be apparent at a blastocyst stage but present later as glycogen stores, or other macromolecules that require glucose as a substrate for synthesis, become depleted. Studies assessing pyruvate and glucose uptake and metabolism at earlier stages and measurements of glycogen content and other macromolecules are necessary to determine the role of GLUT9 at this stage of development.
The developmentally regulated movement of GLUT9 may also be due to differentiation of the embryo at this stage. At an early blastocyst stage, the embryo differentiates from a collection of totipotent cells to an organism consisting of two cell types, trophectoderm (TE) and ICM. From our initial studies in this report, it appeared that GLUT9 was expressed exclusively in TE cells. The metabolic profiles of these two cell types are significantly different, and it is possible that GLUT9 has a specific role in TE cells that differs from its role in the totipotent embryo and thus results in relocalization. Further studies evaluating substrate specificity of the different isoforms, localization of this transporter within the embryo cell types, and determining the metabolic consequences of a decrease in GLUT9 expression with antisense oligonucleotides are necessary to determine the role of GLUT9 at this stage of development.
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Acknowledgments |
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Footnotes |
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Abbreviations: 2-DG, 2-Deoxy-D-[3H] glucose; E, embryonic day; ICM, inner cell mass; MCT, monocarboxylate transporter; TE, trophectoderm; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.
Received September 22, 2003.
Accepted for publication November 21, 2003.
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