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RESEARCH |
Department of Anatomy and Cell Biology, Martin Luther University Faculty of Medicine, Grosse Steinstrasse 52, D-06108 Halle (Saale), Germany
Correspondence should be addressed to B Fischer; Email: bernd.fischer{at}medizin.uni-halle.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Insulin binds to its cell surface located receptor. The mature receptor is composed of two extracellular 
and two transmembrane ß subunits which are disulfide-linked to an 2ß2 heterotetrameric structure (Lee & Pilch 1994, Czech & Corvera 1999). Following insulin binding to the extracellular -subunit tyrosine kinase domain, the ß subunits undergo a series of intramolecular transautophosphor-ylation reactions, resulting in tyrosine autophosphorylation at multiple sites. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate cellular response. Insulin and IGF-I act as a survival factor by decreasing apoptosis and increasing cell proliferation (Herrler et al. 1998, Spanos et al. 2000). Another important mechanism is the increase of glucose transport via the insulin-dependent translocation of the facilitative glucose transporter 4 (GLUT4) from intracellular storage vesicles (Rea & James 1997) to the plasma membrane in insulin target tissues, primarily striated muscle and adipose tissue (Pessin et al. 1999, Patki et al. 2001). Currently, the GLUT family comprises 13 members, GLUT1–12 and HMIT (H+ coupled myoinositol-transporter) (Joost et al. 2002, Stuart Wood & Trayhurn 2003). Each GLUT isoform consists of 12 helical transmembrane-spanning domains, an extra-cellular glycosylated hydrophilic segment, an intracellular loop and intracellular located amino- and carboxyl-terminals (Mueckler et al. 1985, Cope et al. 1994).
The expression pattern of glucose transporters in preimplantation embryos has been studied in the mouse (Hogan et al. 1991, Aghayan et al. 1992, Morita et al. 1992, Chi et al. 1993, Pantaleon et al. 1997), rabbit (Robinson et al. 1990), bovine (Lequarre et al. 1997, Wrenzycki et al. 1998, 2003, Navarrete Santos et al. 2000, Augustin et al. 2001) and human (Dan-Goor et al. 1997). In mice, GLUT1 was found in all preimplantation stages. GLUT3 was detected from the eight-cell stage onwards (Pantaleon et al. 1997). While expression of the recently described insulin responsive GLUT8 was shown in mouse blastocysts (Carayannopoulos et al. 2000), the other insulin-sensitive isoform, GLUT4, was not found in mice preimplantation or early postimplantation stages (Hogan et al. 1991, Aghayan et al. 1992). In preimplantation embryos of other species, however, GLUT4 has been detected (bovine: Navarrete Santos et al. 2000, Augustin et al. 2001, rat: Korgun et al. 2001). Therefore, the question arose whether and which compounds of the insulin–GLUT signaling cascade are expressed in embryos and which function they may execute during preimplantation development in mammals. Here we show that IR and both insulin-responsive isoforms, GLUT4 and GLUT8, are expressed in rabbit preimplantation embryos in a developmentally regulated manner. Rabbit blastocysts also express GLUT1 and GLUT3, but not GLUT2 and GLUT5.
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Materials and Methods |
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Embryo recovery
Embryos were collected from sexually mature rabbits (hybrid strain Zika) which had been stimulated by 100 I.U. follicle-stimulating hormone (Ovagen, Immuno-Chemical Products, Auckland, New Zealand). Mating, embryo recovery and embryo culture were performed as described before (Kietz & Fischer 2003). Morulae were flushed on day 3 and blastocysts on days 4 and 6 post coitum; they were washed three times in PBS, pooled and randomly divided among the experimental groups. For molecular analysis embryos were stored until use at –80 °C in TRIzol reagent and in RIPA buffer for RNA and protein isolation respectively.
In order to investigate spatial expression of GLUT4 and GLUT8 in trophoblast (TE) and embryoblast cells (inner cell mass, ICM), blastocyst coverings were mechanically removed and the embryonic disks were microdissected from the trophoblast under a stereomicroscope. Isolated ICM and TE from each blastocyst (total number 11 blastocysts) were stored separately at –80 °C. For immunohistochemistry embryos were fixed overnight in Bouin fixative containing 75% (v:v) aqueous-saturated picric acid solution, 20% (v:v) formalin, and 5% (v:v) acetic acid and/or in 4% (v:v) paraformaldehyde/PBS overnight. Bouin-fixed embryos were dehydrated and embedded in paraffin. Sections (5 µm) were prepared with an ultramicrotome. Paraformaldehyde-fixed embryos were dehydrated and stored in methanol at –20 °C until whole mount staining.
RNA extraction
Preparation of total RNA from whole embryos was performed by using 1 ml TRIzol reagent according to the previously described protocol (Koerber et al. 1998). Total RNA from tissues was extracted as described by Chomczynski and Sacchi (1987). RNA was treated with DNAse for 1 h. The amount of total RNA was determined spectrophotometrically at 260 nm. The mRNA extraction from separated trophoblast and embryoblast was performed with DYNABEADS (Dynal, Oslo, Norway) in order to collect sufficient amounts from the small number of cells.
Cloning of rabbit IR, GLUT4 and GLUT8 sequences
Rabbit IR, GLUT4 and GLUT8 cDNA sequences were determined by PCR amplification with degenerated primers derived from human sequences (IR accession no. (acc.) X02160
[GenBank]
.1, GLUT4 acc. M91463
[GenBank]
, GLUT8 acc. Y17801
[GenBank]
, primers shown in Table 1
) and 3' RACE-PCR (3/5 RACE kit, Roche Diagnostics) using specific rabbit primers (Table 1) on reverse transcribed rabbit skeletal muscle and liver mRNA for GLUT4 and IR, and GLUT8 respectively. The amplified PCR products were purified by separation in a preparative 1.8% agarose gel and extracted by a Gel Extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Fragments were cloned into the pGEM-T vector and transformed into competent E. coli XL1blue cells. Recombinant plasmid DNA was analyzed by restriction analysis and sequenced using the ABI Prism Ready Reaction Dyedeoxy Terminator Sequencing kit (Amersham Biotech, Freiburg, Germany) and T3 and T7 sequencing primers in an ABI 373 automated sequencer. The sequenced cDNA was screened for homology in the GenBank EMBL using the BLASTN search modus and for amino acids using the BLASTP search modus.
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RT-PCR of IR, GLUT1–5 and 8 mRNA
Primers used for RT-PCR are listed in Table 1
. One microgram total RNA was reverse transcribed in a volume of 20 µl containing 0.5 mM dNTPs, 10 mM dithiothreitol (DTT), 200 units superscript II, 20 units RNAse inhibitor, 1 µl random primer and 2 µl reverse transcriptase buffer at 42 °C for 1 h, followed by an incubation at 90 °C for 5 min. As a control for DNA contamination, 1 µg RNA was PCR amplified without reverse transcription reaction. This control reaction was performed for each primer combination and in all PCR amplifications. PCR amplification was carried out with 1 µl cDNA in a 50 µl volume containing 5 µl dNTP, 2.5 units Taq polymerase, employing the primer combinations listed in Table 1. Resulting PCR products were separated by electrophoresis on 1.8% agarose gel and stained with ethidium bromide.
Semiquantitative RT-PCR on the separated trophoblast and embryoblast tissue
Trophoblast and embryoblast mRNA were directly transcribed into cDNA with the RNA-PCR Core kit (Perkin-Elmer Roche, Boston, USA). Reverse transcription reaction was performed in a thermocycler (Biometra, Göttingen, Germany) under the following conditions: 10 min at 25 °C, 1 h at 42 °C, 5 min at 99 °C. Afterwards 40 µl H2O were added. The quality of tissue separation was controlled by cytokeratin 18 PCR (see Fig. 5A). Only embryos with clearly higher transcript numbers of cytokeratin 18 in the separated trophoblast and low expression in embryo-blast tissues were used for semiquantitative PCR on GLUTs. To equalize for different RNA amounts of individual embryos, first a PCR on ß-actin was performed. This housekeeping gene was used as an internal standard for GLUT transcript numbers (Kietz & Fischer 2003). All PCR reactions were carried out in 50 µl volume containing 2 µl cDNA, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 U Taq polymerase (Life Technologies, Eggenstein, Germany) and 150 ng of the primer combination, rabActinp1/p2 and rab-GLUT1p1/p2, rabGLUT3p1/p2, rabGLUT4p1/p2 and rab-GLUT8p1/p2 (Table 1) for GLUT1, GLUT3, GLUT4 and GLUT8 respectively. The amplification profile was as follows: 5 min at 94 °C, 28 (ß-actin), 32 (GLUT1, GLUT3, GLUT8) and 35 (GLUT4, cytokeratin 18) cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C, and a final extension period for 5 min at 72 °C. Gels were photographed and the product bands were quantified by densitometric analysis employing the software BIO-Profile 1D (LTF-Labortechnik, Wasserburg, Germany). The relative amount of GLUT1, GLUT3, GLUT4 and GLUT8 mRNA was calculated as a ratio of the specific product and the housekeeping gene band volume (ß-actin). All PCR reactions were performed three times.
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Protein preparation and immunoblotting
Blastocysts were washed three times with PBS after culture and transferred to a 1.5 ml Eppendorf tube. They were homogenized in 100 µl cold RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) with a protease inhibitor cocktail (Sigma, St Louis, MO, USA). The samples were centrifuged at 5000 g for 10 min. The supernatant was stored at –80 °C until use. The total protein content was determined using the BIO-RAD Protein Assay (Bio-RAD, München, Germany). Twenty micrograms embryonic protein and 40 µg of reference tissues were heated at 70 °C for 5 min before solubilizing in Laemmli buffer containing 200 mM DTT and electrophoresed on 8% SDS-PAGE. Proteins were electro-transferred to nitrocellulose membranes. Membranes were blocked for 1 h in Tris-buffered saline containing 0.1% Triton (TBST) with 5% BSA (for detection of IR) or 5% non-fatty milk powder (for GLUT4 and GLUT8) at room temperature. Blots were incubated in TBST containing 5% BSA with monoclonal mouse anti-IR (ß-subunit, Ab-4, Oncogene Research Products Calbiochem, Darmstadt, Germany) at a dilution of 1:200, in 5% nonfat milk powder/TBST with monoclonal mouse anti-GLUT4 antibody (1:6000, DPC Biermann, Bad Nauenheim, Germany) or rabbit anti-GLUT8 antibody (1:400, Alpha Diagnostics International, San Antonio, TX, USA) overnight at 4 °C. Blots were subjected to three 20-min washes in TBST and incubated for 1 h with goat anti-mouse IgG (1:25 000) or goat anti-rabbit IgG (1:20 000) conjugated to horseradish peroxidase (Dianova, Hamburg, Germany) in 5% BSA/TBST or 5% non-fatty milk powder/TBST at room temperature. Afterwards, the immunoreactive signals were visualized by enhanced chemiluminescence detection (ECL Plus, Amersham Biotech). Apparent molecular weights were determined by comparison with standard molecular weight markers (high range marker, Promega Corp., Mannheim, Germany).
Immunohistochemistry (IHC)
The GLUT4 antigen was localized on embryonic sections and whole blastocysts. Bouin-fixed, paraffin-embedded day 6 rabbit blastocysts were sectioned at 5 µm. Sections were mounted on silanized slides, deparaffinized in xylene and rehydrated through a series of graded alcohols. Various antigen retrieval methods were tested on the paraffin-embedded rabbit tissues for IR antigen detection. With the monoclonal mouse anti-IR (ß-subunit, Ab-4, Oncogene Research Products Calbiochem) we could not detect any immunoreactions on fixed tissues (rabbit liver, muscle) or embryo sections. For whole mount-IHC the par-aformaldehyde-fixed blastocysts were rehydrated through a series of graded alcohols. The neozona was removed mechanically before peroxidase blocking. Endogenous peroxidase was quenched by treatment with 3% hydrogen peroxide in methanol for 30 min. Non-specific antibody binding was blocked with 10% normal goat or donkey serum in PBS at room temperature for 1 h and incubated with the primary antisera overnight at 4 °C in a humidified chamber. GLUT4 antibody (mouse anti-GLUT4 antibody 1:2500, DPC Biermann) was diluted in PBS with 1% BSA. Sections and whole blastocysts were rinsed with PBST and incubated with the peroxidase-labeled secondary antisera (DAKO EnVision +/HRP-goat-anti mouse IgG, DAKO, Hamburg, Germany) for conventional light microscopy. The antigen was visualized with the diaminobenzidine (DAB, WAK-Chemie Medikal, Bad Soden, Germany) substrate. The development of DAB was stopped in water after 5 min. Sections were counterstained with hematoxylin, dehydrated and cleared in xylene. The slides were mounted in DPX and examined under bright-field microscopy with an AH-3 microscope (Olympus, Hamburg, Germany). For whole mount confocal microscopy the GLUT4 protein was localized by immunofluorescence detection with the secondary antibody fluorescein (FITC)-conjugated AffinPure donkey-anti-mouse IgG (1:300, Jackson ImmunoResearch Lab. Ltd, Cambridgeshire, UK). The nuclei were counterstained with 7-amino-actinomycin. Whole blastocysts were scanned and examined by confocal laser microscopy with Leica TCS SP (Bensheim, Germany). The specificity of immunostaining was demonstrated by the absence of signals in sections and whole embryos incubated with control mouse IgG (DAKO) or in sections processed after omission of the primary antibody. Only reactions with negative controls were included in the study. At least 5 embryos, pooled from different donor animals, were examined per group. IHC reactions of the same specimens were repeated three times.
Accession numbers
The GeneBank accession numbers for rabIR, rabGLUT2, rabGLUT4 and rabGLUT8 were AY339877
[GenBank]
, CB814983
[GenBank]
, AY339876
[GenBank]
and BF146289
[GenBank]
respectively.
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, 2A). The partial rabbit GLUT8 cDNA sequence with a length of 230 bp (Fig. 1B, rabGLUT8 – accession number BF146289
[GenBank]
) was amplified from rabbit liver tissues. All sequences show a high homology to human and other mammalian sequences at both the DNA and protein level (Table 2).
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). The GLUT4 5' coding region and the putative start of translation were amplified by human huGLUT4p3 and rabGLUT4p4, whereas the 3' terminus was determined by 3'-RACE with specific rabGLUT4p3 and a poly(A) specific anchor primer (primers listed in Table 1). The resulting cDNA contains an open reading frame of 1527 nucleotides encoding a protein of 509 amino acids (aa) with a calculated molecular mass of 54.84 kDa and an estimated pI of 7.77. The TGA stop codon is followed by 825 bases of 3' non-coding region. The rabGLUT4 3'-flanking region shows partial homology with the horse, bovine, mouse and human GLUT4 genes and is specific for rabGLUT4. The specific rabbit primers rabGLUT4p1 and rabGLUT4p2 (Table 2, Fig. 1A) were designed for PCR amplification on rabbit embryos yielding a PCR product of 398 bp. The primer design avoided genomic amplification due to two bridged introns.
The new rabbit IR cDNA (accession number AY339877
[GenBank]
) sequence contains the complete region for the - and ß-subunits of the receptor (Fig. 2). The insulin receptor sequence is highly conserved in mammalian species (Fig. 2). The insulin binding region and the amino acids phosphorylated by tyrosine kinase are identical in the rabbit and human sequences. The complete coding region was cloned. Analysis of a 4026 bp cDNA revealed an open reading frame coding for a propolypeptide of 1341 aa with a molecular mass of 152.4 kDa. The protein sequence between rabbit and human IR differed only in 44 aa. In the -subunit the amino acids involved in insulin binding are present. In the ß-subunit the tyrosine residues representing the autophosphorylation consensus sequence were identical to the human ones (Ottensmeyer et al. 2000) (Fig. 2). These molecular features prove that the rabIR gene product is a member of the IR family and represents the insulin receptor from Oryctolagus cuniculus. The identity of the rabbit IR to the receptor molecule is higher than with other members of the IR family such as IGF-IR.
Expression of GLUT isoforms and IR in rabbit embryos
The glucose transporter isoforms 1, 3, 4, 8 and IR are expressed in expanded rabbit blastocysts (day 6) (Figs 3 and 4). While transcripts of the GLUT4 gene were detectable in rabbit morulae and blastocysts, IR and GLUT8 were not found at the morula stage. First transcripts for IR and GLUT8 mRNA were detectable in early blastocysts recovered on day 3 and in day 4 blastocysts respectively (Fig. 4A). GLUT4 and 8 transcript numbers were significantly higher in the trophoblast than in the embryoblast (P < 0.05). Such cell lineage-specific effects were not found for GLUT1 and 3 (Fig. 5B). The GLUT isoforms 2 and 5 were not found in day 6 blastocysts (Fig. 3, rab-GLUT2 – accession number CB814983
[GenBank]
).
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). Molecular weight and signal specificity of the antibody were verified by employing target tissues such as rabbit heart muscle for GLUT4, GLUT8 and liver for IR in the same blot (Fig. 4B, C, D). GLUT4 immunoreactivity was confirmed by IHC on day 6 blastocysts (Fig. 6). Antibodies for GLUT8 and IR, however, were not immunoreactive on rabbit embryo and target tissues (liver, heart muscle).
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). Immunohistochemical staining revealed a different subcellular localization of the protein. Whereas the outer trophoblast layer showed a membrane and perinuclear staining, the extra-embryonic endoderm exhibited an intense staining for cytoplasmic and perinuclear compartments. Confocal laser scans showed a closed localization of GLUT4 with nuclear membranes in the extraembryonic endoderm. In embryoblast cells, GLUT4 was localized mainly perinuclearly in the cytoplasm. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Sequence analyses of the new rabbit genes prove that the rab IR encoded protein is a member of the IR family and that rabGLUT4 and 8 belong to the facilitative glucose transporters. The amino acid sequence responsible for insulin binding is almost identical with the human insulin receptor sequence. Insulin initiates its effects through interaction with the high-affinity cell surface glycoprotein receptors, consisting of two (135 kDa) and two ß (95 kDa) subunits each. The human insulin receptor is expressed in two isoforms, A and B, which are generated by alternate splicing (Ebina et al. 1985, Ullrich et al. 1985, Seino & Bell 1989). The two mature receptor proteins differ in the absence or presence of 12 aa in the C terminus of the extracellular subunit. This insertion is encoded by the 36 nucleotide exon 11 of the receptor gene (Seino et al. 1989). The A and B isoforms have different tissue distributions (Moller et al. 1989, Seino & Bell 1989) and functional properties (Yamaguchi et al. 1991, 1993, Kosaki et al. 1995). The subunit in the rab IR was found to be highly homologous with the subunits of human IR isoform A without insertion of exon 11. A more detailed analysis regarding the presence of two IR isoforms in rabbit tissues was not performed in the present study.
The IR protein-tyrosine kinase has been implicated as the mediator of most, if not all, effects of insulin. IR deficiency in IR–/– mice caused a number of major metabolic alterations and led to the death of the newborns within one week after birth (Accili et al. 1996). At birth, mouse IR–/– mutant pups could not be distinguished from other littermates, contrary to human patients with mutations in IR, which usually have a severe intrauterine growth retardation (Lamothe et al. 1998).
During embryo development insulin exerts a number of important actions. (i) It accelerates the uptake of amino acids and proteins by preimplantation embryos (mouse: Dunglison & Kaye 1993; pig: Lewis et al. 1992), (ii) it promotes blastocyst formation and increases the number of embryonic cells (Matsui et al. 1995, Herrler et al. 1998, Augustin et al. 2003), in some species specifically of ICM cells (mouse: Harvey & Kaye 1990, Gardner & Kaye 1991, Smith et al. 1993, bovine: Sirisathien et al. 2003), and (iii) it prevents apoptosis (rabbit: Herrler et al. 1998, bovine: Augustin et al. 2003). The mechanism of insulin action in mammalian embryos is still unclear. Also, the effects on glucose transport and metabolism need further consideration. For mouse blastocysts it has been shown that insulin and IGF-I act via IR to increase glucose uptake (Gardner & Leese 1988, Harvey & Kaye 1991, Pantaleon & Kaye 1996, Carayannopoulos et al. 2000). Compared with IGF-I, glucose uptake, measured by uptake of 3-o-methyl-D-glucose, was clearly more stimulated by the IGF-IR/ IGF-I system than by insulin (Pantaleon & Kaye 1996). An insulin-dependent increase in glucose uptake via translocation of GLUT4, as described for differentiated myocytes and adipocytes, has not yet been shown in preimplantation embryos. First screening studies of glucose transporter isoform expression in mammalian preimplantation embryos, performed in mice, failed to prove GLUT4 expression (Hogan et al. 1991, Aghayan et al. 1992). The expression of the insulin responsive GLUT4 isoform has been shown for the first time in bovine blastocysts (Navarrete Santos et al. 2000). During bovine preimplantation development GLUT4 was expressed in 8-day-old in vitro-derived blastocysts and in day 14 elongated in vivo-grown blastocysts (Augustin et al. 2001). Another isoform demonstrated to be insulin responsive in blastocysts, GLUT8, has recently been described in mouse blastocysts (Carayannopoulos et al. 2000). GLUT8 was found to change its intracellular localization and to be involved in increased glucose uptake after insulin treatment in murine blastocysts (Carayannopoulos et al. 2000). Inhibition of GLUT8 translation and translocation enhanced the rate of apoptosis in mouse blastocysts (Pinto et al. 2002). Functional studies in other species expressing this isoform (bovine, rabbit) are needed to clarify the exact role of GLUT8 for embryo development. It is remarkable and may be indicative of different functions of GLUT isoforms in different species that mice and rabbits express GLUT8 only at the blastocyst stage (Carayannopoulos et al. 2000, present study) while during bovine embryogenesis GLUT8 mRNA is present from oocytes throughout preimplantation development (Augustin et al. 2001).
In mouse blastocysts, the high affinity isoform 3, localized in the outer apical cell membrane of the trophoblast cells, mediates glucose transport from the uterine fluid into the blastocyst (Pantaleon et al. 1997). GLUT1, situated at the basal and basolateral trophoblast cell membranes, accomplishes the supply of the ICM (Pantaleon & Kaye 1998). In the present study, both isoforms were also found in the rabbit, pointing to a similar mechanism for glucose uptake and supply as in mice. The experimental proof for GLUT2 expression in mammalian preimplantation embryos is controversial. Transcripts were reported for mice 8-cell/compacted morulae (Schultz et al. 1992) and blastocysts (Harvey & Kaye 1991). The protein was found in blastocysts (Aghayan et al. 1992). However, in the present study, as in others (mouse: Morita et al. 1992, Tonack et al. 2004, cattle: Augustin et al. 2001), GLUT2 expression could not be verified in rabbit blastocysts.
The diversity of glucose transporter expression in mammalian embryos presumably reflects the importance of glucose as the major metabolic energy substrate. Diverse glucose transporters have evolved to allow an efficient, stage- and cell-specific uptake and utilization. Glucose concentration in human serum is maintained around 5 (4.4 to 6.6) mM. The early human embryo is exposed to lower (3.15 mM; Gardner et al. 1996) or almost the same (Casslen & Nilsson 1984) glucose concentrations in utero as those present in serum. The oxygen level in this organ, however, is significantly lower than in blood (Fischer & Bavister 1993). This specific constellation and its physiological implications may have led to the more complex furnishing with glucose transporters in embryos than in differentiated muscle, fat or neuronal cells. Considering glucose uptake by preimplantation embryos, which is in a pmol per embryo per hour range (rabbit: Robinson et al. 1990, rat: Brison & Leese 1994, mouse: Martin & Leese 1999, bovine: Donnay & Leese 1999), and assuming a
Different results have been reported on the developmental implications of an altered glucose supply for embryos. In mice, glucose deprivation affected trophoblast cells more than the ICM. In deprived blastocysts cell numbers in the trophoblast, but not in the ICM, were statistically significantly decreased (Leppens-Luisier et al. 2001). Also high glucose concentrations are reported to exert detrimental effects on embryo development. Blastocysts from diabetic rats showed an impaired growth of both cell types with cell numbers being more affected in the ICM than in the trophoblast (Dufrasnes et al. 1993). Blastocysts from diabetic mice have lower intraembryonic glucose concentrations (Moley et al. 1998) and an increase in the rate of apoptosis (Chi et al. 2000).
GLUT4 has been localized in the cytoplasm of trophoblast, embryoblast and extraembryonic endoderm cells in close association with membranes and nuclei. A GLUT4 shuttling is well investigated in insulin responsive adipocytes and myocytes (for review see Zorzano et al. 1998, Watson & Pessin 2001). In these cells GLUT4 is associated with cytoplasmic vesicles (so-called GLUT4 storage vesicles, GSV; Rea & James 1997) in several morphologically distinct localizations. Ultrastuctural studies have shown that GLUT4 is present in tubulovesicular structures distinct from lysosomes. As in rabbit blastocysts in the present study, GLUT4 has also been found in the perinuclear compartment which is in close vicinity to the trans-Golgi network (Hudson et al. 1992, Jhun et al. 1992, Lee et al. 1999). Insulin increases the rate of GLUT4 translocation from the cytoplasm to the cell membrane so that the proportion of GLUT4 at the cell surface increases from <10% in the absence of insulin to 35 to 50% in its presence (adipocytes: Bogan et al. 2001). In the rabbit blastocyst, the cells in the outer trophoblast layer showed a comparable subcellular localization of GLUT4 as insulin sensitive tissues. The membrane localization of GLUT4 in trophoblast cells can be regarded as good evidence that the transporter is active in rabbit blastocysts. The more intense staining of the embryoblast and the extraembryonic endoderm may indicate another functional state or a different function of GLUT4. The perinuclear localization and the association of GLUT4 with the nuclear membranes, not described in adult tissues so far, support the view of a function of GLUT4 other than glucose transport in embryonic cells. Recently, a nuclear localization has been described for GLUT1 in mouse oocytes and early cleavage stages (Pantaleon et al. 2001) stressing the need for a more detailed analysis of potential functions of the various glucose transporter isoforms during early embryogenesis.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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