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Endocrinology Vol. 141, No. 2 802-808
Copyright © 2000 by The Endocrine Society

ARTICLES

Retinoic Acid Synthesis and Expression of Cellular Retinol-Binding Protein and Cellular Retinoic Acid-Binding Protein Type II Are Concurrent with Decidualization of Rat Uterine Stromal Cells1

Wen Li Zheng, Elaine Sierra-Rivera, Jing Luan, Kevin G. Osteen and David E. Ong

Departments of Biochemistry (W.L.Z., D.E.O.), Obstetrics and Gynecology (E.S.R., K.G.O.), and Cell Biology (J.L.), Vanderbilt School of Medicine, Nashville, Tennessee 37232

Address all correspondence and requests for reprints to: David E. Ong, Ph.D., Department of Biochemistry, 610 MRB-I, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. E-mail: david.e.ong{at}vanderbilt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decidualization of stromal cells at the site of embryo implantation in the rat uterus is accompanied by expression of cellular retinol-binding protein and cellular retinoic acid-binding protein [CRABP(II)], whose presence has been shown to correlate with gain of ability to synthesize retinoic acid in other cells. Here we examined whether decidual cells also acquired the ability to synthesize retinoic acid, which would have important implications for understanding the implantation process. Decidual cells were isolated from the uterus on day 8 of pregnancy and cultured. When provided with retinol, they indeed synthesized and released retinoic acid to the medium. To follow acquisition of this ability more closely, artificial induction of decidualization was exploited. Ovariectomized rats were placed on a hormonal regimen that allows decidualization to occur in vivo, with oil stimulation, or in vitro, if cells are isolated on day 5 of the regimen and then cultured. Decidualization in vivo reproduced the expression of cellular retinol-binding protein and CRABP(II) seen during pregnancy. Stromal cells isolated on regimen day 2 synthesized little retinoic acid and expressed little alkaline phosphatase, a marker of decidualization. Stromal cells isolated on regimen day 5 had elevated levels of alkaline phosphatase, increasing during the 3 days of culture examined. The ability of the stromal cells to synthesize retinoic acid showed the same pattern: a substantially elevated production from that previously observed, on day 2, with production increasing significantly over the next 2 culture days. Thus, expression of CRABP(II) was correlated with gain of ability to synthesize retinoic acid. Retinoid signaling may be an important part of the process of embryo implantation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE VITAMIN A alcohol (retinol) is essential for proper functioning of the female reproductive system and maintenance of pregnancy. Deficiency of retinol leads to irregular estrous cycles, morphological changes in the uterine epithelium, failure to establish or complete pregnancy, and fetal malformations (1, 2). Retinoic acid, one of the hormonal forms of vitamin A, can restore and maintain normal estrous cycles and frequency of mating in animals deprived of its precursor, retinol. Retinoic acid administration delays the onset of embryonic degeneration and supports the invasion of allantoic blood vessels into the labyrinth and thus the formation of the chorioallantoic placenta (3, 4). However, when retinoic acid is provided in place of retinol, fetuses are resorbed on about the 15th day of gestation. This condition can be prevented if a very small amount of retinol (2 µg) is provided on about the 10th day of gestation. If retinoic acid supplementation (in the absence of additional retinol) is continued, the pregnancy proceeds to parturition (5). This evidence suggests that retinoic acid can fulfill most, but not all, of the vitamin A requirement for a successful pregnancy.

During early pregnancy, the uterine endometrium responds to an implanting blastocyst with extensive growth and differentiation of endometrial stromal cells into decidual cells at the site of implantation (6, 7, 8). This process is controlled by both maternal hormones and signals from the implanting embryo and is essential for successful implantation. We previously have observed that expression of two of the cellular retinoid-binding proteins, cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein type II [CRABP(II)] is initiated at the site of decidualization (9). Further, we have established that CRABP(II) expression is associated with the ability of several cell types to synthesize retinoic acid from retinol (10, 11). These observations suggested that the retinoic acid necessary for the maintenance of pregnancy might arise from the decidual cells themselves. Here we report that the process of decidualization does indeed lead to the acquisition of retinoic acid-synthesizing ability, suggesting that part (or all) of the requirement for retinoic acid is met by local synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female ovariectomized Sprague Dawley rats (200–225 g) were housed in a temperature- and light-controlled room (21 ± 1 C; lights on, 0700–1900 h). Rats were fed rat chow (Ralston Purina Co., St. Louis, MO), provided with water ad libitum, and allowed to acclimate for 1–2 weeks before use in these experiments. Timed pregnant rats were ordered 1 day before use in the experiments. These studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the oversight of veterinarians and our local institutional animal care and use committee.

Treatment of animals
To obtain rats with uteri sensitized for decidualization, estrogen and progesterone in sesame oil were administrated sc into ovariectomized rats, as previously described (12) (see Fig. 1Go). Briefly, 2 weeks after ovariectomy, rats were placed on a hormonal regimen of estrogen and progesterone that maximized uterine sensitivity to a decidual stimulus. The hormonal regimen consisted of 0.2 µg estrogen in the morning for 3 days, 0.2 µg estrogen and 1 mg progesterone in the afternoon of day 0, 4 mg progesterone in the afternoons of days 2 and 3, 0.3 µg estrogen and 4 mg progesterone in the afternoon of day 4, and 0.1 µg estrogen and 4 mg progesterone in the morning of day 5. In the afternoon of day 5, some of the rats were killed, and uteri were collected for cell isolation. The remaining rats were given an intrauterine injection of sesame oil at the ovarian end of the uterus to induce a decidual response. Injections of 0.1 µg estrogen and 4 mg progesterone were continued for 4 more days (days 9–12). Each group has at least six female Sprague Dawley rats.



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  		Figure 1. A schematic diagram of the hormonal treatment protocol administrated to obtain rats sensitized for decidualization. Hormones were dissolved in sesame oil. Intrauterine stimulation was accomplished by injection of 100 µl sesame oil at the ovarian end of the uterine horn. OVX, Ovariectomized adult rats (2 weeks after surgery); E2, estradiol; P4, progesterone.

 
Cell preparations from the uteri of pregnant rats
Uteri were collected from rats on day 8 of pregnancy (4 rats). After embryonic tissue was removed, decidual balls were dissected from the uteri. Decidual tissue was minced into 1- to 2-mm fragments immediately, as described for the isolation of endometrial stromal cells (13). The fragments were washed in HBSS (Life Technologies, Inc./BRL, Gaithersburg, MD) and resuspended in solution consisting of 0.4% type IV collagenase (Worthington Biochemcial Corp., Freehold, NJ), 0.02% deoxyribonuclease (Sigma), and 2% chicken serum (Sigma) for release of cells. Fragments were placed at 4 C for 30 min, followed by incubation for 1 h in a 37 C shaker water bath. Isolated decidual cells were separated from fragments by filtration through an 88-µm filter (Swinex) unit. Red blood cells present in the cell suspension were removed by centrifugation on a 66% Percoll gradient. Cell viability was determined by trypan blue exclusion. Cells were plated at a density of 2.5 x 105/well (24-well plate). Cultures were maintained in DMEM-Ham’s F-12 supplemented by 20% FBS (He-Clone), 1% ITS+ (Life Technologies, Inc./BRL), and 0.1% Excyte (Miles Scientific, Elkhark, IN) at 37 C in a humidified atmosphere with 95% air-5% CO2.

Cell preparation from the uteri of hormone-treated ovariectomized rats
Uterine cell separation was accomplished by modification of our previous method of sequential enzymatic dissociation, filtration, and differential sedimentation (13). In brief, the uteri were slit longitudinally and placed in medium containing 0.4% type IV collagenase (Worthington Biochemical Corp.), 0.02% deoxyribonulcease (Sigma), and 2% chicken serum (Sigma) for 30 min at 4 C. This was followed by incubation at 37 C for 1 h. The uteri were vortexed (six times) at medium speed for 30–40 sec on a Vortex mixer (Fisher Scientific, Pittsburg, PA). The supernatant liquid, containing stromal and epithelial clumps, was collected. The stromal cells were isolated from epithelial clumps by sequential filtration through 88- and 22-µm filter units, respectively.

To prepare stromal cells, the effluent from filtration was washed and placed in a 66% Percoll gradient to remove red blood cells. Stromal cells migrated to the interface between the medium and the Percoll. After collection, stromal cells were washed with serum-free medium and resuspended in 5 ml growth medium (serum containing).

Epithelial clumps were further digested in HBSS (Ca2+ and Mg2+ free) containing 0.4% collagenase, 0.1% hyaluronidase, and 0.1% pronase at 37 C for 20–30 min until a large number of single epithelial cell had separated. Isolated cells and glands were washed in serum-free medium and resuspended in serum-containing medium. Cells were counted with a hemocytometer, and viability was assessed via trypan blue. Stromal cells were cultured on a coating of type I rat tail collagen (Vitrogen 100, Collagen, Inc., Fremont, CA) in a 24-well tissue culture plate. Epithelial cells were cultured in Matrigel (Collaborative Biomedical Products, Bedford, MA)-coated 24-well tissue culture plates.

Immune reagents and immunohistochemistry
Preparation of specific IgG antibody preparations against CRABP(II) and CRBP has been previously described (14, 15). Tissue samples for immunolocalization were immersion fixed for approximately 24 h in 20% isopropyl alcohol, 4.0% (wt/vol) paraformaldehyde, 2.0% (wt/vol) trichloroacetic acid, and 2.0% (wt/vol) zinc chloride and then were transferred to 70% ethanol. Paraffin embedding and slide sectioning were carried out by the Vanderbilt histopathology department. Our immunohistochemical procedures have been described previously (15). Primary antibody incubations [OD280 = 0.37, diluted 1:150 for CRBP; OD280 = 0.36, diluted 1:1000 for CRABP(II)] were carried out in a humidified chamber at 4 C overnight. The IgG populations not retained by the recombinant CRBP and CRABP(II) affinity columns used to prepare the specific IgG reagents were used as the negative controls for the immunolocalization studies.

In situ hybridization
In situ hybridizations were carried out using a complementary RNA CRABP(II) probe labeled with [{alpha}-35S]UTP (NEN Life Science Products, Boston, MA). Antisense or sense riboprobes to CRABP(II) 3'-untranslated region were prepared by RT using SP6 or T7 polymerase (Ambin, Ambion, Inc., Austin, TX). Paraffin sections were deparaffinized and treated with proteinase K (20 µg/ml) for 8 min at room temperature. After 15 min in 4% paraformaldehyde, sections were put in 0.25% acetic anhydride and 0.1 M triethanolamine (pH 8.0) for 10 min, followed by hybridization overnight at 50 C with 4 x 105 cpm/ml 35S-labeled probe in 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 0.01 M Tris-HCl (pH 7.4), 5 mM Na-EDTA (pH 8), 0.2% Ficoll 400, 0.2% polyvinyl pyrrolidone, 50 mM dithiothreitol, and 100 µg/ml yeast transfer RNA.

Sections hybridized with riboprobes were washed with 2 x SSC (standard saline citrate)-20 mM ß-mercaptoethanol at 50 C for 15 min, then washed twice with wash buffer A (4 x SSC, 50% formamide, and 20 mM ß-mercaptoethanol) at 55 C for 30 min each time and washed twice with wash buffer B (4 x SSC, 20 mM Tris-HCl (pH 7.5), and 2 mM EDTA, pH 8.0) at 37 C for 10 min each time. Sections were then treated with 20 µg/ml ribonuclease A (Sigma) in wash buffer B at 37 C for 30 min. Two additional washes were carried out in wash buffer B with 20 mM ß-mercaptoethanol at 30 C for 10 min each time, and another two washes were performed in wash buffer A at 55 C for 30 min each time. Then sections were washed in water twice and air-dried. Slides were dipped in Kodak NTB-2 emulsion (Kodak), developed after 1–2 weeks, and counterstained with hematoxylin.

Measurement of retinoic acid production
Cell cultures were supplied with 8 µM BSA/2 µM retinol. After incubation for 12 h, the medium was removed to a 50-ml conical tube for extraction of retinoic acid. The procedures used for retinoic acid analysis have been described in detail previously (10).

Measurement of alkaline phosphatase activity (ALP)
At various times throughout the culture period the medium was removed, and cells were washed with PBS. Cells of wells that were to be assayed for ALP activity received 150 µl 0.25% sodium deoxycholate and were stored at -70 C until assayed (16). Activity is expressed as nanomoles of substrate, p-nitrophenol phosphate (Sigma), hydrolyzed per 30 min.

Statistical analysis
The data are presented as the mean ± SEM of either triplicate or quadruplicate observations from a single experiment. Each experiment was performed at least twice on different cell preparations. Because of significant differences between experiments, the data from different cell preparations have not been pooled. In the long term culture experiments, there was consistently an increase in ALP activity between day 1 and day 3 in culture conditions, but the magnitude of the increase varied, possibly reflecting differences in the ability of cell preparations to undergo decidualization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retinoic acid production by decidual cells isolated from uteri on day 8 of pregnancy
We have previously reported that expression of CRABP(II) and CRBP was observed in decidual cells at the implantation cite on the antimesometrial side of the uterus during normal pregnancy (9). Coexpression of these proteins is not seen in the stromal cells of the cycling uterus (15), and stromal cells in culture do not synthesize retinoic acid (10). To determine whether the appearance of these binding proteins correlated with a gain of ability to synthesize retinoic acid, as we have observed for other cell types (10, 11), decidual cells from the uteri of rats on the eighth day of pregnancy were isolated and placed in culture with and without the addition of estrogen and/or progesterone. After a 1-day recovery period, the cultured cells were provided with 2 µM retinol and 8 µM BSA, and the medium was analyzed for the appearance of retinoic acid after a 12-h incubation. In contrast to our previous observations of cultured nondifferentiated uterine stromal cells (10), retinoic acid production by these cells was observed (Fig. 2Go). No significant difference was observed in the amount of retinoic acid recovered between cells cultured with or without ovarian steroid hormones. This indicated that these hormones were not required to maintain synthesis activity, at least in vitro.



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  		Figure 2. Synthesis of retinoic acid by cultured decidual cells. Decidual cells isolated from the uterus on day 8 of pregnancy were cultured under different hormone conditions in 24-well plates (105 cells/well) and provided with 2 µM all-trans-retinol and 8 µM BSA. After 12 h, retinoic acid was extracted from the medium and measured by HPLC. P4, Progesterone (10-8 M); E2, estradiol (10-8 M).

 
CRBP and CRABP(II) expression in the decidualized uterus of the ovariectomized animal
To follow more easily the onset and progression of binding protein appearance, we next examined whether the model of decidualization induced by an artificial stimulus also produced the same uterine response. Ovariectomized rats were injected with hormones as described (Fig. 1Go), and on day 5, three animals received an intrauterine injection of 100 µl sesame oil to induce decidualization of the stromal cells. The other three unstimulated animals were continued with the same protocol as the stimulated animals. Animals were killed on days 6 and 8, and the uteri were removed and prepared for immunohistochemical analysis of the presence of CRBP and CRBP(II).

On day 6, just 1 day after the intrauterine stimulation, CRBP expression was evident as stromal cells on the antimesometrial side of the uterus began to differentiate into decidual cells. No comparable expression was seen for the unstimulated animal (Fig. 3Go, A and B). Expression of CRBP in the smooth muscle was present in both groups. On day 8, the area of expression of CRBP had expanded as more stromal cells differentiated into decidual cells; again, this was most prominent on the antimesometrial side (arrow in Fig. 3DGo). In contrast, there was no CRBP expression or differentiation of stromal cells into decidual cells in the nonstimulated uteri (Fig. 3CGo). Smooth muscle had faint staining.



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  		Figure 3. Demonstration of the induction of CRBP expression in the decidualized rat uterus by immunohistochemistry. A, Day 6 uterus from hormone-treated ovariectomized rat without intrauterine stimulation. No decidualization occurred, and CRBP expression is seen only in the smooth muscle; B, day 6 uterus from hormone-treated ovariectomized rat with intrauterine stimulation showing strong staining for CRBP in the decidual cells (arrow). Inset, High power view of the antimesometrial side. C, Day 8 uterus from an ovariectomized rat without intrauterine stimulation. CRBP expression remains as before. D, Day 8 uterus from an ovariectomized rat with intrauterine stimulation. CRBP staining is clearly evident in the decidual cells and is greater on the antimesometrial side (arrows).

 
CRABP(II) expression mirrored that seen for CRBP (Fig. 4Go). Staining of CRABP(II) was noted at the beginning of the decidualization of stromal cells on the antimesometrial side of uterus on day 6 (arrow in Fig. 4AGo). As more stromal cells underwent decidualization by day 8, increased expression of CRABP(II), primarily on the antimesometrial side, was noted (arrows in Fig. 4Go, B and C). However, for uteri from the unstimulated animal, no staining for CRABP(II) was noted in these areas (Fig. 4DGo).



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  		Figure 4. Demonstration of the induction of CRABP(II) expression in the decidualized rat uterus. Intrauterine stimulation was performed on day 5. A, On day 6, staining for CRABP(II) begins on the antimesometrial side of the uterus (arrow). B, On day 7, increased staining for CRABP(II) is evident as more stromal cells undergo decidualization. C, Extensive staining for CRABP(II) is evident on day 8 in the stimulated uterus. D, No staining was observed for CRABP(II) in the stroma on day 8 for rats not receiving intrauterine stimulation. Expression in the uterus on day 8 was confirmed by in situ hybridization of CRABP II messenger RNA using a35S-radiolabeled antisense RNA probe (E) and a sense RNA probe (F).

 
As a further check of the induction of CRABP(II), in situ hybridization was performed for the day 8 in vitro decidualized uteri, using a 35S-radiolabeling antisense RNA probe (Fig. 4EGo) and a sense RNA probe (Fig. 4FGo). CRABP(II) messenger RNA was highly expressed in the decidual cells and smooth muscle cells, correlating well with the immunohistochemical results.

These observations demonstrated that the decidualization induced by this procedure was comparable to the decidualization that occurs during normal pregnancy with respect to induced expression of the retinoid-binding proteins and suggested that the procedure could be used to follow acquisition of the ability to synthesize retinoic acid as well.

Retinoic acid production by cells cultured from the uteri of hormone-treated ovariectomized rats
If endometrial stromal cells are isolated from the uteri of the hormone-treated ovariectomized rats on the fifth day, when intrauterine stimulation induces decidualization, the cells will differentiate into decidual cells in culture without the need for further stimulation (17, 18). It has been shown that the ability of stromal cells to undergo decidualization in vivo is dependent on uterine sensitization, which is time and hormone dependent (19). If isolated at an earlier time (e.g. the second day), the cells will not undergo decidualization. This provided a means to follow acquisition of the ability to synthesize retinoic acid during the differentiation process.

One of the characteristic of decidualization is an increase in endometrial ALP activity. Histochemical localization of enzyme activity demonstrates its presence within the stromal tissue and its relative absence in the epithelium and myometrium (20), suggesting that an increase in ALP activity is specific to those stromal cells that have differentiated into decidual cells. Thus, ALP activity appears to be a convenient marker of decidualization (21).

Both stromal and epithelial cells were isolated from the uteri of rats on days 2 and 5 of the hormonal regimen. Retinoic acid synthesis and ALP activity were determined through 3 days of culture. From the uteri of rats killed on day 2, both stromal cells and epithelial cells synthesized small amounts of retinoic acid and displayed low ALP activity over the 3 days, indicating that these stromal cells have little ability to decidualize, as expected (Fig. 5Go).



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  		Figure 5. Demonstration of increased ability to synthesize retinoic acid as cells underwent decidualization in culture. Ovariectomized rats undergoing hormone treatment were killed on day 2 or day 5, and stromal and epithelial cells were isolated for culture. Medium was provided with 2 µM all-trans-retinol and 8 µM BSA on the days indicated and then collected after 12 h for measurement of retinoic acid content. ALP activity was determined as an indication of the degree of decidualization. Little retinoic acid synthesis was noted for cells isolated on day 2, and no increase was noted with continued culture. Cells cultured from uteri on day 5 showed a significantly greater synthesis of retinoic acid that increased with days in culture, paralleled by an increase in ALP activity.

 
However, stromal cells cultured from the uteri of rats killed on day 5 of treatment underwent differentiation into decidual cells, as indicated by the increase in ALP activity over the 3 days of culture. In concert with this increase there was a substantial increase in retinoic acid production (Fig. 5Go). On the first day of culture, the cells synthesized significantly higher levels of retinoic acid (P < 0.001) than did the stromal cells isolated on day 2. After 2 or 3 days in culture, a significant increase in retinoic acid production by the decidualizing stromal cells was observed compared with that by cells cultured for 1 day (P < 0.001). Epithelial cells were also examined and were found to synthesize substantially less retinoic acid than did the decidualizing cells (data not shown). Because there was an increase over the 3 days of culture, we cannot eliminate the possibility that this synthesis actually was by stromal cell contamination.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The successful establishment of pregnancy requires synchronized growth and differentiation of the preimplantation embryo and the uterus. These events are coordinated by the ovarian steroids, estrogen and progesterone. Estrogen, acting on a background of progesterone, is required for initiating uterine receptivity to an implanting embryo or artificial stimulus (6, 22). Here, we report that decidual cells isolated from the uterus on day 8 of pregnancy synthesized retinoic acid when provided with retinol. This is a gain of function, as uterine stromal cells are unable to synthesize retinoic acid (14). This indicates that retinoic acid signaling is part of the complex hormonal interplay necessary for successful implantation of the embryo. The exact role(s) of retinoic acid in this process may well be difficult to identify because of the current lack of methods to either inhibit retinoic acid synthesis or antagonize its action. It has been demonstrated that administered retinoic acid decreases both progesterone and estrogen receptor-mediated transcriptional activation and also can inhibit estrogen-induced uterine stromal and myometrial cell proliferation in vivo (23, 24). Thus, the finding here that decidual cells synthesize retinoic acid supports the idea that regulation of pregnancy and implantation involves interplay between the actions of steroid hormones and those of retinoic acid.

The ability to induce decidualization artificially, both in vivo and in vitro, allowed the demonstration that the expression of CRBP and CRABP(II) and the acquisition of retinoic acid-synthesizing ability were independent of any embryo-derived signal. Further, the amount of retinoic acid synthesis observed correlated well with the degree of decidualization, as measured by increase in ALP activity. It is of interest that artificial induction of decidualization faithfully reproduces an important part of the normal physiological process, a part that was previously unknown. In addition, the specific immunostaining for the binding proteins was stronger in the decidual cells on the antimesometrial side of the uterus, mirroring our previous observations of the normal pregnant rat uterus (9) and observations by others (25). This provides additional evidence of the value of artificial decidualization as a research tool.

Although decidual cells have both retinoic acid receptors and retinoid X receptors (25), the site of action of this locally generated retinoic acid may well be the neighboring stromal cells, rather than the decidual cells themselves. We have demonstrated that CRABP(II) is restricted to the cytoplasmic compartment (i.e. nuclear excluded), which may well prevent the retinoic acid from reaching the nuclear receptors of the synthesizing cells (26). It has been shown that retinoic acid can suppress in vitro decidualization of human endometrial stromal cells (27). This suggests that retinoic acid may be acting in a paracrine manner to inhibit differentiation of neighboring stromal cells. However, this idea does not preclude the possibility that retinoic acid is also functioning as a paracrine factor to mediate decidual-trophoblast interactions.

In summary, we have shown that the expression of CRBP and CRABP(II) and the synthesis of retinoic acid are linked with decidualization, implicating retinoic acid as an important signal during implantation of the embryo. In addition, this is the third example of an association of CRABP(II) expression with the ability to synthesize retinoic acid (10, 11). Thus, the appearance of CRABP(II) appears to mark the beginning of production of retinoic acid in some systems, perhaps those in which synthesis of retinoic acid only occurs at particular times, rather than constitutively.


   Acknowledgments
 
We thank Prof. Thomas Kennedy (Department of Physiology and Obstetrics and Gynecology, University of Western Ontario, London, Ontario, Canada) for graciously providing the tissue for analysis of binding protein expression during in vivo decidualization.


   Footnotes
 
1 This work was supported by NIH Grants DK-32642, HD-25206 (to D.E.O.), and HD-28128 (to K.G.O.) and the immunohistochemistry core facilities of the Clinical Nutrition Research Unit (HD-26657). Back

Received June 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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