Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8+/+, Slc2a8+/−, and Slc2a8−/− mice, respectively) resulting in a deviation (p = 0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.


Introduction
Facilitated diffusion of glucose into mammalian cells along a concentration gradient is catalyzed by specific carriers (GLUT proteins). The GLUT family comprises 14 members and, according to sequence similarities, can be divided into three classes (Joost & Thorens 2001). GLUT8 belongs to the class 3 transporters and is an intrinsic membrane protein that catalyzes the facilitative transport of glucose and probably also fructose and galactose (Doege et al. 2000, Ibberson et al. 2000, Joost & Thorens 2001. A unique feature of class 3 transporters is the presence of endosomal targeting motifs that dictate their intracellular retention (Lisinski et al. 2001, Augustin et al. 2005, Schmidt et al. 2006. So far, translocation of class 3 transporters to the plasma membrane in response to a stimulus has only been demonstrated for GLUT8 in blastocysts (Carayannopoulos et al. 2000) and for the H ' / myo-inositol transporter HMIT which belongs to the class 3 transporters due to its sequence similarities (Uldry et al. 2004).
GLUT8 is expressed predominantly in testis; smaller amounts of GLUT8 mRNA were detected in most other tissues including brain (cerebellum, brain stem, hippocampus, and hypothalamus), spleen, liver, and insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissue. GLUT8 was detected in preimplantation embryos, in which it was reported to be essential for the development and survival of the embryo (Pinto et al. 2002). In testis, the GLUT8 protein was found to be associated with the acrosomal region of mature spermatozoa (Schü rmann et al. 2002a), whereas others detected GLUT8 in differentiating spermatocytes of type 1 stage but not in mature spermatozoa (Ibberson et al. 2002). GLUT8 may also play a role in adipocyte metabolism because its expression increases markedly during fat cell differentiation, and is sensitive to prolonged hypoxia and glucose deprivation (Scheepers et al. 2001). However, in adipose cells, GLUT8 does not undergo insulin-stimulated translocation to the plasma membrane as described for the GLUT4 isoform (Lisinski et al. 2001). In liver, GLUT8 was detected in perivenous hepatocytes, indicating that GLUT8 might have a function in the regulation of glycolytic flux (Gorovits et al. 2003). Recombinant GLUT8 expressed in COS-7 cells exhibits glucose transport activity when reconstituted into lecithin liposomes (Doege et al. 2000). In addition, high-affinity glucose transport activity (K m 02 mM) was observed in Xenopus oocytes after injection of GLUT8 mRNA carrying a mutation of the amino-terminal dileucine motif (Ibberson et al. 2000). This activity was specifically inhibited by Dfructose and D-galactose, indicating that GLUT8 might be a multifunctional sugar transporter (Ibberson et al. 2000).
In order to analyze the specific role of the intracellular GLUT8 in glucose metabolism and function of sperm cells we generated a null mutant lacking Slc2a8 and examined its phenotype with the focus on number and motility. While our study was in progress, it was described that targeted disruption of the Slc2a8 gene in mice caused mild alterations in brain and heart, such as an increased proliferation of hippocampal cells and a slightly impaired transmission of the electrical wave through the atrium leading to a reduction in P-wave (Membrez et al. 2006). Here we report data demonstrating that GLUT8 is required for maintaining mitochondrial membrane potential and motility of spermatozoa. This study indicates that the intracellular compartment in which GLUT8 is expressed is needed for transport of metabolites required for energy production.

Antibody
A polyclonal antibody against two GLUT8-specific peptides corresponding to a sequence of the big intracellular loop (WGSEEGWEEPPVGAEG) and of the C-terminus (KGRTLEQVTAHFEGR) of mouse GLUT8 was raised in rabbit.

Inactivation of the Slc2a8 gene
To generate a floxed Slc2a8 allele we constructed a targeting vector (see Figure 1A). Exons 5 and 7 of Slc2a8 were flanked with two loxP sites, and a PGKneo/HSVtk cassette (Neo/tk) with a third loxP site which was introduced downstream of the flanked exon 7. To verify a single introduction of the targeting construct in the homologously recombined ES cell clone, we blotted and hybridized the Xba III-digested ES cell DNA with the indicated probe giving a single band of 12.5 kb band for the wild type. Homologously recombined ES cell clones containing the targeted allele were transiently transfected with pCre to generate ES clones carrying a deleted allele and clones carrying the floxed Slc2a8 allele. The two types of ES cell clones were identified by PCR with specific primers matching to a 5?flanking region of exon 5 and the 3?-flanking region of exon 7. ES cells carrying the deleted allele were used for a morula aggregation. Blastocysts were then transferred into a pseudopregnant (day 2.5) female mouse. Male chimeric mice were mated with C57BL/6 females. Offspring carrying the transgene were backcrossed on to C57BL/6 3 times and subsequently intercrossed. The littermates of this intercross were used for the phenotypic characterization. Genotyping was performed by PCR (forward primer: 5?-CATCTTCTGTGCAGTCC-ATC-3?, reverse primer: 5?-GGTACCAAAGGCA-CTCATACTG-3?).

Animals
The animals were housed in air conditioned rooms (temperature 20928C, relative moisture 50Á60%) under a 12 h/12 h light/dark cycle. They were kept in accordance with the NIH guidelines for the care and use of laboratory animals, and all experiments were approved by the ethics committee of the Ministry of Agriculture, Nutrition and Forestry (State of Brandenburg, Germany).

Southern blot analysis
DNA of ES cell clones was digested with XbaI, separated on a 0.7% agarose gel, and blotted onto a Hybond-N ' -nylon membrane (Amersham Pharmacia Biotech). To verify a homologous recombination of the targeting construct in the ES cell clones, a 522 bp fragment of the Slc2a8-gene, external to the targeting construct, was used for hybridization after labelling with [a 32 P]dCTP by using a random priming kit (Amersham Pharmacia Biotech). An 8.8 kb band of the wild-type allele and a 12.5 kb band of the floxed allele indicated a homologous recombination of the targeting construct (data not shown). For identification of a single introduction of the targeting construct in the homologous recombined ES cell clone, a 500 bp sequence that corresponds to the HSVtk-cassette was labelled and a single integration site of the targeting construct was indicated by a single band of 12.5 kb ( Figure 1A).

RNA preparation and first strand cDNA synthesis
RNA was extracted from different tissues and cDNA synthesis was performed as previously described (Buchmann et al. 2007). Quality of cDNA was controlled performing a PCR with murine GAPDH primers (forward: 5?-ACC ACA GTC CAT GCC ATC AC-3?; reverse, 5?-TCC CAC CAC CCT GTT GCT GTA-3?).

Quantitative real-time PCR
Quantitative real-time PCR analysis (qRT-PCR) was performed with the Applied Biosystems 7300 Real-Time PCR System as described previously (Buchmann et al. 2007). The TaqMan gene expression assay (Mm00444635_g1) was used to detect the GLUT8-mRNA expression. The assay amplifies the region between exons 5 and 6, which is deleted in  Slc2a8 (/( mice. For the determination of other GLUT encoding genes, the following TaqMan gene expression assays were used: GLUT1 (Mm0044 1473_m1), GLUT3 (Mm00441483_m1), GLUT5 (Mm00600311_m1), GLUT6 (Mm00554217_m1). Data were normalized referring to Livak & Schmittgen (2001), whereas a b-actin expression assay (Mm00607939_si; Applied Biosystems) was used as endogenous control.

Immunohistochemical detection of GLUT8
Paraffin sections of testis from Slc2a8 '/' and Slc2a8 (/( males were dewaxed in toluene and rehydrated, antigen demasking was performed by heat treatment (microwave, 2.5 min, 850 W) in Target Retrieval Solution (ChemMate TM , Dako Cytomation, Hamburg, Germany). Endogenous peroxidase was deactivated with 3% hydrogen peroxide for 10 min. Unspecific binding sites were blocked with DAKO antibody diluent for 30 min. The affinity purified polyclonal anti-GLUT8 antibody in a concentration of 5 mg/ml was applied overnight at 48C in a humid chamber. Specific antibody binding was visualized by biotin-conjugated donkey anti-rabbit IgG (1:800; Dianova, Hamburg, Germany), for 30 min at 378C, followed by incubation with a streptavidin-biotin-horseradish peroxidase complex (StreptAB-complex/POD) for 30 min, and diaminobenzidine as substrate according to the manufacturer's specifications. The sections were dehydrated in graded ethanol and toluene and mounted in Histomount. Microscopic investigation and photo documentation were done with the combined light and fluorescence microscope ECLIPSE E-100 (Nikon, Dü sseldorf, Germany) in combination with the video camera CCD-1300CB (Vosskü hler, Osnabrü ck, Germany) and the Analysis System LUCIA G (Nikon, Dü sseldorf, Germany).
Briefly rinsed spermatozoa were transferred to 1 ml medium supplemented with 5 mg of bovine serum albumin per ml and 15 mM NaHCO 3 , semen was allowed to exclude (15 min at 378C, 5% CO 2 from 3 to 5 small incisions). Cells were diluted to 4 ml and collected twice by sedimentation (400 g, 5 min). Sperm count and motility of the sperm cells were analyzed with a CASA (computer-assisted sperm analyzer, MTG, medeaLAB, Altdorf, Germany) as described by Krause (1995). Parameter settings were used as described by Wennemuth et al. (2003). For all assays sperm cells were kept in the indicated media not longer than 15 min in order to suppress capicitation (Wennemuth et al. 2003). Cytoplasmic calcium levels of spermatozoa were assayed by loading the cells with 1 mM of the fluorescent calcium indicator Fluo3/acetoxymethyl (AM) ester (Gentaur; excitation: 506 nm, emission: 525nm recorded in FL-1, 530930 nm band pass filter, FACSCalibur, BD) for 20 min at 378C/5% CO 2 . After transport into the cells, AM hydrolysis occurs and, thereafter, the dye is trapped within the cell. Upon binding to calcium Fluo-3 exhibits a marked increase in fluorescence intensity (Perl et al. 2006). The mitochondrial calcium levels were estimated with 4 mM Rhod2/AM ester, which is compartmentalized into the mitochondria (Perl et al. 2006). Sperm cells were incubated with the dye for 20 min at 378C/5% CO 2 and emission was recorded in FL-2 (585942 nm band pass filter).
The survival rate of spermatozoa was determined by staining with membrane-permanent nucleic acid dye 50 nM SYBR 14 according to manufacturer's instructions (Molecular Probes) for 10 min at 378C/ 5% CO 2 . Stained and unstained sperm were assayed by flow cytometry (excitation 488 nm, emission 520 nm allocated in the FL1 channel; FACSCalibur, BD).
Mitochondrial transmembrane potential (DC m ) of spermatozoa was likewise analysed by flow cytometry. 5,5?,6,6?-tetrachloro-1,1?,3,3?-tetraethylbenzimidazolocarbocyanine iodide (JC-1) is a potential dependent J-aggregate forming lipophilic cation, which accumulates in mitochondria and indicates the membrane potential by an emission shift from green (527 nm) to red (590 nm, high transmembrane potential). Sperm where incubated with 2 mM JC-1 for 20 min at 378C. JC1 fluorescence excited at 488 nm was collected with a 530930 nm filter in the FL1 channel (green fluorescence) and a 585942 nm filter in the FL2 channel (orange fluorescence). A preliminary staining with 50 mM carbonyl cyanide 3chlorophenylhydrazone (CCCP) for 10 min was used as a positive control to disrupt the DC m .
The ATP values of spermatozoa where determined by using the ATP Bioluminescense Assay Kit CLS II (Roche, Mannheim, Germany). The results were referred to the protein concentration of the samples.

Serum parameters
Blood glucose levels, plasma insulin, triglycerides and free fatty acids were analyzed as previously described (Buchmann et al. 2007).

Glucose uptake
Uptake of [ 14 C]-2-deoxyglucose by sperm of 12 weeks old Slc2a8 '/' and Slc2a8 (/( mice was determined as described (McLean et al. 1997). Briefly, isolated sperm were diluted to 0.5 )10 6 sperm/ml with 50 mM TES, pH7.4, containing 130 mM NaCl and were centrifuged (700 g). Washed sperm were diluted in the above buffer containing 1 mM deoxy-D-glucose, and 2 mM CaCl 2 and 1 mCi/ ml [ 14 C]-2-deoxyglucose and incubated for 15 min at 378C. Glucose uptake was stopped by addition of a cold 0.1 M glucose solution and centrifugation at 12000 g for 5 min. Radioactivity of pellets was determined in a Beckman LS 6000LL scintillation counter.

Statistical analysis
Statistical significance was determined by two-tailed Student's t-test or by Pearsons's chi-square test with 2 degrees of freedom for fertility mating. All values are the mean9SEM. Statistical significance was set at pB0.05.

Inactivation of GLUT8 by targeting the Slc2a8 gene in mice
For generation of GLUT8 knockout mice (Slc2a8 (/( mice) we used the Cre/loxP recombination system which allows both the conventional deletion of the gene and the generation of tissuespecific null mutants. ES cells were transfected with the targeting construct ( Figure 1A), and homologous recombinants were re-transfected with crerecombinase to generate deletion of the neo and tk cassettes (floxed allele) or of the full segments (deleted allele). For the generation of Slc2a8 (/( mice, we performed a morula fusion with ES cells carrying the deleted allele as shown in Figure 1A (lower left panel). Male chimeric mice were mated with C57BL/6 females, and F1 progeny carrying the transgene were backcrossed 3 times on to the C57BL/6 background. Quantitative real-time PCR failed to detect full-length mRNA of Slc2a8 in all investigated tissues of Slc2a8 (/( mice ( Figure 1C, upper panel). Furthermore, no upregulation of GLUT1, GLUT3, GLUT5, or GLUT6 was observed in testis ( Figure 1C, lower panel). Immunohistochemistry of testis sections performed with a specific anti-GLUT8 antibody raised against sequences corresponding to the intracellular loop 6 and the C-terminus of GLUT8 confirmed the absence of GLUT8 protein in testis of Slc2a8 (/( mice while a positive staining of GLUT8 was detected in spermatocytes, spermatides, and mature spermatozoa of Slc2a8 '/' littermates ( Figure 1D). As shown in the lower panel of Figure 1D also in spermatozoa GLUT8 is located in an intracellular compartment rather than in the area of the acrosome as we have previously described (Schü rmann et al. 2002a).
Slc2a8 (/( mice were viable with normal growth and no apparent abnormality. In sections from several tissues (liver, skeletal muscle, and adipose tissue) from Slc2a8 (/( mice, no morphological or pathological abnormalities were observed (data not shown). Body weight development ( Figure 2A) and body composition of Slc2a8 '/' and Slc2a8 (/( mice were identical ( Figure 2B). In addition, no differences in serum glucose, triglyceride, free fatty acid or insulin levels were observed between fed wild-type and knockout mice (Table I). Due to these results we conclude that GLUT8 does not play an important role for the regulation of the energy balance and glucose homeostasis.
In the F2 progeny which was used for characterization of the phenotype, we obtained Slc2a8 '/' , Slc2a8 '/( , and Slc2a8 (/( mice in a ratio of 30.5:47.3:22.1 (Table II). Since the number of homozygous knockout mice was significantly lower (p 00.0024) than the expected 25%, breeding pairs of wild-type males and females, Slc2a8 (/( males and females as well as Slc2a8 (/( males and Slc2a8 '/' females were formed, and mean litter size and number of pups born over a time period of 6 months were compared. No significant differences in the average litter size or the number of live pups were  obtained between the indicated crossings (Table III), indicating that loss of GLUT8 does not cause a reduction in fecundity.
Intracellular and mitochondrial calcium levels are not altered in Slc2a8 (/( spermatozoa The high expression levels of GLUT8 in testis and the slightly altered genotype distribution observed with heterozygous breeding pairs suggested an impaired function of mature spermatozoa in the absence of GLUT8. We therefore analyzed different parameters which have been associated with defective fertility, such as structural abnormalities (Escalier & David 1984, Lalouette et al. 1996, alterations of intracellular (Perl et al. 2006) or mitochondrial calcium concentrations (Okunade et al. 2004) or reduced mitochondrial membrane potential (Perl et al. 2006). Analysis of testis sections revealed no histopathology or indications that spermatogenesis was abnormal ( Figure 1D). In addition, no differences in intracellular ( Figure 3A) or mitochondrial Ca 2' concentration ( Figure 3B) were detected with specific fluorescent calcium indicators in spermatozoa isolated from Slc2a8 '/' and Slc2a8 (/( mice. ATP levels and mitochondrial membrane potential are reduced in Slc2a8 (/( spermatozoa We next measured the ATP levels of isolated control and Slc2a8 (/( spermatozoa and detected significantly (p 00.037) reduced ATP levels of knockout sperm cells (0.3990.11 mM/mg protein) in comparison to that of wild-type litter mates (0.7890.15 mM/mg protein; Figure 3C). Beside glycolysis the mitochondrial phosphorylation plays crucial roles for the energy supply required for sperm motility (Turner 2006, Marchetti et al. 2002. Therefore, we analyzed the mitochondrial membrane potential (DC m ) of Slc2a8 '/' and Slc2a8 (/( spermatozoa by using the potentiometric fluorescent dye JC-1. As shown in Figure 3D DC m was reduced by more than 20% in Slc2a8 (/( spermatozoa. JC-1 staining as shown in Figure 3E detects mitochondria within the mid-piece of wild-type sperm. This staining was much lower in Slc2a8 (/( spermatozoa. In addition, sperm viability was monitored by staining with Sybr 14 (Garner & Johnson 1995) but viability of Slc2a8 '/' and Slc2a8 (/( spermatozoa was not different (33.593.5 vs 31.992).

Glucose uptake into Slc2a8 (/( sperm is not altered
In order to evaluate the effects of Slc2a8 disruption on glucose transport into spermatozoa we measured uptake of labeled deoxy-glucose of isolated sperm cells from wild-type and Slc2a8 (/( sperm. As expected from the intracellular localization of GLUT8 ( Figure 1D) we did not detect differences in glucose transport between the two genotypes indicating that a reduced energy supply is not responsible for altered ATP levels of Slc2a8 (/( sperm.
Electron microscopic analysis of Slc2a8 '/' and Slc2a8 (/( testis To determine whether structural abnormalities are responsible for impaired motility of Slc2a8 (/( sperm cells we examined testis of Slc2a8 '/' and Slc2a8 (/( mice by electron microscopy. As shown in Figure 6, in early and late spermatides of wildtype mice, normal development of acrosome and surrounding membranes was visible ( Figure 6A, B) whereas development of mitochondria was not consistent within the epithelium of Slc2a8 (/( testis. We observed a lack of condensation of mitochondria in some late spermatides ( Figure 6E, F) but also late spermatides with normally developed mitochondria ( Figure 6D). In addition, we stained sections of cauda epididymus with Pisum sativum agglutinine which detects the acrosome. As shown in Figure 6G no differences between wild-type and knockout sperm could be observed indicating that the epididymal maturation of Slc2a8 (/( sperm was not affected.

Discussion
The present data show that the facilitative glucose transporter GLUT8 is required for maintenance of the mitochondrial transmembrane potential (DC m ) of spermatozoa. Ablation of GLUT8 reduces ATP levels of sperm and sperm motility which results in a slight but significant deviation of offspring from the Mendelian distribution of genotypes. However, sperm number and survival rate as well as glucose transport of sperm cells were unaffected by deletion of GLUT8. Our data give new evidence for an intracellular function of GLUT8, presumably the transfer of sugars or metabolites through membranes in order to maintain energy flow.
Motility and fertility of spermatozoa are regulated in a complex way. The role of glycolysis for ATP production is controversially discussed in the literature. An important study by Narisawa et al. (2002) has shown that under conditions of defective mitochondrial phosphorylation the sperm is still able to generate ATP and to fertilize. In addition, targeted deletion of sperm-specific GAPDH, an enzyme located in the fibrous sheath resulted in male sterility which was associated with severe aberrations in sperm motility (Miki et al. 2004). On the other hand, Ford (2006) demonstrated that glycolysis is not required to support normal sperm function. Inhibition of glycolysis by a-chlorohydrin did not alter mouse sperm motility. Therefore, beside energy provided by glycolysis sperm depend on production of reactive oxygen intermediates (Fisher & Aitken 1997) and maintenance of DC m (Marchetti et al. 2002(Marchetti et al. , 2004. Male mice lacking the enzyme transaldolase (TAL) which indirectly contributes to the generation of NADPH are sterile because sperma-tozoa lost DC m and mitochondrial integrity (Perl et al. 2006). The effects on DC m and ATP levels in Slc2a8 (/( males did not lead to infertility; Slc2a8 knockout sperm were able to fertilize Slc2a8 wildtype oocytes. In addition, no differences in the number of litters or in the number of pups born after crossing Slc2a8 (/( males with Slc2a8 '/' females were observed in comparison to an intercross of Slc2a8 '/' mice (Table III). However, the reduced number of motile Slc2a8 knockout sperm resulted in a moderate but significant reduction of Slc2a8 '/( (47.3 instead of 50%) and Slc2a8 (/( (22.1 instead of 25%) offspring in the intercross of heterozygous mice when Slc2a8 wild-type and Slc2a8 knockout sperm competed for the oocytes (Table II). Altered mitochondria condensation as detected by ultrastructural analysis (Figure 6) might participate in the reduction of motility. We hypothesize that knockout sperm cells which exhibit defective mitochondrial condensation during development are those that are immobile after maturation. For the detection of mitochondrial membrane potiential and ATP levels all sperm cells were used including immotile sperm cells. Thereby reduced DC m and ATP levels of the Slc2a8 (/( spermatozoa were detected, indicating that the energy supply required for sperm motility was impaired. However, we did not detect differences in glucose uptake between Slc2a8 '/' and Slc2a8 (/( sperm (Figure 4). In addition, spermatozoa express the high-affinity glucose transporter GLUT3 at the cell surface (Haber et al. 1993, Burant & Davidson 1994. Therefore, it seems unlikely that deletion of GLUT8 which is primarily localized to intracellular membranes of all cell types (spermatocytes, spermatides, mature spermatozoa) impairs glucose uptake in Slc2a8 knockout cells. Rather, we propose that GLUT8 functions in a different, intracellular transport process which is Slc2a8 +/+ Slc2a8 -/-sperm motility sperm count p=3.3⋅10 -9 Figure 5. GLUT8 is required for normal sperm motility. Sperm count and sperm motility from Slc2a8 '/' and Slc2a8 (/( mice. Spermatozoa were isolated from epididymal tissues of 10Á12 weeks old Slc2a8 '/' and Slc2a8 (/( mice and counted (left panel), and their mobility was analyzed as described in Materials and Methods (means9SEM from 6 mice of each genotype). Figure 6. Electron microscopy of testis of Slc2a8 '/' (AÁC) and Slc2a8 (/( mice (DÁF) and analysis of cauda epididymal sperm with Pisum sativum agglutinine. (A) Early spermatides, (B, D, E) late spermatides, (C, F) cross section of a late spermatide, cut in the mid-piece region of the tail. Arrows depict the mitochondria (magnification approx. A and D)18,000; B and E)12,000; C and F)40,000). (G) Sections of the cauda epididymis of Slc2a8 '/' and Slc2a8 (/( males at the age of 10Á12 weeks were stained with Pisum sativum agglutinine and analyzed by confocal laser scanning microscopy. Nuclei were co-stained with DAPI. required for mitochondrial function and generation of ATP.
The role of GLUT8 in the regulation of the energy homeostasis and the metabolism is so far poorly understood. Recently, Membrez et al. (2006) described a phenotypical characterization of Slc2a8 (/( mice and observed normal embryonic and postnatal development, glucose homeostasis and response to mild stress, respectively. Here we confirmed the results of Membrez et al. (2006) who observed no differences in body weight between Slc2a8 '/' and Slc2a8 (/( mice kept on a standard chow diet. We therefore conclude that GLUT8 does not play a major role in the regulation of energy balance. GLUT8 can be expressed in blastocysts and translocated to their surface in response to insulin (Carayannopoulos et al. 2000). In addition, suppression of GLUT8 expression by antisense mRNA caused increased apoptosis of blastocysts, suggesting that GLUT8 plays an essential role in early embryogenesis (Pinto et al. 2002). However, our data do not confirm this conclusion. Since mating of knockout mice produced viable, normally developing offspring in numbers comparable to those of a wildtype intercross (Table III).
In summary, our data indicate that deletion of GLUT8 results in reduced sperm motility due to impaired mitochondrial function in spermatozoa, suggesting that GLUT8 is involved in the regulation of energy metabolism of sperm cells.