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b Department of Medical Biochemistry and Microbiology, Biomedical Centre, SE-751 23 Uppsala, Sweden
c Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala Biomedical Centre, Uppsala University, S-751 24 Uppsala, Sweden
d Anatomy, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
ABSTRACT
Retinol and retinoic acid that are potent modulators of gene expression are vital for development and growth of the conceptus. Apart from being transported across the placenta, retinol and retinoic acid may also be active in the placenta per se. Three proteins involved in 1) serum transport of retinol (retinol binding protein [RBP]), 2) cellular transport and metabolism of retinol (cellular RBP [CRBP] I), and 3) retinoic acid (cellular retinoic acid binding protein [CRABP] I), respectively, have been located by immunohistochemistry during gestation in the porcine placenta. This is a diffuse epitheliochorial placenta composed of areolar-gland subunits, where transport of larger molecules takes place, and interareolar regions, where gas-exchange and trophoblast absorption of hemotroph occur. Immunoreactive-RBP (ir-RBP) as well as CRBP I (ir-CRBP) was detected in uterine glands and in areolar trophoblasts, suggesting that RBP-retinol is secreted by the glands and absorbed by the trophoblasts. Both proteins were present also at the interareolar regions, with ir-CRBP in both the uterine epithelium and the apposing trophoblasts, but ir-RBP only in the former. The localization of ir-CRABP was, in contrast, strictly limited to interareolar trophoblasts. Together these findings suggest that 1) the areolar gland subunits are important for transport of retinol and retinol-RBP, and 2) retinoid binding proteins are involved in the development and growth of the porcine placenta.
female reproductive tract, placenta, placental transport, uterus
INTRODUCTION
An intriguing fact of placentation is the extraordinary variability of placental structures that can be observed throughout the vertebrate species [1–3], and it is thus no surprise that also the manner by which similar placental functions are executed may vary between species [4]. One important role for all placentas is in placental transfer and possibly also metabolism of vitamin A (retinol). Retinol, and its active metabolite retinoic acid, are unstable hydrophobic compounds that are indispensable for cellular differentiation and growth in general [5] and for placental and embryonic and fetal development in particular [6–8]. The transport and metabolism of retinol and retinoic acid in the body, adult as well as embryo, is tightly regulated by the retinoid binding proteins [5, 9]. These include the 21-kDa plasma retinol binding protein (RBP), which is the retinol transport vehicle in serum [10], the cellular RBPs (CRBP I and II), and cellular retinoic acid binding proteins (CRABP I and II) that are approximately 16-kDa proteins capable of binding retinol and retinoic acid, respectively, with high affinity [11–13]. The CRBPs are considered to be involved in cellular transport of retinol and its metabolism into retinyl esters for storage or into retinoic acid (only CRBP I). The CRABPs have been proposed to be involved in many events in the retinoic acid signaling pathway, including regulation of the availability of retinoic acid to the nuclear receptors [14] and modulation of retinoic acid metabolism [12]. Finally, there are nuclear receptors for retinoic acid (RARs and RXRs) that confer the transcriptional activity of retinoic acid [15, 16].
The present study is part of a long-term study of the localization of RBP, CRBP I, and CRABP I in various types of placentas. One of the aims is to see whether these patterns are homologous, i.e., are the same retinoid binding proteins expressed in cells proposed to have similar functions in different types of placentas. Another is to see whether the number of cell layers present between maternal and fetal circulations is reflected in the presence of retinoid binding proteins, i.e., is the expression of retinoid binding proteins more extensive in placentas with many layers than in placentas with few layers of the interhemal barrier. Previous studies of the hemochorial placentae of mice and man have pointed out differences between these species, emphasizing the importance of the yolk sac placenta in retinol transport in mice [8, 17–21]. One characteristic feature of the retinoid binding proteins is the high degree of sequence homology found between species [9]. Previously, antibodies against human RBP have been used successfully in pigs [22, 23], antibodies against rat CRBP I in mice [19] and humans [20], and antibodies against bovine CRABP I [24, 25] in both mice [14, 26] and humans [27]. The object of the present study, the porcine placenta, is a diffuse folded epitheliochorial placenta with highly specific regions along the materno-fetal interface, the so-called interareolar and areolar-gland regions. These are quite disparate both regarding their structure and function (see Fig. 1). Previously, the relative amounts of RBP transcripts as well as the location of immunoreactive RBP (ir-RBP) have been studied in homogenates and sections, respectively, of the porcine placenta [22, 23, 28–32], but in none of these investigations were areolar and interareolar regions specifically studied and compared with respect to expression patterns. In the present study, ir-RBP, immunoreactive CRBP I (ir-CRBP), and CRABP I (ir-CRABP) have been specifically located by immunohistochemistry, at the materno-fetal interface in regular areolar and interareolar regions in porcine placentae of three developmental stages: early pregnancy (Days 16–35), midpregnancy (Days 36–69), and late pregnancy (Days 70–97).
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MATERIALS AND METHODS
Animals and Collections of Tissues
Material from animals of gestational ages 16 to 35 were collected at a research farm, ages 36 and older were collected at a slaughterhouse. The total of 23 animals was pooled into three groups of different gestational stages, where the early stages of gestation (up to Day 35) were determined from the time of natural insemination. The later stages were estimated from the fetal C/R length [33]. All animals were free of apparent maternal and placental disease. From each placenta, tissue was sampled from the mesometrial side close to the developing embryo or fetus.
The first group (13 gilts) consisted of embryos of 16–35 days of gestation (dg), which represents the period of embryogenesis and initial placental growth and development into the characteristic architecture [34, 35]. The placental regular areola can be observed histologically already on the 15 dg [36]. Two individual implantation sites, 16 dg and 17 dg (two gilts at each stage), and one each from 19, 20, 21, 23, 25, 26, 28, 32, and 35 dg were perfusion-fixed or in situ-fixed [34, 35] with 4% buffered formalin.
In the second group, 36–69 dg, one implantation site from each of six different sows (embryos aged 39, 45, 53, 55, 60, and 67 dg) was studied. These ages represent a period of placental maturation [36, 37] and moderate fetal growth.
The third group, 70–97 dg, consisted of one placenta each from four sows (embryos aged 70, 78, 84, and 97 dg). During this period, the fetus grows rapidly but there is little or no placental growth [37]. Implantation sites from these two latter groups were perfusion- or immersion-fixed with 4% buffered formalin.
Embedding and Sectioning
All the collected tissues were postfixed by immersion into fresh fixative for an additional 24 h at 4°C. The tissues were then dehydrated through a graded series of ethanol and xylene, embedded in paraffin (melting point, 58°C), sectioned at 5–7 µm, and mounted on Superfrost Plus slides (Mentzel-Gläser, Hounisen Risskov, Denmark). Sections were chosen so as to contain regular areolar regions as well as interareolar regions.
Immunocytochemical Localization of RBP, CRBP I, and CRABP I
Antibodies In the present study, the following antibodies were used: two types of polyclonal rabbit anti-human RBP (from [38], and from Dako, Glostrup, Denmark), rabbit anti-rat CRBP I [24, 25], and rabbit anti-bovine CRABP I [24, 25]. The Dako antibody against human RBP has been shown to recognize all four isoforms of RBP present in the porcine uterus [29] and has previously been used for biochemical as well as immunohistochemical studies of the pig placenta [22, 23].
Immunohistochemistry All incubations were done at room temperature unless otherwise stated. Sections were cleared in xylene and rehydrated through descending concentrations of ethanol, washed with PBS, incubated in 0.5% hydrogen peroxide (v/v) in PBS with 0.03% Triton X-100 (v/v) (PBST) for 30 min to block endogenous peroxidase activity, washed in PBS, incubated for 1 h in a solution containing 4% BSA (grade V) in PBS with 1–5% of swine serum, and then in the same solution but with primary antibody at 4°C overnight. The polyclonal antibodies against human RBP were used at a concentration of 4–6 µg/ml [38] or 1:700 (Dako), both diluted in 1% swine serum; the polyclonal antibodies against CRBP I at 0.1–0.3 µg/ml with 5% swine serum; and the polyclonal antibodies against CRABP I at 5 µg/ml with 1% swine serum. Controls included incubation with normal rabbit immunoglobulin G (IgG) with similar concentrations of swine serum.
After the overnight incubation sections were washed vigorously in many changes (more than four) of PBST and PBS, respectively, incubated with swine anti-rabbit biotin-conjugated antibodies, diluted 1:500 in 4% BSA in PBS, for 30 min, washed again as above, before incubating for 30 min with an avidin-biotinylated horseradish peroxidase complex (ABComplex, Dakopatts, Glostrup, Denmark), diluted 1+1:125 in PBS, and washing with PBS. To visualize the antigen-antibody complex, we used a peroxidase substrate solution containing 0.007% hydrogen peroxide and 0.06% 3,3'-diaminobenzidine-tetrahydrochloride (Merck, Rödovre, Denmark) in PBS. After washing, the sections were weakly counterstained with Mayer's hematoxylin, dehydrated, and mounted with DPX (BDH Laboratory Supplies, Poole, England).
The immunoreaction data as used in Table 1 are quantified as follows: no positive cells observed (-), some positive cells observed (+), half of the cell population was positive (++), most of the cells observed were positive (+++), all of the cells observed were positive (++++), and changes from negative to positive or vice versa during a period are indicated by /.
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Controls Normal rabbit IgG was used instead of primary antibodies to check for nonspecific binding of antibodies. The antibodies against CRBP I and CRABP I have previously not been used on pigs, and sections of porcine embryos were thus included as positive controls and the staining patterns obtained in, e.g., the developing neural system were compared to those described for such structures in mouse embryos (CRBP I [26]; CRABP I [39]).
RESULTS
General Comments
Only results regarding the materno-fetal interface in interareolar regions and in the regular areolar regions of the areolar gland-complex will be given (Fig. 1). It should be noted that the staining patterns were heterogeneous, i.e., localized to individual cells or groups of cells in all tissues examined (Table 1), but unless otherwise stated below, at least approximately half of the cells in a specific tissue were positive for the protein in question. Any changes in localization patterns observed within a given age group are pointed out in the text and are also noted in Table 1, but not in the summary shown in Figure 1.
Controls
Staining was very low or undetectable in sections incubated with normal (nonimmune) rabbit IgG. A number of representative controls are shown in Figures 2, b, d, f, h, and m, 3c, and 4d. It should be noted that Figure 4d is control for 4, a–c.
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Positive controls, using the antibodies against CRBP I and CRABP I on sections of pig embryos, resulted in staining of structures also described positive in mouse embryos [26, 39], such as, e.g., neural structures (data not shown).
Early Pregnancy (Days 16–35)
Immunoreactive RBP was detected in the uterine epithelium throughout this period (Fig. 2a), including the epithelial proliferations, whereas the uterine epithelium in areolar regions was nonreactive (not shown). Most of the glandular epithelium contained ir-RBP, sometimes with a particularly strong staining of fluid present in the glandular lumen (Fig. 2c).
In the embryonic part of the placenta, some trophoblasts stained for ir-RBP (Fig. 2e). The staining was mainly located in granules of varying sizes, and in general a number of adjacent trophoblasts contained granules. From around Day 25, only the high columnar areolar trophoblasts contained ir-RBP (Fig. 2g), whereas the interareolar trophoblasts appeared void of ir-RBP-positive granules (not shown). In the areolar trophoblasts, ir-RBP-positive granules were observed both in a supranuclear and infranuclear position (Fig. 2g).
A granular staining for ir-CRBP was found in the uterine epithelium, mainly located around the nucleus (Fig. 2i). In cells of the epithelial proliferations observed around 16 dg, however, the granules were oriented as streams running through the uterine epithelial cells (not shown), an impression that could be due to the shape of the epithelial proliferations. Similarly to ir-RBP, the uterine epithelium at areolae and in glands appeared nonreactive for ir-CRBP (not shown).
Immunoreactive CRBP was also present in the trophoblasts. In interareolar trophoblasts, ir-CRBP was present in apical granules (Fig. 2i), whereas in areolar trophoblasts a heavy staining was observed throughout the cytoplasm of the cells (Fig. 2j). The distribution of ir-CRABP was much more restricted than that of ir-CRBP, as ir-CRABP was detected only in interareolar trophoblasts, and only from around Day 25 and then throughout the period investigated (Fig. 2k). The staining was present throughout the cytoplasm and often appeared to be present also in the nucleus of the cells.
Midpregnancy (Days 36–69)
The staining patterns of ir-RBP, ir-CRBP, and ir-CRABP in the uterine-placental compartment remained basically the same as those observed during the previous period. There was, however, one striking change: the appearance of ir-CRBP in the glandular epithelial cells. The staining was granular and these granules were mainly located in a supranuclear position (Fig. 2l).
In contrast to the regular areolar regions (not shown), dramatic histological changes could be observed in the interareolar regions. Here the materno-fetal interface had developed the very characteristic folds where trophoblasts are low and irregular at the top and sides of the chorioallantoic ridges and cuboidal to columnar at the fossae between them. In both the uterine epithelium and in trophoblasts, ir-CRBP was present in epithelial cells of the complementary ridges and fossae (Fig. 3a). The reactivity was still granular and the location similar to that of early pregnancy (Figs. 2i and 3a). Immunoreactive CRABP was still present in trophoblasts, and the reactivity was present throughout the cytoplasm and sometimes also in the nuclei of trophoblasts (Fig. 3b).
Late Pregnancy (Days 70–97)
No major changes in staining patterns were observed during this period or compared to midpregnancy. The interareolar region continued to develop with the interhemal barrier becoming strikingly thinner. The low uterine epithelium that now contained many intraepithelial capillaries remained positive for ir-RBP (Fig. 4a), as well as ir-CRBP. The latter was present in granules located around the nucleus of the uterine epithelium and in apical granules in the well-vascularized apposing trophoblasts (Fig. 4b), and ir-CRABP was present throughout the cytoplasm of the trophoblast and also in most of their nuclei (Fig. 4c).
DISCUSSION
In the present study, the immunohistochemical localization of RBP, the serum transport protein for retinol, and of CRBP I and CRABP I, involved in cellular transport and metabolism of retinol and retinoic acid, respectively, was determined at the materno-fetal interface of the porcine placenta throughout gestation. For the first time the regular areolar-gland subunits and interareolar regions have been studied as separate units in order to clarify possible transfer routes for retinol. The distribution of the investigated proteins was basically as follows. Immunoreactive-RBP was found in the interareolar uterine epithelium, in uterine glands of areolae, and in areolar trophoblasts. Immunoreactive-CRBP was found in granules in the interareolar uterine epithelium, from midpregnancy in granules in uterine gland epithelium, and in both interareolar and areolar trophoblasts, but as apical granules in the former and as a homogeneous staining in the latter. Finally, ir-CRABP was detected solely in the interareolar trophoblasts (see Fig. 5 for a schematic summary and Table 1 for a more detailed summary of the results).
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Areolar-Gland Subunits
The detection of RBP transcripts in the uterine endometrium, of ir-RBP also in glands, and of RBP and retinol in uterine secretions [22, 23, 28–32] have suggested that the uterine endometrial glands of the pig placenta secrete RBP-retinol into the uterine lumen. These results were confirmed by the present investigation, which added the important information that ir-RBP is absent from areolar uterine epithelium and present in the fetal interface of the areolar-gland subunit in the areolar trophoblasts. In two studies by Harney and collaborators [22, 23] trophoblasts were found to be positive for ir-RBP by 15 dg, but in contrast to the present study, not from 30 dg and onward. Moreover, ir-RBP was described as being present in the entire uterine epithelium [23], in contrast to its reported absence from areolar uterine epithelium (present study). One possible explanation to the apparent discrepancies in results is that areolar regions were not specifically studied by Harney et al. Areolas are histologically discernible already by Day 15 [36] but may easily be overlooked as they are scattered along the vast (1–1.5 m) highly folded materno-fetal interphase. It is also possible that differences in immunohistochemical techniques (although the antibody against RBP came from the same source in these three investigations) have yielded slightly different results. These discrepancies in results, however, further emphasize the need for more detailed descriptions of the material investigated when studying placentas with such apparent regional differences as the pig. In conclusion, results from the present study together with those of Harney and collaborators [22, 23] suggest that as the areolar region develops, ir-RBP becomes predominantly located to the areolar trophoblasts, and by 30 dg, only to these cells.
In the present study, the reactivity of RBP in areolar trophoblasts was localized to granules of varying sizes. These granules were present throughout the cytoplasm of the cells, from the apical to the basal side. Together with a well-developed apical tubular system, they have been described ultrastructurally in areolar trophoblasts and suggested to mirror a capacity for transcytosis of molecules [40, 41]. Moreover, the localization pattern of ir-RBP is strikingly similar to that of uteroferrin, a 35-kDa glycoprotein that functions as an iron transporter to the developing porcine conceptus [42, 43], and iron, complexed to uteroferrin in uterine glands, is secreted into the areolar cavity [40, 42–46]. The similar staining and secretion patterns for ir-RBP and uteroferrin may suggest that RBP-retinol is also absorbed by areolar trophoblasts before transcytosis and release into chorioallantoic capillaries for further transport via the umbilical vessels to the embryo or fetus. Previously, RBP mRNA has been isolated from homogenates of pig chorioallantoic membranes [23], which could suggest that retinol is coupled to newly synthesized RBP in the trophoblast. The CRBP I, which was also present in areolar trophoblasts (present study), could have a role not only in storage of retinol but also as an intracellular transporter of retinol from maternal to trophoblast RBP. Such a transport mechanism has been suggested for the yolk sac endoderm of mice [17]. The glandular component of the areolar-gland subunit also contained ir-CRBP, but only from around midgestation. This is a time when RBP-retinol secretion from uterine glands has been observed to decrease in in vitro studies [47], which may indicate that CRBP I has a role in the storage of retinol that is not secreted, presumably in the form of retinyl esters [12].
Interareolar Regions
The role for RBP in retinol transport includes transport of retinol to target cells in the adult as well as in the embryo or fetus [10]. Uteroferrin in contrast, appears to be involved only in transfer of iron to the uterine lumen of cyclic animals and to the conceptus during gestation [45]. This functional difference may explain why uteroferrin is present only in areolar-gland subunits [42], whereas ir-RBP was also present in interareolar regions (present study). Paracrine transport of retinol from the uterine epithelium to the trophoblasts has previously been suggested to be of importance during growth and differentiation of the pig placenta [23, 31], and retinol has been shown to be one among other factors needed to secure mouse placental development [6, 7, 21]. Retinoic acid is a potent modulator of cell growth and differentiation [48], and the effectors of retinoic acid action, the RARs and RXRs [16, 49, 50], have previously been detected in porcine pregnant endometrium and conceptus tissues of early pregnancy [31, 32]. In the present study, both ir-CRBP and ir-CRABP were detected in porcine trophoblasts throughout gestation (ir-CRABP from 25 dg). The function of interareolar trophoblasts has been suggested to depend on their location along complementary folds that develop at the materno-fetal interface during placentation [51, 52]. The trophoblast cells at the base of the folds are high columnar and only sparsely vascularized, whereas trophoblasts found on the ridges and their sides are irregular with many intraepithelial capillaries [51, 52]. The diffusion distance for, e.g., gases is much shorter at the ridges and their sides. It has therefore been suggested that gaseous exchange occurs here [51], whereas the trophoblast at the base of the fetal ridges is suggested to be involved mainly in uptake and metabolism of substances less diffusible than gases [51, 52], one of which could be retinol and/or retinoic acid. Immunoreactive CRBP and ir-CRABP were, however, not only present in the columnar trophoblast but rather in all types of trophoblast cells along the interareolar region. This may suggest that the role for these proteins is one in placentation rather than in materno-fetal transfer of retinol or retinoic acid, which would be in accordance with results presented regarding mouse placentation [21, 53]. Interestingly, ir-CRABP reactivity was limited to trophoblasts of the interareolar region, suggesting that these cells have a particular need to regulate the level of free retinoic acid [12, 14].
Species Differences in Expression Patterns of Retinoid Binding Proteins
The localization of ir-RBP, ir-CRBP, and ir-CRABP were quite different in the pig placenta compared to those observed previously in the hemochorial placentae of the mouse and human in that it was much more extensive. The porcine trophoblasts contained either 1) ir-RBP and ir-CRBP or 2) ir-CRBP and ir-CRABP, whereas mouse trophoblasts contained only ir-CRBP [19], and human trophoblasts were reactive only for ir-RBP and the above-mentioned RBP receptor [20]. In mice, however, a functional yolk sac placenta composed of trophoblasts, parietal and visceral endodermal cells is present and functional throughout gestation [54, 55], and the visceral endoderm contained ir-RBP, RBP-receptor like antigens, as well as ir-CRBP [19], and ir-CRABP (see Fig. 3b in Gustafson et al. [26]). Also in the rat, a functional yolk sac placenta is present that synthesizes RBP [56]. A histological comparison between the porcine areolar trophoblast and the visceral endoderm of mouse yolk sac reveals that both types are high columnar cells with many absorptive vesicles [41, 54, 55]. Thus, not only are these two cell types functionally and histologically similar, they also contain the ir-RBP and ir-CRBP, which may suggest that they play similar roles in placental vitamin A transport and metabolism. Interestingly, in animals where the yolk sac regresses during early pregnancy—humans, sheep, and pigs—the yolk sac expresses RBP during its relatively short life span (unpublished observations) [23, 57].
A number of investigations have studied the expression of RBP in sheep and bovine placentas, but nothing is known about the expression of CRBP I and CRABP I in these species. The sheep chorioallantoic membrane has been found to express RBP mRNA [58], and ir-RBP was localized to the ectodermal cells of chorion and amnion, respectively, and to the endodermal cells of the allantois [59]. However any regional differences that correlate to histologically separate units, as observed in the present study, were not reported. Also, the bovine chorioallantoic membrane has been found to express RBP mRNA as well as ir-RBP, and in one investigation the mRNA was specifically located to the trophectodermal cells of the bovine cotyledons [60–62], which are comparable to the interareolar areas of the pig. Finally, similar to the porcine placenta, ir-RBP was also detected in bovine uterine glands.
Conclusions
The results from the present study suggest that ir-RBP, ir-CRBP, and ir-CRABP are present in the developing pig placenta to an extent that exceeds that of the species thus far investigated by immunohistochemistry (mouse and human). It also suggests that the areolar-gland subunit plays an important role in transfer of retinol and retinol-RBP from mother to fetus, and that the porcine placenta, like, e.g., the mouse placenta, may utilize retinol during its development and growth. Finally, the early stages of development, 16–25 dg, do not only represent a time of morphological change but also a time when changes in localization and possible expression of retinoid binding proteins occur, thus being the period of highest interest for future studies.
ACKNOWLEDGMENTS
Mrs. Hanne Holm, Gunnel Holden, and Inge Bjerring are thanked for excellent technical assistance and Dr. Ulf Eriksson for the generous gift of antibodies.
FOOTNOTES
First decision: 4 February 2000.
1 Correspondence: Vibeke Dantzer, Anatomy, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Bulowsvej 13, DK-1870 Frederiksberg C, Denmark. FAX: 45 35 28 25 47; vibeke.dantzer{at}iaf.kvl.dk 
Accepted: August 15, 2000.
Received: December 31, 1999.
REFERENCES
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, maternal intraepithelial capillary; T, trophoblast. b) A granular staining for ir-CRBP was most pronounced in the uterine epithelium, but also in apical granules (
) of trophoblasts (T). Staining was observed in fetal connective tissue (arrowheads). c) Reactivity for ir-CRABP was detected in the cytoplasm as well as in many nuclei of trophoblasts (T). The uterine epithelium (UE) showed no reaction. *Fetal intraepithelial capillary; 