BIOLOGY OF REPRODUCTION 58, 963-970 (1998)
Spatial and Temporal Patterns of Expression of Cellular Retinol-Binding Protein and Cellular Retinoic Acid-Binding Proteins in Rat Uterus during Early Pregnancy' Wen Li Zheng and David E. Ong 2 Department of Biochemistry, Vanderbilt School of Medicine, Nashville, Tennessee 37232 ABSTRACT Retinoic acid, perhaps the most potent hormonal form of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of normal pregnancy and embryo development. However, little is known about the specific sites of action and metabolism in the uterus during pregnancy. In this study we describe the pattern of temporal and cell-specific expression of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding proteins type I and type II (CRABP and CRABP[II], respectively) in the rat uterus during the periimplantation period (Day 1 to Day 7 of pregnancy; Day 1 = presence of vaginal plug). Immunohistochemical studies showed that there were dramatic and rapid changes in expression pattern of the retinoid-binding proteins after mating as early as Day 1, as well as a differential expression of these proteins when the mesometrial side and antimesometrial side of the uterus were examined during the periimplantation period. CRABP(II), whose presence has been previously shown to correlate with retinoic acid synthesis in the uterine epithelium, was specifically localized to the luminal epithelium at Day 1, being stronger on the mesometrial side, and then fell to lower levels. CRBP was also expressed in the luminal epithelium on the mesometrial side at Day 1 as well as in some stromal cells, declining at these sites over the next several days. CRABP was localized to some of the stromal cells at Day 1, overlapping CRBP expression. Embryonic implantation was accompanied by the appearance of CRBP and CRABP(II) in the decidual cells. CRBP and CRABP were also present in both smooth muscle layers of the uterus. The changes in the temporal and cell-specific distribution of retinoid-binding proteins imply a multifunctional role of vitamin A in uterine cell proliferation, differentiation, and embryonic implantation. The presence of CRABP(II) suggests that local generation of retinoic acid is important in these processes. INTRODUCTION The importance of vitamin A (retinol) and its active metabolite retinoic acid in the process of reproduction has long been recognized. Long-term vitamin A deficiency produces a complete loss of spermatogenesis and atrophy of the seminiferous tubules in male rats. In females, deficiency produces irregular estrous cycles, morphological changes in the uterine epithelium, and reproductive failure, with pregnancies typically ending in fetal death and resorption [1]. Retinoic acid can restore normal uterine epithelial cell differentiation and maintain fertility in retinol-deficient animals [2]. However, retinol itself is specifically required for successful parturition [1, 3]. The production and the biological effects of retinoic acid on the expression of responsive genes are mediated via two types of proteins: cytoplasmic binding proteins involved in
retinoid transport and/or metabolism, and nuclear receptors that act as ligand-dependent transcriptional regulators. There are two groups of cytoplasmic binding proteins: cellular retinol-binding protein type I and type II (CRBP and CRBP[II]), and cellular retinoic acid-binding protein type I and type II (CRABP and CRABP[II]). CRBP(II) directs the reduction of retinal to retinol and the subsequent esterification of retinol in the intestine [4], while CRBP directs the esterification/deesterification of retinol and the subsequent oxidation of that retinol to retinal and then retinoic acid [5-7]. CRABP is involved in the metabolism of retinoic acid to more polar metabolites and in limiting the access of retinoic acid to the nucleus, blocking cells from the retinoic acid signal [6, 8-11]. The expression of CRABP(II), however, has been found recently to be associated with cells that synthesize relatively large amounts of retinoic acid, perhaps indicating a retinoic acid paracrine system [12]. Two distinct families of nuclear receptors, known as the RAR and RXR families, have been found to mediate the effects of retinoic acid. They are ligand-dependent transcriptional regulators and modulate the transcriptional activity of specific target genes by binding specific DNA sequences contained in the regulatory regions of these genes [13]. We have previously described the variable expression and cell-specific location of cellular retinoid-binding proteins in the uterus during the normal estrous cycle of the rat [14]. We have also observed that estrogen provided to immature female rats leads to a marked increase in the expression of both CRABP and CRABP(II) in the uterus [15]. This suggests that interactions between vitamin A and steroid hormones are involved in the female reproductive system. There is other evidence that supports this suggestion: in retinol-deficient rats, serum and ovarian progesterone concentrations are decreased [16, 17]; in vitro, both retinol and retinoic acid can increase progesterone production by rat luteal cells [18]; retinoic acid decreases progesterone and estrogen receptor-mediated transcription [19]; and retinoic acid can inhibit estrogen-induced uterine stromal and myometrial cell proliferation in vivo [20]. It is well known that there are changing steroid hormone levels during pregnancy. Here we have investigated the expression patterns and distribution of cellular retinoid-binding proteins during the periimplantation period and found rapid and specific changes suggesting that retinoic acid is being produced at several sites at different times in the uterus during early pregnancy. MATERIALS AND METHODS Animals Female Sprague-Dawley rats (225-250 g on delivery) were housed in a temperature- and light-controlled room (21 + 1C, 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 wk before use
Accepted December 2, 1997. Received September 5, 1997. 'This work was supported by NIH grant HD-25206. 2 Correspondence. FAX: (615) 343-0704; e-mail:
[email protected]
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in these experiments. Female rats were exposed to fertile males overnight. Day 1 of pregnancy was determined by the presence of vaginal sperm on the following morning. Rats (3-4) were killed between 0900 and 1000 h on the specific days of pregnancy. Uteri were removed and frozen or immersion fixed for later processing. When possible, pregnancy was also confirmed by flushing embryos from the oviduct or uterus after killing the animal. 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. Preparation of Cytosolic Fractions Frozen uteri were weighed and immediately homogenized at 18 000 rpm for two 30-sec bursts, in a 4:1 (v:w of tissue) ratio of 10 mM Tris, 1 mM EDTA solution (pH 7.4). The homogenates were first centrifuged at 20 000 x g for 30 min at 4C in a J2-21 Beckman (Palo Alto, CA) centrifuge. The supernatant liquid was then centrifuged at 100 000 x g for 60 min at 4C in a Beckman ultracentrifuge. Protein quantification of the resultant cytosols was performed using the BCA protein analysis kit (Pierce, Rockford, IL). The cytosolic fractions were aliquoted and frozen at -70 0C. Preparation of Immune Reagents and Western Analysis Antibodies to CRABP(II) were those generated as described previously [15]. The preparation of the immune serum for rat CRABP has also been described [21]. For detection of CRBP, we used immune serum that had been raised against human CRBP [22]. The total IgG fractions of these antisera were isolated by use of protein A columns (Pierce), followed by passage over affinity columns of either recombinant rat CRBP or CRABP bound to Sepharose 4B to isolate the binding protein-specific IgG populations. The specificity of these antibodies for the binding proteins against which they were generated has been reported elsewhere [14, 23]. The specificity of antibody against CRBP was analyzed by Western blot and dot blot. Secondary antibody incubation and subsequent visualization steps were performed according to protocols in the Amersham enhanced chemiluminescence (ECL) kit (Amersham, Arlington Heights, IL). There was no apparent cross-reaction with CRABP or CRABP(II). A slight reaction was seen with CRBP(II), visible after a long exposure of the blots. However, CRBP(II) is not present in the uterus. For both Western blots and immunolocalization studies, the specific IgG populations were used at a dilution of 1:150 for CRBP (initial A280 = 0.35), 1:100 for CRABP (initial A280 = 0.30), and 1:1000 for CRABP(II) (initial A280 = 0.36). Western blots were prepared by separating 50-}pg aliquots of uterine cytosols on 12% SDS-polyacrylamide gels using a mini-gel system (Hoefer, San Francisco, CA). Proteins were transferred to Immobilon-P (Millipore Corp., Bedford, MA) paper for 1 h at 1.0 amp in 25 mM Tris, 192 mM glycine, and 20% methanol. Blots were blocked for 30 min in 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.05% Tween 20, and 5% (w:v) Carnation (Los Angeles, CA) instant milk. Primary antibody (using the same dilution as in immunohistochemistry) incubation was performed overnight at 4°C in blocking solution. Blots were washed three times in wash solution (blocking solution without milk). Secondary antibody incubation and subsequent visualiza-
FIG. 1. Western blot analysis of CRBP (A), CRABP (B), and CRABP(II) (C) in the rat uterus during the periimplantation period. Lanes 1-7, proteins from Days 1 to 7 of pregnancy, respectively.
tion steps were again performed according to protocols in the Amersham ECL kit (Amersham). Immunohistochemical Localization of CRBP, CRABP, and CRABP(II) Tissue samples for immunolocalization were immersion fixed for approximately 24 h in 20% isopropyl alcohol, 4.0% (w:v) paraformaldehyde, 2.0% (w:v) trichloroacetic acid, and 2.0% (w:v) zinc chloride and then were transferred to 70% EtOH. Paraffin embedding and slide sectioning were carried out by Vanderbilt histopathology. After removal of the paraffin with xylene and equilibration of the tissue sections in Tris-buffered saline (TBS, pH 7.6), the slides were blocked for 30 min in TBS containing 3% (w: v) crystalline BSA. This blocking agent was also used to
FIG. 2. Immunohistochemical localization of CRBP in the rat uterus during the periimplantation period. The affinity-purified IgG fraction that recognizes CRBP (see Fig. 1) was used for analysis of the expression of CRBP in the uterus. The brown color indicates the presence of immunoreactivity. All sections were lightly counterstained with hematoxylin to reveal cellular detail. A) Longitudinal section of the uterus at Day 1 of pregnancy. The antimesometrial side is to the left. Inset) Higher-power view of the mesometrial side. Note the strong staining of the mesometrial epithelial cells (filled arrowheads), staining of smooth muscle on both sides (open arrows), and lighter staining of some stromal cells on the mesometrial side (open arrowhead). B) Section of the uterus at Day 3. Note lack of staining of the epithelium and darker staining of the smooth muscle cells on the mesometrial side (empty arrows). C) Uterus at Day 4. Note the increased staining of the luminal epithelium (filled arrowhead) compared to Day 3. D) Uterus at Day 6 showing strong staining of decidual cells (filled arrow, antimesometrial side). The embryo is visible in the central lumen. E)Section of the uterus at Day 7 showing staining of primary (filled arrow) and secondary (double star) decidual cells on the antimesometrial side. F) Similar section at Day 7 of the mesometrial side showing de-
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creased staining in the smooth muscle cell layers (empty arrow; compare to the smooth muscle in E) and decidual cells (double star). G) Cross section of uterus at diestrus in the normally cycling rat. Note the staining of stromal cells underlying the epithelium (open arrowhead). A very few epithelial cells also may be stained. H) Longitudinal section of the uterus at estrus showing that most of the epithelial cells are now positive (filled arrowhead). All bars = 100 tlm, except inset of A, where bar = 10 Lm.
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FIG. 3. Immunohistochemical localization of CRABP in the rat uterus during the periimplantation period. The affinity-purified IgG fraction that recognizes CRABP (see Fig. 1) was used for analysis of the expression of CRABP. All sections were lightly counterstained as above. A) Longitudinal section of the uterus at Day 1 of pregnancy. The antimesometrial side is to the left. Inset) Higher-power view of the mesometrial side. Note the strong staining on the mesometrial side of stromal cells underlying the epithelium (open arrowheads) and the lack of staining of the epithelium (filled arrowhead). The smooth muscle cell layers also stained for CRABP (empty arrows). B) Section of the uterus at Day 3. Note darker staining of the smooth muscle cells on the mesometrial side (empty arrow). C) Uterus at Day 4. Note darker staining of the smooth muscle on the mesometrial side (empty arrow). D) Uterus at Day 6. Note darker staining of the smooth muscle on the mesometrial side (empty arrow) and no staining in decidual cells (filled arrow). E)Section of uterus at Day 7 showing lack of staining of primary (filled arrow) and secondary (double star) decidual cells on the antimesometrial side. F) Longitudinal section of the uterus at estrus, showing that stromal cells underlying the epithelium are positive (empty arrowhead). All bars = 100 Ipm, except inset of A, where bar = 10 ILm.
RETINOID-BINDING PROTEINS IN THE PREGNANT UTERUS dilute the primary antibodies. Primary antibody incubations were carried out in a humidified chamber at 40C overnight. After rinsing in TBS, the slides were incubated with goat anti-rabbit IgG antibody conjugated to biotin (Jackson ImmunoResearch Labs, West Grove, PA) for 1 h. The slides were rinsed and incubated with an antibiotin antibody linked to alkaline phosphatase (Jackson ImmunoResearch Labs) for another 1 h. Staining was produced by incubation for 20-30 min with an alkaline phosphatase substrate (Dimension Labs, Mississauga, ON, Canada). Tissues were counterstained with hematoxylin and preserved with aqueous mountant (Serotec, Washington, DC). IgG fractions that were not retained by the recombinant CRBP, CRABP, and CRABP(II) affinity columns were used as the negative controls for the immunolocalizations. Antibody preparations preadsorbed with recombinant protein for 3 h at room temperature in PBS (with slow end-over-end mixing) also exhibited no staining on positive control slides. RESULTS Cellular Retinoid-Binding Protein Expression Levels in the Uterus of the Pregnant Rat To determine the relative levels of expression of each of the proteins during early pregnancy, cytosols were prepared from frozen uteri (3-4 uterine horns were pooled) collected at various times during the periimplantation period. Equal amounts of cytosolic protein from each sample (50 g) were separated on SDS-PAGE gels, transferred to a polyvinyl difluoride membrane, and probed with antibodies previously shown to be specific for the binding proteins against which they were generated. The expression of CRBP increased after Day 1, reaching its highest level at Day 3-4, and then decreased to levels seen at Day 1 (Fig. A). CRABP levels decreased to a lower level on Day 2 and Day 3, then increased to higher levels than seen at Day 1 (Fig. 1B). The change in expression of CRABP(II) was the most dramatic. It was expressed at its highest level on Day 1 but decreased dramatically by Day 2. A small rebound was apparent on Day 4 and Day 5 (Fig. 1C). These changes in expression level were not quantified because it became clear from the subsequent work that the changes were due to a combination of factors such as disappearance at one site coupled with appearance at a new site, or a change for the same cell type that was region dependent. Thus any change in total uterine content of the binding proteins simply reflected the dynamic nature of retinoid processing during early pregnancy, and no specific conclusions could be drawn from the degree of change. The sites and changes of expression indicated by the Western analysis were examined by immunohistochemical localization of the binding proteins for several samples (3-4) of half uteri representing each day of the periimplantation pregnancy. Cell-Specific Localization of CRBP Incubation of uterine sections with anti-CRBP resulted in staining of both the inner and outer muscle layers throughout the periimplantation period (Fig. 2). The intensity was uniform for both layers of smooth muscle at Day 1. However, after Day 1, the staining intensity of the smooth muscle became region dependent. In some areas, the staining for CRBP was stronger on the mesometrial side than on the antimesometrial side (Fig. 2B). At Day 1, in addition to the staining of smooth muscle cells, strong
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staining was noted in many of the cells of the luminal epithelium and also some stromal cells, localized to the mesometrial side of the uterus (Fig. 2A). Scattered and fewer cells of the luminal epithelial and in the stroma were stained on the antimesometrial side (Fig. 2A). There was no apparent staining of the glandular epithelial cells (data not shown). The epithelial staining disappeared after Day 1; then weaker staining of the luminal epithelium was noted on Day 4 and Day 5 (Fig. 2C and data not shown). Human endometrium has also been found to express CRBP in both stroma and epithelial cells [24]. After blastocyst attachment, from Day 6 onward, distinct immunoreaction for CRBP was observed in the primary decidual cells around the implantation chamber. On Day 7, the smooth muscle cell layers and the secondary decidual cells at the antimesometrial side, the side of implantation, expressed higher levels of CRBP than the other side (Fig. 2, D-F). Because we had not seen epithelial and stromal cell staining in our previous study of the normal estrous cycle [14], we reexamined this question with the better antibody preparation used in this study. With the more avid antibody preparation we now detected CRBP staining in the luminal epithelial cells at estrus (coincident with the expression of CRABP[II]) and detected stromal cell staining during diestrus. Only faint or no staining was noted for CRBP at these sites during proestrus and metestrus (Fig. 2, G and H, and data not shown). The intensity of CRBP staining in the normally cycling animal was less than that seen in the luminal epithelium at Day 1 of pregnancy, which perhaps contributed to the failure to detect staining with the weaker antibody preparation (data not shown). Cell-Specific Localization of CRABP CRABP expression was primarily in the layers of smooth muscle. In some areas of the uterus, the intensity of the staining was stronger on the mesometrial side than on the antimesometrial side (Fig. 3 and data not shown). This staining pattern was noted for all days examined. The intensity of staining decreased after Day 1 (Fig. 3, A-C). On Day 1, in addition to the muscle staining, some of the stromal cells on the mesometrial side were positive for CRABP, including the stromal cells that had been positive for CRBP (Fig. 3A and data not shown). No specific staining was detected in the stromal cells on the antimesometrial side (Fig. 3A). The stromal staining for CRABP was very region dependent. Stronger staining for CRABP was seen for stromal cells on the mesometrial side near the oviduct than was seen in the middle uterus or near the cervical end (data not shown). No positive staining was seen in the decidual cells on Day 6 and Day 7 (Fig. 3, D and E); but on Day 7, some stromal cells in the implantation chamber on the mesometrial side showed weak immunostaining for CRABP (data not shown). We previously had not noted CRABP expression in the stromal cells of normal cycling rats, but in those studies the uteri were examined only in cross section [14]. In order to determine whether the temporal and region-dependent expression of CRABP in the stromal cells was unique to the pregnant uterus, we prepared new sections of the normal cycling uterus, now cut longitudinally. We observed that some of the stromal cells on the mesometrial side stained positively for CRABP, but only during the estrous stage (Fig. 3F). Further, cells that were positive for CRABP were considerably fewer in number than for the Day 1 preg-
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FIG. 4. Immunohistochemical localization of CRABP(II) in the rat uterus during the periimplantation period. The affinity-purified IgG fraction that recognizes CRABP(II) (see Fig. 1) was used for analysis of the expression of CRABP(II) in the uterus. A) Longitudinal section showing the antimesometrial side of the uterus at Day 1. Light staining of the epithelium can be seen (filled arrowhead). Inset) Higher-power view of the antimesometrial side.
RETINOID-BINDING PROTEINS IN THE PREGNANT UTERUS nant uterus. The staining was found to have the same regional dependence described above. The cross sections stained for the estrous state in our previous study came from the middle region. Cell-Specific Localization of CRABP(II) In the normal estrous cycle, CRABP(II) is found to be highly expressed in the luminal epithelial cells, but only during estrus; only faint staining was noted the day after estrus [14]. Here strong staining for CRABP(II) was observed for the luminal epithelium at Day 1 of pregnancy, 1 day after estrus. The intensity of CRABP(II) staining was also noted to be stronger on the mesometrial side than on the antimesometrial side. This was observed only on Day 1 (Fig. 4, A and B). Visible, but much less significant, staining was detected after Day 1. On Day 4 and Day 5, there was an increased staining for CRABP(II) in the luminal epithelium; also a visible, but weaker, staining appeared in the glandular epithelium (Fig. 4, C-E). These results correlated well with the estimation of CRABP(II) expression by Western blot (Fig. 1) and also matched the pattern seen for CRBP at these sites. Immunoreaction for CRABP(II) was then observed in the primary decidual cells around the implantation chamber on Day 6 and Day 7 (Fig. 4, F-H). On Day 7, some staining was also noted in the secondary decidual cells on the antimesometrial side, but much less than seen on the other side (Fig. 4, G and H). DISCUSSION The observations we have reported here indicate that the local generation of retinoic acid is an important signal in the preparation of the uterus for implantation of the embryo. Although we did not determine retinoic acid production directly, we believe that we can infer, from the expression of CRABP(II) in certain cells, that those cells are producing retinoic acid as a paracrine, and perhaps, autocrine hormone. This inference is based on our previous work with the pseudopregnant rat model. We established that uterine epithelial cells synthesize retinoic acid in culture only if isolated at the time of CRABP(II) expression. That such retinoic acid synthesis also occurs in the intact animal was verified by the demonstration that high levels of retinoic acid could be extracted from isolated uteri only at the time when CRABP(II) was expressed [12]. Thus, we assume that the same correlation between CRABP(II) expression and retinoic acid synthesis exists for the uterus of the pregnant animal. In the normally cycling animal, CRABP(II) is present in
B) Longitudinal section of the mesometrial side of the uterus at Day 1. Note the stronger staining in the epithelial cells at this side (filled arrowhead), and no staining in the glandular epithelium (empty arrow). Inset) Higher-power view of the mesometrial side. C) Uterus at Day 2. Note the decreased staining of the luminal epithelium compared to Day 1. D) Uterus at Day 4 showing increased staining of epithelium (filled arrowhead) and light staining of glandular epithelium (empty arrow). Inset) Higherpower view of the glandular epithelium. E) Section of uterus at Day 5. Note the strong epithelial cell staining. F) Section at Day 6 of the antimesometrial side showing that decidual cells (filled arrow) are positive. The embryo is visible in the central lumen. G) Section of uterus at Day 7, showing staining of primary (filled arrow) and secondary (double star) decidual cells on the antimesometrial side. H) Similar section of the uterus at Day 7 of the mesometrial side showing faint or no staining in the secondary decidual cells. All bars = 100 gi, except insets of A and B, where bars = 10 lm.
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the uterine epithelium primarily at the estrous stage, declining quickly when estrus ends [14]. It was interesting to see that as rapidly as 1 day after mating, retinoid processing in the uterus had already responded to the new physiological state. Instead of the decline that would have been observable by this time in an unmated animal, the level of CRABP(II) had actually increased. This increase was seen only on the mesometrial side, an asymmetry never observed for the uterus at estrus. Coincident with this was the coexpression of CRBP, the carrier of the retinol that is the precursor of retinoic acid synthesis. This suggests that the retinoic acid signal is part of the mechanism by which the uterus prepares for the coming implantation. On Day 6-7 during the postimplantation period, CRABP(II) was expressed in the decidual cells in the implantation chamber. CRBP was also found in the same area. Preliminary data in this laboratory have shown that both human and rat uterine stromal cells that are induced to decidualize in vitro gain the ability to produce retinoic acid from retinol (unpublished results). Recently it was reported that decidual cells have both RARs and RXRs [25]. This suggests that retinoic acid, locally generated from retinol, could be involved in the differentiation of stromal cells to decidual cells and/or that it may function in the process of implantation and/or early embryo development. Previous work has demonstrated that retinol-deficient pregnant rats frequently resorbed embryos and that this was accompanied by obvious placental lesions [1]. It is believed that CRABP is involved in control of the level of retinoic acid available to its nuclear receptors, via sequestration and increased catabolism [8-10]. The expression of CRABP in the smooth muscle cells and temporal expression in the stromal cells on Day may then be explained by CRABP's serving a role as a protector of these cells at that time from the effects of retinoic acid produced by the epithelium. The presence of CRBP in those cells also expressing CRABP(II) is consistent with its role in the production of retinoic acid [6]. CRBP was also present in the smooth muscle cell layers that expressed CRABP There its role is less clear. Smooth muscle cells from the uterus of the pseudopregnant rat were found not to synthesize retinoic acid [12]. It is generally accepted that CRBP also mediates retinol esterification as well as production of retinoic acid. We have observed that the smooth muscle of the small intestine possesses large stores of retinol esters and that it contains both CRBP and the enzyme activity responsible for the esterification of retinol, lecithin-retinol acyltransferase, suggesting that the muscle may serve as a local store of vitamin A [7, 26]. We have recovered significant retinol ester in extracts of total uterus (unpublished results). The muscle cell may serve a similar role as a site of storage of retinol in the uterus, since the uterus appears to carry out extensive retinoid processing. The mechanism for mobilization of the esters to produce retinol or active retinoids has not been elaborated, although the smooth muscle of the intestine does contain a potent retinol ester hydrolase activity [26]. Implantation occurs on the antimesometrial side of the uterus during early pregnancy. The decidualization of stroma cells begins on the antimesometrial side and later spreads to the mesometrial side. The greater expression of CRBP, CRABP, and CRABP II on the mesometrial side at Day 1 suggests that the retinoic acid signal is involved in establishing the functional differences of the uterus that lead to successful implantation. Later, with decidualization, the binding proteins appeared first on the antimesometrial
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side, suggesting that the actual step of implantation also involves the retinoic acid signal. In summary, we suggest that the spatial and temporal cell-specific expression of the retinoid-binding proteins during the periimplantation period relates to their roles in controlling retinoic acid production and its availability to the nuclear receptors in certain cells. However, the factors that produce this pattern are not known. In the prepubertal rat uterus [15] and adult rat cervix [27], expression of the CRABPs and CRBP was found to be dynamically regulated by the steroid hormone estrogen. It has been reported that retinoic acid decreases both progesterone and estrogen receptor-mediated transcriptional activation [19] and that retinoic acid can inhibit estrogen-induced uterine stroma and myometrial cell proliferation in vivo [20]. Moreover, retinoic acid can either induce or repress expression of several members of the transforming growth factor 3 superfamily in various cell lines and alter the expression of fibroblast growth factor family members [28-31]. These polypeptide hormones are expressed in the uterus [32, 33], and consequently the varying synthesis of retinoic acid suggested by this study may indicate that such interplay occurs during pregnancy. Clearly there are many factors and hormones participating in the maintenance of pregnancy. The expression pattern of retinoid-binding proteins defined here indicates that retinoic acid is one of those factors. REFERENCES 1. Thompson JN, Howell JM, Pitt GAJ. Vitamin A and reproduction in rats. Proc R Soc Lond 1964; B159:510-535. 2. Bo WJ, Smith MS. The effect of retinol and retinoic acid on the morphology of the rat uterus. Anat Rec 1966; 156:5-9. 3. Wellik DM, DeLuca HE Retinol in addition to retinoic acid is required for successful gestation in vitamin A-deficient rats. Biol Reprod 1995; 53:1392-1397. 4. Ong DE. Retinoid metabolism during intestinal absorption. J Nutr 1993; 123(suppl 2):351-355. 5. Napoli JL, Posch KP, Fiorella PD, Boerman MH. Physiological occurrence, biosynthesis and metabolism of retinoic acid: evidence for roles of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) in the pathway of retinoic acid homeostasis. Biomed & Pharmacother 1991; 45:131-143. 6. Napoli JL. Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis. J Nutr 1993; 123(suppl 1):362-366. 7. Ong DE. Cellular transport and metabolism of vitamin A: roles of the cellular retinoid-binding proteins. Nutr Rev 1994; 52:S24-31. 8. Boylan JE Gudas LJ. Overexpression of the cellular retinoic acid binding protein-I (CRABP-I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells. J Cell Biol 1991; 112:965-979. 9. Boylan JE Gudas LJ. The level of CRABP-I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells. J Biol Chem 1992; 267:21486-21491. 10. Maden M, Ong DE, Summerbell D, Chytil E Spatial distribution of cellular protein binding to retinoic acid in the chick limb bud. Nature 1988; 335:733-735. 11. Zheng WL, Bucco RA, Schmitt MC, Wardlaw SA, Ong DE. Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis. Endocrinology 1996; 137:50285035. 12. Bucco RA, Zheng WL, Davis JT, Sierra-Rievra E, Osteen KG, Chaudhary AK, Ong DE. Cellular retinoic acid-binding protein(II) presence in rat uterus epithelial cells correlates with their synthesis of retinoic acid. Biochemistry 1997; 36:4009-4014.
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