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Endocrinology Vol. 140, No. 9 4236-4243
Copyright © 1999 by The Endocrine Society

ARTICLES

Maternal Epidermal Growth Factor Deficiency Causes Fetal Hypoglycemia and Intrauterine Growth Retardation in Mice: Possible Involvement of Placental Glucose Transporter GLUT3 Expression1

Yoshimasa Kamei, Osamu Tsutsumi, Akio Yamakawa2, Yoshitomo Oka, Yuji Taketani and Junko Imaki

Department of Obstetrics and Gynecology (Y.K., O.T., Y.T.), The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; CREST (O.T.), Japan Science and Technology, Kawaguchi, Saitama 350, Japan; Department of Biosignal Research (A.Y.), Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173, Japan; Third Department of Internal Medicine (Y.O.), Yamaguchi University, Ube, Yamaguchi 755, Japan; and Department of Anatomy (J.I.), Nippon Medical School, Bunkyo-ku, Tokyo 113, Japan

Address all correspondence and requests for reprints to: Osamu Tsutsumi, M.D., Ph.D., Department of Obstetrics and Gynecology, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. E-mail: osamut-tky{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the physiological role of epidermal growth factor (EGF) in fetal growth in mice in which midgestational sialoadenectomy induced maternal EGF deficiency. Sialoadenectomy decreased the fetal weight significantly, indicating that maternal EGF deficiency caused intrauterine growth retardation. The weight of the fetal liver in the sialoadenectomized mice was reduced in proportion to the decrease in body weight (82.7 ± 10.2 vs. 70.9 ± 10.9 mg), whereas the brain weight was not reduced. Sialoadenectomy significantly decreased the glucose concentration in fetal plasma (86.0 ± 13.0 vs. 63.0 ± 11.8 mg/dl) without affecting the maternal plasma level of glucose. Transplacental transfer of 3H-2-deoxyglucose was significantly decreased by sialoadenectomy (5.17 ± 1.25 vs. 2.94 ± 1.02%), but transfer of 14C-aminoisobutyric acid was not affected. Northern blot analysis and in situ hybridization of glucose transporter isoform GLUT1 and GLUT3 messenger RNAs (mRNAs) in placenta revealed that sialoadenectomy significantly reduced the expression of GLUT3 mRNA without affecting GLUT1 mRNA levels. Administration of anti-EGF antiserum enhanced the effects of EGF deficiency, which were almost completely corrected by EGF supplementation. These results indicate that EGF plays an important role in fetal growth by regulating the transplacental supply of glucose via GLUT3 expression in the placenta.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRAUTERINE GROWTH RETARDATION is a major obstetrical problem because a fetus with a below-normal weight is at increased risk of death or of physical and/or mental impairments (1). Fetal growth retardation can be symmetrical or asymmetrical (1). Asymmetrical growth retardation most often results from uteroplacental dysfunction during the latter stage of pregnancy. Normal growth of the head or preferential sparing of the brain at the expense of the liver and other viscera is characteristic of this type of growth retardation (2, 3). Recent animal and epidemiological studies have suggested that malnutrition of the fetus in the middle and late gestational stages, which leads to asymmetrical intrauterine growth retardation, is associated with increased risks of cardiovascular disease and noninsulin-dependent diabetes mellitus in the adult (4, 5, 6, 7). Therefore, an understanding of the mechanisms by which intrauterine fetal growth retardation develops is clinically important.

Glucose is the major fuel for energy metabolism and growth during embryogenesis. Because the mammalian embryo is unable to generate glucose until the late stage of development (8, 9), the transfer of glucose from the maternal circulation to the conceptus is of major importance for mammalian development (10). Indeed, fetuses suffering from growth retardation are often hypoglycemic (11), and impaired placental glucose transport is thought to result in asymmetrical growth retardation (1). Recent studies have revealed that glucose transportation is mediated by a family of integral membrane glycoproteins. At least five different facilitative glucose transporter isoforms (GLUT1~5) have been identified and characterized (12, 13, 14, 15). GLUT1 and GLUT3, which are believed to mediate basal glucose uptake (12), are expressed in the placenta (12, 16, 17, 18, 19, 20, 21, 22). GLUT1 is responsible for supplying glucose for use as a placental fuel and GLUT3 is important for glucose transfer to the embryo (20, 22). However, the physiological role of placental glucose transporters in fetal development is not well defined, and the molecular mechanisms of placental glucose transport and its regulation remain to be elucidated.

Epidermal growth factor (EGF), a polypeptide with 53 amino acids, was first recognized for its mitogenic action on epidermal and mesodermal cells (23). A large amount of EGF is produced in the submandibular glands in mice (24). EGF has important roles in placental growth and function: it is involved in embryonal implantation (25), stimulates syncytiotrophoblast differentiation in vitro (26), and modulates placental endocrine functions (26, 27, 28, 29). We previously reported that midgestational sialoadenectomy (surgical removal of bilateral submandibular glands), which reduces maternal circulating EGF levels, caused asymmetrical growth retardation without affecting other maternal conditions or the placental weight in mice (30). In the present study, we used midgestational sialoadenectomy to produce a model of maternal EGF deficiency to investigate the possible role of EGF in glucose transfer between the maternal and fetoplacental compartments and in the regulation of placental expression of GLUT1 and GLUT3 messenger RNAs (mRNAs).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal experiments
The protocol of the animal experiment was approved by the ethics committee at our institution. C3H/HeN virgin female mice 8 to 10 weeks old were mated with mature C3H/HeN male mice of proven fertility. They were maintained under controlled temperature (25 C) and lighting conditions (lights on from 0800 to 2000 h). Each female mouse was checked every morning for the presence of a vaginal plug, and its day of detection was designated as day 1 of pregnancy. Pregnant mice were caged individually, and they had free access to food and water during the experimental period. In the morning on day 13 of pregnancy, mice were anesthetized with ether and underwent a sialoadenectomy (removal of the submandibular glands) or a sham operation (skin incision and manipulation of the submandibular glands). An osmotic minipump (Alzet osmotic minipump, model 1007D, Alza Corp., Palo Alto, CA) for the delivery of various agents was inserted sc on the animal’s back. To sham-operated mice, PBS was administered via the osmotic minipump. Sialoadenectomized mice received PBS, antimouse EGF antiserum (Collaborative Research, Waltham, MA), or mouse EGF (receptor grade, 1 µg/day, Collaborative Research). The antiserum was capable of binding 200 µg of mouse EGF per 100 µl, as determined by a modification of the Ouchterlony double-immunodiffusion method (31). The osmotic minipump delivered agents in a constant volume of 0.5 µl/h. Animals recovered quickly from surgery without any abnormalities, as judged by their general behavior, eating and drinking habits, and weight gain. Anti-EGF and EGF infusion also did not affected the maternal food intake and weight gain. In the evening on day 19 of pregnancy, the mouse was killed by cervical dislocation. Blood was collected in a heparinized syringe via the inferior vena cava and immediately chilled to 4 C. Blood samples were centrifuged at 2,000 x g for 5 min at 4 C to separate plasma, which was stored at -80 C until use. Fetuses and placentas were obtained via hysterectomies, and fetuses were assessed for viability. After weighing the fetus and placenta, we collected the fetal blood in a heparinized microsyringe by cardiopuncture. The plasma was obtained and stored in the same way as maternal plasma. Fetal organs were then dissected and weighed.

EGF RIA
The plasma concentration of EGF was determined by RIA, as described previously (32). This assay has a sensitivity of 0.1 ng/ml, and the intraassay and interassay coefficients of variation are 6.5% and 9.9%, respectively. We purchased 125I-mouse EGF from NEN Life Science Products (Boston, MA).

Glucose assay
Fetal organs were washed in PBS three times and homogenized in PBS in a Potter-Elvehjem glass-Teflon type homogenizer at 4 C. The glucose concentrations in maternal and fetal plasma and homogenized fetal organs (the brain and liver) were determined fluorometrically by the method of Lowry et al. (33). Briefly, the reaction mixture consisted of 100 mM of Tris-HCl, 1 mM of MgCl2, 0.5 mM of dithiothreitol (DTT), 300 µM of ATP, 30 µM of NADP, 1 µg/ml of hexokinase, and 0.02 U/ml of glucose-6-P dehydrogenase. The sample (1 µl) was mixed with 1 ml of reagent, and the mixture was incubated for 3 min at room temperature. A blank was run by incubating only the reaction mixture and adding 10 mM of NADPH as the standard. The fluorescence was read in a fluorometer (Ratio fluorometer-2, Farrand Optical, New York, NY), and the glucose content was estimated from the amount of NADPH. The protein content in homogenized fetal organs was determined by the method of Lowry et al. (34) using BSA (Sigma, St. Louis, MO) as the standard. ATP, DTT, NADP, hexokinase, glucose-6-P dehydrogenase and NADPH were purchased from Roche Molecular Biochemicals (Mannheim, Germany).

Transport assay of 3H-2-deoxyglucose (3H-2DG) and 14C-aminoisobutyric acid (14C-AIB)
Pregnant mice were anesthetized with a sc injection of 0.2 ml of a pentobarbital sodium solution (50 mg/ml, Abbott Laboratories, North Chicago, IL) on day 19 of pregnancy. A laparotomy was performed, and the inferior vena cava was exposed. A mixture of 3H-2DG and 14C-AIB (NEN Life Science Products) diluted in PBS was injected into the vein using a 30-gauge needle connected to a 100-µl Hamilton syringe. The mixture consisted of 50 µl of the 3H-2DG solution and 50 µl of the 14C-AIB solution; the specific activity of both solutions was 2 µCi/100 µl. The mixture was delivered over 5 sec, and the needle was left in place to prevent hemorrhage. The animals were killed 5 min after injection, and fetuses were dissected by hysterectomy. Fetuses were homogenized in 1 ml of PBS in a Polytron homogenizer (Kinematica, Luzerne, Switzerland) at maximum speed for 2 min at 4 C. The homogenate was completely dissolved in 15 ml of Aquasol II (NEN Life Science Products) and decolored with 1 ml of 30% hydrogen peroxide. Tracer concentrations were measured simultaneously with a liquid scintillation counter (Aloka, model LSC-903, Tokyo, Japan). The transfer ratio of the radioactivity represents the percentage of the fetal radioactivity, i.e. each fetal radioactivity divided by the total radioactivity of the agent injected to the mother.

RNA isolation and Northern blot analysis
Total RNA isolation and Northern blot analysis of GLUT1 and GLUT3 mRNA expressions were performed as previously described (35). Mouse GLUT1 complementary DNA (cDNA) inserted in a pUC9 plasmid and mouse GLUT3 cDNA inserted in a pGEM4Z plasmid were kindly provided by Dr. S. Nagamatsu (36). Briefly, 20 µg of total RNA were separated by electrophoresis on a 1% agarose gel containing 6% formaldehyde and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). DNA fragments of mouse GLUT1 and GLUT3 were labeled using the Random Primed DNA Labeling Kit (Roche Molecular Biochemicals) and was used as a probe. After prehybridization for 2 h, hybridization was carried out for 16 h in buffer containing 5 x SSC, 50% formamide, 5 x Denhardt’s solution, 0.2% SDS, 100 µg/ml alkali-denatured salmon sperm DNA, and 10% dextran sulfate at 42 C. The blots were washed three times in buffer containing 2 x SSC, 0.1% SDS at room temperature, and twice with 0.1 x SSC, 0.1% SDS at 55 C. Signals were detected by autoradiography with BioMax MS film (Eastman Kodak Co., Rochester, NY). To quantify the signals from each band, BAS-2500 (Fuji Photo Film Co., Ltd. Films, Tokyo, Japan) image analyzer and Mac BAS version 2.4 (Fuji Photo Film Co., Ltd. Films) were used. The same membranes were probed sequentially with mouse GLUT1, mouse GLUT3, and human ß-actin.

In situ hybridization of uteroplacental GLUT1/GLUT3
Placentas obtained on day 19 of pregnancy were fixed with 4% paraformaldehyde in 0.1 M of phosphate buffer at room temperature and postfixed in the same fixative containing 20% sucrose for 2 days. Specimens were cut into 10 µm-thick frozen sections with a cryostat and mounted on poly-L-lysine-coated slides. In situ hybridization was performed as described by Simmons et al. (37). Briefly, sections were vacuum dried, digested by proteinase K (10 µg/ml) for 5 min, and acetylated with 0.25% acetic anhydride in 0.1 M of triethanolamine. The sections were dehydrated in an ascending ethanol series and air-dried. The probe (3 x 106 dpm/ml) was dissolved in a buffer containing 50% formamide, 10% dextran sulfate, 1 x Denhardt’s solution, 12 mM of EDTA (pH 8.0), 10 mM of Tris-HCl (pH 8.0), 30 mM of NaCl, 0.5 mg/ml of yeast transfer RNA, and 10 mM of DTT; 100 µl of the probe solution were applied to each slide. Slides were coverslipped and incubated at 55 C overnight. Coverslips were then removed, and the slides were rinsed in 4 x SSC, digested with RNase A (20 µl/ml) for 30 min at 37 C, and rinsed sequentially in 2 x SSC, 1 x SSC, 0.5 x SSC, then for 30 min in 0.1 x SSC at 55 C, before again being dehydrated. The sections were exposed to XAR 5 film (Kodak) for 4 days, dipped in NTB2 nuclear emulsion (1:1 with water; Kodak) and stored with desiccant at 4 C for 2 weeks, developed, and then stained with hematoxylin and eosin for microscopic evaluation. Radioactive cRNA copies were synthesized using T7 polymerase and mouse GLUT1/GLUT3 cDNA (36) with [{alpha}-35S]UTP (NEN Life Science Products), as described previously (37). The specific activity of the probe was approximately 1.0 x 108 dpm/µg. As a control for nonspecific labeling, a sense-oriented probe generated by SP6 polymerase was applied to adjacent sections. All other molecular reagents were obtained from Promega Corp. (Madison, WI).

Quantification of in situ hybridization
Hybridization was quantified using nuclear emulsion-dipped slides, as reported previously (38). Briefly, radial sections of the uteroplacental unit were chosen at random. We counted all the grains within the grid area (2.5 x 2.5 µm) on the fetal side and the maternal side of the placenta for GLUT1 and GLUT3 and in the decidua for GLUT3 under dark-field illumination at x400 magnification.

Statistical analysis
Results are presented as the mean ± SD. Statistical analysis was performed using the Kruskal-Wallis test. A level of P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal plasma level of EGF
Sialoadenectomy performed on day 13 of pregnancy markedly reduced the plasma concentration of EGF, which fell to below the level of sensitivity on day 19 of pregnancy (Table 1Go). EGF replacement in the sialoadenectomized mice increased the EGF level to 0.30 ± 0.11 ng/ml.


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  		Table 1. Effects of midgestational sialoadenectomy and various agents on the EGF content of maternal plasma and fetal characteristics

 
Fetal characteristics
Sialoadenectomy with PBS did not affect the litter size, but the percentage of pups born alive was significantly lower in the sialoadenectomy plus PBS group (80.3%) compared with PBS-treated sham-operated control mice (96.2%) (Table 1Go).

The placental weight in PBS-treated sialoadenectomized mice was essentially the same as in PBS-treated sham-operated mice (Table 1Go). However, the average weight of pups was significantly lower in the PBS-treated sialoadenectomized mice than in the PBS-treated sham-operated control mice.

The liver weight in PBS-treated sialoadenectomized mice was reduced in proportion to the fetal body weight (Table 2Go). However, sialoadenectomy did not significantly affect the weight of the fetal brain. Thus, the ratio of the fetal brain weight to the total body weight was significantly higher in the sialoadenectomized mice than in sham-operated controls.


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  		Table 2. Effects of midgestational sialoadenectomy and various agents on the weight and glucose content of fetal organs

 
Administration of antimouse EGF antiserum to sialoadenectomized pregnant mice further exacerbated the above-mentioned fetal conditions, which were restored by EGF replacement in the sialoadenectomized mice.

There were no differences in fetal characteristics between the sham-operated mice treated with normal rabbit serum (Life Technologies, Inc., Grand Island, NY) or antimouse EGF antiserum and sham-operated mice treated with PBS (data not shown). Normal rabbit serum had no effect on fetal characteristics in the sialoadenectomized mice compared with the sialoadenectomized mice treated with PBS (data not shown).

Glucose content in plasma and fetal organs
There was no significant difference in the maternal plasma level of glucose among groups (Fig. 1Go). However, the fetal plasma level of glucose was significantly lower in PBS-treated sialoadenectomized mice (63.0 ± 11.8 mg/dl) than in the control mice (86.0 ± 13.0 mg/dl). Administration of antimouse EGF antiserum to the sialoadenectomized mice further reduced the fetal plasma concentration of glucose (42.2 ± 11.5 mg/dl). EGF supplementation in sialoadenectomized mice increased the fetal plasma concentration of glucose to 81.3 ± 15.3 mg/dl, which was not significantly different from the level in control mice.



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  		Figure 1. Effects of midgestational sialoadenectomy, anti-EGF antibody, and EGF on the plasma glucose content. Results are shown as the mean ± SD. C, PBS-treated sham-operated control mice; Sx, PBS-treated sialoadenectomized mice; Ab, anti-EGF antiserum-treated sialoadenectomized mice; R, EGF-treated sialoadenectomized mice. *, P < 0.05, {ddagger}, P < 0.01 vs. group C; §, P < 0.05 vs. group Sx; ||, P < 0.05, ¶, P < 0.01 vs. group R.

 
Sialoadenectomy significantly reduced the weight and glucose content of the fetal liver and decreased the glucose content of the fetal brain without affecting its weight (Table 2Go). Anti-EGF treatment further reduced the weight and glucose content of both organs; EGF replacement reversed these changes to the level of the sham-operated mice.

Placental transfer of 3H-2DG and 14C-AIB
Sialoadenectomy significantly reduced the transplacental transfer ratio of 3H-2DG to 2.94 ± 1.02% compared with sham-operated control mice (5.17 ± 1.25%) (Fig. 2Go). The ratio was further reduced by anti-EGF treatment in the sialoadenectomized mice (to 1.61 ± 0.50%) and was improved by EGF replacement (5.17 ± 1.79%). The transplacental transfer ratio of 14C-AIB was not affected by sialoadenectomy, anti-EGF treatment, or EGF replacement.



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  		Figure 2. Effects of midgestational sialoadenectomy, anti-EGF antibody, and EGF on the transplacental transfer of 3H-2DG and 14C-AIB. The data represent the percentage of the fetal radioactivities, i.e. each fetal radioactivity divided by the total radioactivity of the agent injected to the mother, and showed as mean ± SD. Abbreviations are explained in Fig. 1Go. *, P < 0.05, {ddagger}, P < 0.01 vs. group C; §, P < 0.05 vs. group Sx; ||, P < 0.05, ¶, P < 0.01 vs. group R.

 
Expression of GLUT1 and GLUT3 mRNA in the placenta
Northern blot analysis of mRNA isolated from placenta showed that sialoadenectomy decreased the GLUT3 mRNA expression in placenta to 70.1% of control mice, without affecting the GLUT1 mRNA expression (Fig. 3Go). Anti-EGF antibody administration to sialoadenectomized mice further reduced the GLUT3 mRNA content (by 52.0%), which was restored by EGF replacement (to 94.2%).



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  		Figure 3. Effects of midgestational sialoadenectomy, anti-EGF antibody, and EGF on the GLUT1 and GLUT3 gene expressions in the placenta. A, Northern blot analysis of GLUT1 (top), GLUT3 (center), and ß-actin (bottom) mRNA in the placenta. Size of the molecular weight markers is given in kb on the right. B, Comparison of GLUT1 and GLUT3 mRNA contents in the placenta. The signals were densitometrically quantified using BAS-2500 image analyzer and Mac BAS version 2.4. The optical density of each band was corrected for ß-actin, and the results were expressed as a percentage of the value obtained for control ones and showed as mean ± SD of six placentas. Abbreviations are explained in Fig. 1Go. *, P < 0.05, {ddagger}P < 0.01 vs. group C; §, P < 0.05 vs. group Sx; ||,P < 0.05, ¶, P < 0.01 vs. group R.

 
Regional distribution of GLUT1 and GLUT3 mRNAs in the placenta
GLUT1 mRNA was abundantly and diffusely expressed in the placenta, and there was no significant difference in its expression among groups (Figs. 4Go and 5Go). However, GLUT3 mRNA levels were decreased in the PBS-treated sialo-adenectomized mice in both the labyrinth and the yolk sac compared with the control mice (by 64.2% and 62.9%, respectively) (Figs. 4Go and 5Go). Antimouse EGF antiserum treatment in the sialoadenectomized mice further reduced the mRNA expressions (by 51.6% in the labyrinth and by 41.6% in the yolk sac); EGF replacement restored these levels to 100.2% and 83.4%, respectively. Control hybridization of serial tissue sections with a sense riboprobe did not produce any significant expression (data not shown).



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  		Figure 4. Effects of midgestational sialoadenectomy, anti-EGF antibody, and EGF on the regional distribution of GLUT1 and GLUT3 gene in the placenta. Brightfield photomicrographs showing GLUT1 mRNA (A, C, E, and G) and GLUT3 mRNA (B, D, F, and H) hybridizations in PBS-treated sham-operated control mice, PBS-treated sialoadenectomized mice, anti-EGF antiserum-administered sialoadenectomized mice, and EGF-treated sialoadenectomized mice, respectively. There were no significant differences in the GLUT1 mRNA levels in both the labyrinth and the yolk sac between groups. In contrast, GLUT3 mRNA expression was significantly decreased by sialoadenectomy and further decreased by anti-EGF antiserum administration and increased by EGF replacement in both the labyrinth and the yolk sac. L, Labyrinth; Y, yolk sac. Other abbreviations are explained in Fig. 1Go. Scale bars, 2.5 mm.

 


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  		Figure 5. Relative GLUT1 and GLUT3 mRNA levels in the placenta. The granule density is expressed as a percentage of that measured in group C. Results are shown as the mean ± SD of six different animals in each group. Abbreviations are explained in Fig. 1Go. *, P < 0.05, {ddagger}, P < 0.01 vs. group C; §, P < 0.05 vs. group Sx; ||, P < 0.05, ¶, P < 0.01 vs. group R.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rate of fetal growth and differentiation is determined by the interaction between genetic factors and various stimulatory substances, such as hormones and growth factors (1). Genetic control predominates in the first half of pregnancy and gives rise to a relatively narrow range of fetal growth patterns. In the second half of pregnancy, stimuli become increasingly important and bring about a greater diversity of growth and maturation. EGF has been shown to be increased in the submandibular glands and plasma during pregnancy in mice (32) and thus is one of the potential regulators of fetal development. Moreover, EGF deficiency induced by pregestational sialoadenectomy has been found to cause abortions in mice (32). We performed midgestational sialoadenectomy to reduce the plasma concentration of EGF (39), to investigate the physiological role of EGF in fetal development. The present and previous (30) results showed that midgestational sialoadenectomy caused significant fetal losses and asymmetrical intrauterine growth retardation. The administration of antimouse EGF antiserum, which neutralizes the action of EGF (40), to sialoadenectomized mice exacerbated these effects. EGF replacement successfully prevented intrauterine fetal death and intrauterine growth retardation. These results suggest that EGF plays a physiological role in normal fetal growth in mice. Previous reports have demonstrated that EGF may also regulate the placental growth (28, 41, 42). In the present study in vivo, we did not observe the effect of EGF on the placental growth in terms of its weight, which may be due to timing of the treatment or other unknown reasons including the difference in species.

Asymmetrical growth retardation most often results from uteroplacental dysfunction during the later stages of gestation (2, 3). It is characterized by normal growth of the head or brain sparing and a reduction in the growth of the liver, which is the organ most severely affected in fetal growth retardation associated with uteroplacental insufficiency. In the present study, the fetal liver weight decreased in association with decreases in body weight in the maternal EGF deficiency model, but the fetal brain weight was not affected. In addition, treatment with antimouse EGF antiserum to sialoadenectomized mice exacerbated growth retardation, whereas EGF replacement prevented these effects. These results suggest that EGF deficiency could cause uteroplacental insufficiency, resulting in asymmetrical intrauterine growth retardation and fetal loss. Alternatively, it is possible that deprivation of maternal EGF by sialoadenectomy or augmentation by EGF administration have direct effect on the fetus, although it has been a controversial subject, still not resolved, as to whether maternal EGF can cross the placenta to the fetus (43, 44). Similarly, antibodies are known to cross the placenta, especially in the latter stages of pregnancy. Synthesis of EGF has been shown for several tissues (45), and the anti-EGF administration to the mother may well inactivate fetally synthesized EGF. This in itself would undoubtedly have a negative effect on fetal growth, and the finding that administration of anti-EGF to the mother had the most severe effect suggests that the antibody acted directly on the fetus, as well as the placenta. In addition, it might be possible that EGF deficiency causes other unidentified critical changes leading to fetal growth retardation.

The fetal glucose pool is derived entirely from maternal glucose pool in most species (8, 9). Although some studies have suggested that placental glucose transport is closely related to the maternal plasma concentration of glucose (8, 46), the observed decrease in maternal glucose levels is too small to account for the severity of fetal hypoglycemia, and there is no significant difference in the relationship between the maternal and fetal blood glucose concentrations (11). In the present study, maternal EGF deficiency resulted in decreased concentrations of glucose in the fetal plasma, brain, and liver but had no effect on the maternal plasma glucose concentration. These results suggest that the major cause of hypoglycemia in the growth-retarded fetuses was a decreased transplacental supply of glucose. We observed a decrease in the transplacental supply of 3H-2DG, nonmetabolized glucose, in sialoadenectomized mice. These results support that maternal EGF deficiency reduces the transplacental supply of glucose.

The transplacental supply of amino acids is also important for fetal protein synthesis and growth (47). The fetal plasma concentrations of maternally transferred FFA is only one sixth to one seventh of the maternal concentrations, and the FFA do not contribute significantly to fetal metabolism (47). It has been reported that EGF might promote the placental amino acid transport (48). Therefore, we investigated the transplacental transfer of 14C-AIB, a nonmetabolized amino acid. In our study, however, maternal EGF deficiency did not affect the transfer of 14C-AIB. Our observations may suggest that, directly or indirectly, EGF selectively regulates the transplacental glucose supply in either a direct or an indirect manner.

Glucose transport across the cell membrane occurs via a stereospecific, saturable, and facilitative diffusion process dependent on glucose transporter proteins. GLUT1 and GLUT3 are two major placental glucose transporter isoforms and the mRNAs and proteins of these isoforms have recently been localized by in situ hybridization and immunohistochemistry (12, 16, 17, 18, 19, 20, 21, 22), suggesting that glucose transport across the placenta is regulated by these transporter isoforms. Ubiquitous expression of GLUT1 in the placenta suggested that it may be responsible for supplying glucose for use as a placental fuel. The specific expression of GLUT3 in the labyrinth, which specializes in nutrient transfer, suggests that GLUT3 may be important for the placental transfer of glucose (22). The precise cellular localization of GLUT3 in the plasma membranes of the maternal side of the syncytiotrophoblast layers suggests that GLUT3 serves a crucial role in placental transfer of glucose (20). However, the mechanism of placental expression of GLUT1 and GLUT3 has not been clarified.

The EGF receptor is strongly expressed in the mammalian placenta (41, 42, 43), and EGF modulates placental endocrine functions (26, 27, 28, 29). GLUT1 mRNA expression is induced by EGF in fibroblast cell lines (49, 50). In the present study, in situ hybridization and Northern blotting revealed that maternal EGF deficiency did not affect GLUT1 mRNA expression. However, EGF deficiency reduced placental GLUT3 mRNA expression in both the labyrinth, in which fetomaternal exchange of substances including glucose occurs, and the yolk sac. Anti-EGF antiserum treatment further inhibited GLUT3 mRNA expression, whereas EGF replacement enhanced GLUT3 mRNA expression. The decreases in GLUT3 expression and transplacental glucose supply induced by maternal EGF deficiency in the absence of effects on GLUT1 expression and the placental weight are consistent with the theory that GLUT1 is responsible for supplying glucose for use as a placental fuel, and that GLUT3 is important for the transplacental transfer of glucose (20, 22). However, it is possible that the reduction of GLUT3 mRNA is not directly affected by EGF. More importantly, because mRNA level does not always equal protein content, immunoblotting of placental protein lysates or immunohistochemistry may provide useful information to draw firm conclusions. As for GLUT1 protein, we measured its expression by Western blotting and confirmed that GLUT1 protein expression is not affected by sialoadenectomy (data not shown). However, unfortunately, antimouse GLUT3 antibody is not available at present. Thus, we cannot evaluate the GLUT3 protein expression.

In conclusion, maternal EGF deficiency during the latter half of pregnancy reduced placental GLUT3 mRNA expression and selectively impaired the transplacental glucose supply, resulting in asymmetrical intrauterine growth retardation in mice. These results suggest that EGF may play an important role in regulating placental function. However, EGF deficiency might also cause other critical changes which affect placental GLUT3 expression or fetal growth.


   Acknowledgments
 
The authors are grateful to Dr. N. Shinozuka for his valuable advice on statistical analysis and to Ms. I. Machida for secretarial work.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and the Ministry of Public Welfare of Japan. Back

2 Present address: Department of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115. Back

Received January 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cunningham FG, MacDonald PC, Gant NF, Levero KJ, Gilstrap III LC, Hankins GDV, Clark SL 1997 Fetal growth restriction. In Cunningham FG, MacDonald PC, Gant NF, Levero KJ, Gilstrap III LC, Hankins GDV, Clark SL (eds) Williams Obstetrics, Appleton & Lange, East Norwalk, pp 839–853
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