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Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan
Address all correspondence and requests for reprints to: Kuniaki Takata, Ph.D., Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan.
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Abstract |
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Introduction |
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Glucose is transferred from the maternal blood to the fetal circulation across the placental barrier, which separates the maternal and fetal circulation. In contrast to the human placenta, where numerous villi are formed (34), a complex of maternal and fetal circulations termed the labyrinth, develops in the rat placenta (35). The labyrinthine part of the placenta is the principal site of maternofetal exchange of substances. The labyrinthine wall, or interhemal membrane, which lies between the maternal blood space and fetal vessels, is composed of three layers of trophoblast (36, 37), i.e. the porous cytotrophoblast layers and syncytiotrophoblast layers I and II, from the maternal to fetal sides. We have previously shown by immunoblotting that GLUT1 protein is abundant in the rat placenta (21, 38, 39). In situ hybridization also confirmed the expression of GLUT1 (28). Immunohistochemical analysis revealed that GLUT1 is present in the syncytial cell layers. Detailed examination revealed the localization of GLUT1 at the plasma membranes of the maternal blood side of syncytiotrophoblast I and the fetal blood side of syncytiotrophoblast II (39). Syncytiotrophoblasts I and II are connected by numerous gap junctions (40, 41, 42). Immunohistochemical examination showed that these gap junctions are made of connexin 26 (Cx26), an isoform of gap junction channel proteins (16). We proposed that GLUT1, connexin 26, and GLUT1 lined in series in the syncytiotrophoblast layers constitute the machinery for the transplacental transfer of glucose through the rat placental barrier (11, 19, 21).
In addition to GLUT1, GLUT3 has been shown to be expressed in the rat placenta; and its possible involvement in the maternofetal transfer of glucose was suggested (28). The precise cellular localization of GLUT3 and comparison of the localization between GLUT1, GLUT3, and connexin 26, however, has not been made in the rat placental barrier. As such information should shed light on the glucose transfer machinery in the placental barrier, in this study we raised antibodies against rat GLUT3 and examined the localization of GLUT3 protein in the rat placental barrier by double-immunofluorescence staining for GLUT1 and GLUT3. We suggest a possible mechanism involving GLUT1, GLUT3, and connexin 26 for the transplacental transfer of glucose across the rat placental barrier.
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Materials and Methods |
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Tissue preparations
Wistar rats at 12, 14, 16, and 18 days of pregnancy were
obtained from Imai Experimental Animal Farm (Gunma, Japan). Days of
pregnancy were designated as previously described (39). The animals
were anesthetized with sodium pentobarbital, and placentas (n =
8–10 from each animal) were removed, cut into pieces, and washed with
PBS to remove blood. Brain (cerebral cortex) was taken from male
4-week-old Wistar rats supplied from the Animal Breeding Facility,
Gunma University (Gunma, Japan).
Immunoblotting
The tissue specimens were homogenized in PBS containing protease
inhibitors (38). Protein content was measured by the BCA Protein Assay
Reagent (Pierce, Rockford, IL). Homogenate (10 µg of protein) was
electrophoresed through SDS polyacrylamide gels and transferred to
membrane filters (Immobilon-PSQ, Millipore, Bedford, MA). The blotted
membranes were sequentially incubated with 3% BSA, guinea pig
anti-GLUT1 or rabbit anti-GLUT3 antibodies diluted at 1:1000 each, and
125I-protein A (New England Nuclear, Wilmington, DE).
Autoradiography was performed for 0.5–18 h with imaging plates, and
the plates were processed with a BAS2000 bioimage analyzer (Fuji Film,
Tokyo, Japan).
Light-microscopic immunohistochemistry
Specimens were fixed in 4% formaldehyde in 0.1
M sodium phosphate buffer, pH 7.4, for 3 h at room
temperature, washed with PBS, infused in 20% sucrose in 0.1
M sodium phosphate buffer, pH 7.4, containing 0.02% sodium
azide, and embedded in OCT compound. Specimens were rapidly frozen in
liquid nitrogen, and then sectioned (6-µm thickness) with a cryostat.
Some of the fixed specimens were cut into small pieces and infused with
2.3 M sucrose in 0.1 M sodium phosphate buffer,
pH 7.4, containing 0.02% sodium azide. Infused tissue blocks were
mounted on stubs and rapidly frozen in liquid nitrogen. Semithin frozen
sections (0.5–1 µm thickness) were cut at -60 C with a Leica
Ultracut S UCT ultramicrotome equipped with an EM FCS cryokit (Leica,
Austria) and glass knives and collected on glass slides.
Immunofluorescence staining was carried out essentially as previously
described (16, 21, 39). For double-immunofluorescence staining for
GLUT1 and GLUT3, sections were covered with 5% normal goat serum in
PBS for 10 min and incubated for 1 h with a mixture of guinea pig
anti-GLUT1 antibody and rabbit anti-GLUT3 antibody, each diluted at
1:500 and 1:1000, respectively. After having been washed with PBS, the
sections were incubated with a mixture of DTAF (dichlorotriazinyl amino
fluorescein)-labeled donkey antiguinea pig IgG (Jackson Immunoresearch,
West Glove, PA), LRSC (lissamine rhodamine sulfonyl chloride)-labeled
donkey anti-rabbit IgG (Jackson Immunoresearch), and DAPI
(4',6-diamidino-2-phenylindole dihydrochloride, Boehringer-Mannheim,
Mannheim, Germany) for 1 h. Specimens were washed with PBS,
mounted using antibleaching mounting medium (21), and examined with a
BX-50 microscope (Olympus, Tokyo, Japan) equipped with epifluorescence
and Nomarski differential interference-contrast optics.
Electron-microscopic immunohistochemistry
Specimens were fixed in 4% formaldehyde in 0.1
M sodium phosphate buffer, pH 7.4, for 3 h at room
temperature, and washed with PBS. They were infused with 20% sucrose
in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.02%
sodium azide, embedded in OCT compound (Miles, Elkhart, IN), and
rapidly frozen in liquid nitrogen. Cryostat sections (10-µm
thickness) were cut and mounted on glass slides coated with
poly-L-lysine. The sections were covered with 5% normal
goat serum in PBS for 10 min and incubated for 1 h at room
temperature with rabbit anti-GLUT3 antibody diluted at 1:1000. After a
rinse with PBS, sections were incubated for 1 h at room
temperature with 1.4-nm gold particles conjugated to Fab' fragments of
antirabbit IgG (Nanogold, Nanoprobes, Stony Brook, NY), diluted at 1:50
(48). After a rinse with PBS, the sections were refixed with 1%
glutaraldehyde for 30 min. The labeled sections were next washed for 30
min with six changes of PBS and incubated with silver enhancement
solution containing 1 mg/ml silver acetate, 14 mg/ml trisodium citrate
dihydrate, 15 mg/ml citric acid monohydrate, and 2.5 mg/ml
hydroquinone, for 3–15 min at room temperature (49). After two quick
rinses with distilled water, the specimens were immersed in 0.05%
sodium acetate for 1 min, and then, after a rinse for 30 min in six
changes of water, were treated with 0.05% chloroauric acid for 2 min
at room temperature. After another rinse for 30 min in six changes of
water, the sections were fixed with 1% osmium tetroxide in 0.1
M sodium phosphate buffer, pH 7.4, for 30 min, dehydrated
through a series of graded ethanols, and embedded in epoxy resin.
Ultrathin sections were cut, stained with uranyl acetate and lead
citrate, and examined with a JEM-1010 transmission electron microscope
(JEOL, Tokyo, Japan).
Immunohistochemical controls
Sections were incubated with normal serum instead of the primary
antibody. The specificity was also checked by incubation with the
primary antibody in the presence of the peptides (5 µg/ml) used to
generate the antibody. These immunohistochemical controls were carried
out side by side with the experimental slides for both
immunofluorescence and immunoelectron microscopic
immunohistochemistry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In the 18-day placenta homogenate, a
40-kDa protein was detected, showing the presence of the GLUT3 glucose
transporter in the rat placenta (Fig. 1b). A 42-kDa protein was
detected in the homogenate of the 18-day placenta with the guinea pig
anti-GLUT1 (Fig. 1c), which confirmed our previous results (21).
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Localization of GLUT3 and GLUT1 was carried out by immunofluorescence
staining of cryostat (Fig. 2) and
semithin frozen sections (Fig. 3) of rat
placentas. We examined the localization of GLUT1 and GLUT3 on days 12,
14, 16, and 18. On day 12, both GLUT3 and GLUT1 were detected in the
labyrinth, whereas only GLUT1 labeling, although weak, was detected in
the junctional zone (Fig. 2, a–f). GLUT3 staining was restricted to
the labyrinth. On day 18, both GLUT3 and GLUT1 were present in the
labyrinth, where maternofetal exchange of substances occurs (Fig. 2, g–i). In the junctional zone, strong labeling for GLUT1 was detected
(Fig. 2h), but no labeling for GLUT3 (Fig. 2g).
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). On
day 12, three GLUT1-positive lines were evident in the
syncytiotrophoblast layers located between the maternal blood space and
fetal capillaries in the labyrinth (Fig. 3b). In addition to
syncytiotrophoblast layers, nonsyncytial undifferentiated cells located
beneath the syncytial cells were positive for GLUT1 (Fig. 3b). GLUT3
labeling in the syncytial cell layer formed two positive lines between
the maternal blood space and fetal capillaries (Fig. 3a). Nonsyncytial
undifferentiated cells were negative or weak for GLUT3 labeling.
Double-exposure images showed three lines along syncytiotrophoblast
layers: yellow, yellow, and green from the
maternal side (Fig. 3c), showing the coexistence of GLUT3 and GLUT1 in
the maternal side of syncytiotrophoblast I and between
syncytiotrophoblast layers I and II. The basal side of
syncytiotrophoblast II was positive for GLUT1 only (Fig. 3c). Neither
GLUT3 nor GLUT1 was detected in cytotrophoblasts directly facing the
maternal blood in the labyrinth (Fig. 3, a–c). Localization pattern of
GLUT3 and GLUT1 in the labyrinthine wall began to change on day 14
where decrease of GLUT1 labeling in between syncytiotrophoblast layers
I and II was evident (Fig. 3, d–f). On days 16 and 18, the labyrinth
was occupied with typical placental labyrinthine structure with a
single cytotrophoblast layer, two syncytial layers, and fetal
endothelium. Undifferentiated trophoblasts were rarely encountered.
GLUT1 exhibited two distinct lines along the double syncytiotrophoblast
layers separating the maternal blood space and fetal blood vessels
(Fig. 3h). Positive staining for GLUT1 was not seen in cytotrophoblast
or endothelial cells of fetal blood vessels. GLUT3 was localized along
double syncytial layers facing the maternal blood space and between the
double syncytial layers, forming two distinct lines (Fig. 3g). Positive
staining for GLUT3 was not seen in cytotrophoblast or endothelial cells
of fetal blood vessels, either. Double-exposure images clearly showed
that GLUT3 and GLUT1 were colocalized in syncytiotrophoblastic layer I
along the cell contour facing the maternal blood space (Fig. 3i).
Between double syncytial layers in the interhemal membrane, only GLUT3
was detected. Only GLUT1 was present along the cell contour of
syncytiotrophoblast layer II facing the fetal blood vessels. Three
anti-GLUT1 antibodies, two of them raised in rabbits and one in a
guinea pig, gave the same staining pattern (data not shown). Other
glucose transporter isoforms tested, i.e. GLUT2, GLUT4, and
GLUT5, showed no positive staining in the labyrinth (data not
shown).
Ultrastructural localization of GLUT3 in 18-day placenta
To examine the ultrastructural localization of GLUT3, we used
Nanogold probes to carry out preembedding electron-microscopic
immunohistochemistry on the 18-day placenta. In the labyrinth, GLUT3
was localized at the plasma membrane of syncytiotrophoblast I facing
the maternal blood space (Fig. 4, a and
b). It was also present at the plasma
membrane of the syncytiotrophoblast II facing syncytiotrophoblast I
(Fig. 4, c and d). The basal side of syncytiotrophoblast II and the
plasma membrane of syncytiotrophoblast I facing syncytiotrophoblast II
were negative for GLUT3.
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, c–f) or a mixture of the primary antibody and 5 µg/ml antigen
peptide, no positive labeling was seen in either immunofluorescence or
immunogold studies (data not shown). In the double-immunofluorescence
studies, replacement of one of the primary antibodies with normal serum
did not affect the staining with the other antibody. These control
experiments confirmed the specificity of the immunostaining. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of GLUT1 alone in the junctional region, but expression of
both GLUT1 and GLUT3 in the labyrinthine part, was reported earlier
(28, 29), and our findings (Fig. 2) are consistent with their results.
Because GLUT1 is ubiquitous in the placenta, it was suggested that
GLUT1 may be responsible for supplying glucose for use as a placental
fuel (28). Specific expression of GLUT3 in the labyrinth, which is
specialized in nutrient transfer, suggested that GLUT3 may be important
for the maternofetal transfer of glucose (28). GLUT3, having a
relatively lower Km value (10, 50) compared with that of
GLUT1, may confer effective transfer of glucose into the
syncytiotrophoblast in the case of hypoglycemia in a similar manner as
in neurons and also serve for retention of the sugar in the
syncytiotrophoblast as a reservoir. In fact, deposition of glycogen in
the syncytiotrophoblast layers was reported by electron microscopy (37, 41). Because detailed examination of the localization of GLUT1 and
GLUT3 in the labyrinthine wall would provide evidence to determine the
validity of this notion, we carried out high resolution
immunofluorescence and immunoelectron microscopic studies. We found
that both GLUT3 and GLUT1 were localized at the plasma membrane of the
syncytiotrophoblast I facing the maternal blood side, a site of entry
into the syncytial layers from the maternal blood side. Both GLUT3 and
GLUT1, especially GLUT3 with its higher affinity for glucose, should
play an important role for the entry of glucose into the syncytial cell
layers. Only GLUT1 was detected at the plasma membrane of
syncytiotrophoblast II facing the fetal blood side, the site of exit
from the syncytial cell layers to the fetal blood side. Therefore,
GLUT3 could not be directly involved in the efflux into the fetal blood
side from the syncytiotrophoblast layers, the site of the placental
barrier. Instead, GLUT1 should play a prime role for the exit of
glucose from the syncytial layers. In addition to GLUT1 and GLUT3,
abundant connexin 26 is present between the two syncytiotrophoblast
layers connected by gap junctions (21). Taking into account the
ultrastructure of the placental labyrinth (35, 40, 41, 42) and the
localization of GLUT3, GLUT1 (39), and connexin 26 (21), we suggest
that transfer of glucose across the placental barrier occurs as
follows: glucose freely passes the cytotrophoblast layer through pores
in it. GLUT3 and GLUT1 in the syncytiotrophoblast I are responsible for
the uptake of glucose into its cytoplasm. Glucose is then transferred
to the cytoplasm of the syncytiotrophoblast II via connexin 26 channels
of gap junctions between the two syncytial cell layers. Glucose leaves
the syncytiotrophoblast II via GLUT1, and enters the fetal circulation
by crossing the endothelial cell through numerous fenestrations. The
function of GLUT3 at the plasma membrane of syncytiotrophoblast II
facing syncytiotrophoblast I remains to be clarified. It may possibly
serve to retrieve glucose that somehow managed to pass the
syncytiotrophoblast I, the first barrier cell layer.
In the placentas of diabetic rats, placental GLUT3 messenger RNA and protein increased, whereas those of GLUT1 remained unchanged (29). A similar increase in GLUT3 was also observed in rats with short-term hyperglycemia achieved by hyperglycemic clamps (29). GLUT3 in the rat placenta, therefore, appears to be highly sensitive to the blood glucose level and to play an important role in the alteration of placental function in the diabetic placenta (29). These results are basically in accord with our model of glucose transfer, that GLUT1 localized at the site of entry and exit in the placental barrier may ensure a basal transfer of maternofetal glucose transfer. Asymmetric distribution of GLUT3 in the syncytiotrophoblast layers may suggest asymmetric kinetics of glucose transfer across the placental barrier. Presence of both GLUT1 and GLUT3 at the site of entry into and of GLUT1 at the site of exit from the barrier would confer the retention of glucose in the fetus and would be beneficial to provide a stable milieu for the fetal development. In hyperglycemia, increases in GLUT3 and glycogen were observed (29), suggesting that overflowed glucose can be transferred and stored as glycogen in syncytiotrophoblasts within placenta through GLUT3 facing the maternal side. Upon maternal hypoglycemia, stored placental sugars may be transferred to the fetal blood circulation through GLUT1 facing the fetal side for fetal development, which transfer would prevent a large fluctuation of the glucose level in the fetal blood. Syncytiotrophoblasts therefore could serve not only as a transfer machinery, but also as a reservoir of nutrients. GLUT3, with its high affinity for glucose, localized at the membrane of syncytiotrophoblast I facing the maternal blood sides, may ensure the influx of glucose into syncytiotrophoblast I under the condition of maternal hypoglycemia.
In the ciliary body epithelium, double-epithelial cell layers composed of pigmented and nonpigmented epithelial cells serve as the structural basis for the blood-aqueous barrier (14, 38, 51). GLUT1 is abundant at the plasma membrane of pigmented cells facing blood vessels and of nonpigmented cells facing the aqueous humor (14, 16). Numerous gap junctions composed of connexin 43 (Cx43) connect the double epithelial cell layers (16). In the rat placenta, where the barrier is also made of double cell layers, abundant gap junctions of connexin 26 connect these double cell layers (21). Connexins, connecting these two layers, can serve as nonspecific hydrophilic channels for relatively small molecules such as glucose. We suggested earlier that the combined action of GLUT1, connexin(s), and GLUT1 in series may serve as the machinery for the transfer of glucose across these blood-tissue barriers (16, 21, 39). The asymmetrical presence of GLUT3 in the rat placental barrier could be important for the efficient transfer of glucose while preventing the loss of glucose at the time of maternal hypoglycemia, thereby providing a stable milieu for the developing fetus.
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Acknowledgments |
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Received March 12, 1997.
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