1 Faculty of MedicineEndocrinology, Memorial University of
Newfoundland, St. John's, Newfoundland A1B 3V6, Canada; and
2 Department of Medicine, University of Melbourne, Austin and
Repatriation Medical Centre, Victoria 3081, Australia
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ABSTRACT |
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The expression of calcitropic genes and proteins was localized within murine placenta during late gestation (the time frame of active calcium transfer) with an analysis of several gene-deletion mouse models by immunohistochemistry and in situ hybridization. Parathyroid hormone-related protein (PTHrP), the PTH/PTHrP receptor, calcium receptor, calbindin-D9k, Ca2+-ATPase, and vitamin D receptor were all highly expressed in a localized structure of the murine placenta, the intraplacental yolk sac, compared with trophoblasts. In the PTHrP gene-deleted or Pthrp-null placenta in which placental calcium transfer is decreased, calbindin-D9k expression was downregulated in the intraplacental yolk sac but not in the trophoblasts. These observations indicated that the intraplacental yolk sac contains calcium transfer and calcium-sensing capability and that it is a probable route of maternal-fetal calcium exchange in the mouse.
parathyroid hormone receptors; fetus; fetal development; placental calcium transfer; calcium receptor; calcitonin; calcitriol; vitamin D receptor; calbindin; vitamin D-dependent calcium-binding protein; calcium-ATPase; trophoblasts; in situ hybridization; immunohistochemistry; gene knockout mice
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INTRODUCTION |
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CALCIUM IS ACTIVELY TRANSPORTED across the placenta in late gestation to meet the needs of the rapidly mineralizing skeleton and to maintain an extracellular level of calcium that is physiologically appropriate for fetal tissues (i.e., for cell membrane stability, blood coagulation, and the like) and that is higher than the maternal calcium concentration (34). In human pregnancy, ~80% of calcium is accreted by the fetal skeleton during the third trimester (16); in rat pregnancy, ~95% of calcium is accreted during the last 5 days of gestation (11), during which time maternal-fetal clearance of calcium increases 72-fold (17). The rate of calcium accretion has not been formally measured in fetal mice; however, because of the slightly shorter gestation period of the mouse (19 vs. 22 days), it is likely that the bulk of calcium transfer occurs in the last 4 days of gestation [embryonic days (ED) 16-19]. On the basis of these observations, it is likely that the factors responsible for regulating placental calcium transfer would be upregulated in the placenta during this time.
The mechanisms by which active calcium exchange occurs across the placenta are not well understood. Analogous to calcium transfer across the intestinal mucosa and kidney (25), it has been proposed that calcium diffuses into calcium-transporting cells through maternal-facing basement membranes, is carried across these cells by calcium-binding proteins (calbindin-D9k and other calbindins), and is actively extruded at the fetal-facing basement membranes by Ca2+-ATPase (8). The placental expression of calbindin-D9k increases 135-fold over the last 7 days of gestation in the rat (17), whereas the expression of the Ca2+-ATPase increases 2-fold over the same interval (17, 49). These observations are consistent with the hypothesis that calbindin-D9k and Ca2+-ATPase are required for maternal-fetal calcium transfer in late gestation.
The factors that regulate placental calcium transfer are also, at best, only partly elucidated. There is evidence from thyroparathyroidectomized sheep (9, 47), and from the Pthrp gene knockout model (35), that midmolecular forms of parathyroid hormone-related protein (PTHrP) stimulate placental calcium transfer. The calcium-sensing receptor (CaSR) also appears to influence the rate of placental calcium transfer (33). Evidence that parathyroid hormone (PTH), calcitriol, and calcitonin might regulate placental calcium transfer is contradictory and less certain (see Ref. 36 and a review in Ref. 34). It is likely that some of the discrepancies are due to the markedly different placental structures in the mammals that have been studied, including humans and other primates, rodents, and sheep.
Murine and human placentas are hemochorial (i.e., maternal blood freely bathes the fetal tissues within the placenta) and have similar function and permeability (31, 46). However, the structures of murine and human placentas do differ in other respects. The murine placenta is trichorial, whereas the human placenta is monochorial. There are three types of trophoblasts in the murine placenta, including the labyrinthine or syncytial trophoblasts, the spongiotrophoblasts, and the giant trophoblasts; in contrast, the human placenta contains syncytiotrophoblasts and cytotrophoblasts. The labyrinthine trophoblasts make up the bulk of the murine placenta and are considered to be the dominant site of maternal-fetal exchange. The giant trophoblasts and spongiotrophoblasts invade the decidual tissue and also express numerous hormones.
An often overlooked structure contained in rodent placenta is the
intraplacental yolk sac (IPYS), which, as the name implies, consists of
part of the primitive yolk sac that later became incorporated into the
placenta (Fig. 1,
A and B) (12). The primitive yolk sac participates in nutrient exchange between the fetal and maternal circulations before the formation of the placenta (12,
26). Like the yolk sac from which it derives, the IPYS is a
bilayered membrane, consisting of tall columnar cells on the visceral
or endothelial side overlying fetal vessels, and smaller parietal or
cuboidal cells on the epithelial side that overlie a thick basement
membrane (Reichert's membrane) and the maternal blood spaces (Fig.
1C). These two layers of the IPYS are separated by a
potential space (sinus of Duval), which communicates with the yolk sac
cavity and, thereby, the uterine lumen. Given its anatomic position
between fetal vessels and maternal blood spaces at the fetal pole of
the placenta, it is well situated for exchange of substances between
mother and fetus (Fig. 1C). The IPYS is found exclusively in
rodent placentas (rat, mouse, gerbil, and hamster) (26); a
corresponding structure has not been described in human, other primate,
or ruminant placentas.
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Since the original description of the IPYS in rodent placenta by Duval
(15), it has been reported that the IPYS contains abundant
expression of calbindin-D9k, greater than that observed in
the surrounding trophoblasts (5). Although the placental expression of calbindin-D9k increases 135-fold during
the time of rapid calcium transfer in the rat (17), the
increase is more marked in the IPYS than in the trophoblasts
(41). The IPYS has also been reported to express
Ca2+-ATPase (3). These observations suggest
that the IPYS may have some role in maternal-fetal calcium exchange,
but this possibility has not been further explored. More recently, it
has been observed that the IPYS does not behave simply as an
incorporated remnant in the placenta; instead, it actively invaginates
and expands in volume during the last 4-5 days of gestation in the
mouse, the time frame of rapid calcium transfer (45). In
the absence of the gene-encoding platelet-derived growth factor
receptor-, the IPYS does not form, and the embryo often dies in
midgestation (45). Whether the lack of IPYS contributes to
the embryonic lethality of that knockout has not been determined. Apart
from these recent observations, the IPYS has been largely ignored and considered to be a nonfunctional remnant of the primitive yolk sac
(12).
Although several calciotropic factors [such as PTHrP, PTH/PTHrP receptor, and vitamin D receptor (VDR)] have been reported to be expressed in the placenta, the specific localization of expression of many of these genes (particularly in the murine placenta) has not been reported. Technical limitations clouded interpretations; for example, radiolabeled calcitriol bound to placenta, suggesting the presence of VDRs (48). However, since the cloning of the VDR gene (Vdr), placental expression of VDR mRNA or protein has not been reported. Determination of the specific intraplacental localization of these calcitropic genes has particular relevance in the definition of the routes along which calcium might be transferred from mother to fetus, and which genes might be involved in that process.
The purpose of this study was twofold. First, we wanted to localize the expression of calcitropic genes within the murine placenta during late gestation (the time frame of active calcium transfer), utilizing placentas from specific knockout mice to rigorously confirm the specificity of the mRNA and proteins detected. Second, we hypothesized that upregulation or downregulation of placental calcium transfer would result in alterations in calcitropic gene and protein expression within placental cells that are involved in maternal-fetal calcium transfer. To test that hypothesis, we systematically examined placentas of mice in which we have previously noted that placental calcium transfer is downregulated (Pthrp-null fetuses) and upregulated [PTH/PTHrP receptor (Pthr1)-null fetuses] (35).
We found that PTHrP, PTH/PTHrP receptor, CaSR, calbindin-D9k, Ca2+-ATPase, and VDR were particularly highly expressed in the IPYS of the murine placenta compared with the trophoblasts. In the Pthrp-null placenta, the expression of calbindin-D9k mRNA and protein was downregulated in the IPYS, but not in the trophoblasts; no such downregulation was observed in the Pthr1-null placentas. The IPYS may be an important route of calcium exchange between mother and fetus in rodents that utilizes a strategy from the process of eggshell calcification.
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METHODS |
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Knockout mice and genotyping. Pthrp (28), Pthr1 (38), Casr (22), Vdr (39), and calcitonin (Ct) (23) gene knockout mice were obtained by targeted disruption of the murine genes in embryonic stem cells, as previously described. Heterozygous mice were mated overnight; the presence of a vaginal mucus plug on the morning after mating marked ED 0.5. Normal gestation in these mice is 19 days. All mice were given a standard chow diet and water ad libitum. All studies were performed with the prior approval of the Institutional Animal Care Committee of Memorial University of Newfoundland.
Genomic DNA was obtained from fetal tails, and genotyping was accomplished by PCR with primers that were specific to the Pthrp, Pthr1, Casr, and Vdr gene sequences (22, 35, 38, 39) and the Ct gene sequence in a single-tube, 36-cycle PCR reaction utilizing a PTC-200 Peltier Thermal Cycler (MJ Research, Cambridge, MA). The Ct genotyping was accomplished in a three-primer system [one in the retained portion of wild-type (wt) allele, one in a deleted portion of the wt allele, and one in the inserted neomycin sequence] that utilized the following specific sequences: CAG GAT CAA GAG TCA CCG CT; GGA GCC TGC GCT CCA GCG AA; and GGT GGA TGT GGA ATG TGT GC.Tissue collection. Wt and null placentas obtained from ED 16.5-18.5 pregnancies were studied, because this time frame corresponds to the interval of rapid maternal-fetal transfer of calcium. Placentas were obtained after placental perfusion with paraformaldehyde to minimize the degradation in mRNA or protein levels that might occur during fixation and processing of the RNase and protease-rich placental tissues (36). After this, the placentas were removed and placed in 10% formalin for standard processing, embedding in paraffin, and sectioning.
Riboprobes and antibodies.
cDNAs used included murine calbindin-D9k (42)
and murine calbindin-D28k (51) (gifts of S. Christakos), human Ca2+-ATPase (37) (gift of
R. Kumar), murine -fetoprotein (gift of Margaret Baron), rat
PTH/PTHrP receptor (2) and rat PTHrP (29)
(gifts of H. M. Kronenberg), murine CaSR (55) (gift
of C. Ho-Pao), murine 57-kDa calcium-binding protein (57-kDa calbindin) (50) (gift of R. S. Tuan), murine VDR (27)
(gift of T. Kawada), murine calcitonin (gift of G. J. Cote),
murine placental lactogen (24) and murine proliferin
(40) (gifts of D. Linzer), and murine nodal
(56) (gift of M. Kuehn). Antibodies included rabbit anti-rat calbindin-D9k (5) (gift of M. E. Bruns), murine anti-human erythrocyte Ca2+-ATPase (Sigma),
rabbit anti-rat PTHrP (30) (gift of J. Moseley and T. J. Martin), mouse anti-CaSR (ADD antibody) (18) [gift of
K. V. Rogers (NPS Pharmaceuticals) and A. M. Spiegel and
P. K. Goldsmith (Metabolic Diseases Branch, NIDDK/NIH)] rabbit
anti-human VDR (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit
anti-human calcitonin (Dako, Carpenteria, CA), and rabbit anti-rat
PTH/PTHrP receptor (Berkley Antibody/Covance Research Products,
Berkeley, CA).
Riboprobe labeling. For in situ hybridization, the plasmids were linearized with appropriate restriction enzymes and labeled with 125 µCi of 35S-labeled UTP using an SP6/T7 Transcription Kit (Promega/Fisher Scientific, Burlington, ON, Canada) and the appropriate polymerase. Unincorporated nucleotides were removed with the NucTrap columns (Stratagene, La Jolla, CA) as per package instructions.
In situ hybridization. In situ hybridization was performed on 5-µm tissue sections, as described previously (32, 36). Slides were then dipped into NTB-2 liquid emulsion, dried, stored in light-tight boxes, and kept at 4°C until developed (3 days to 6 wk). The emulsion was developed using standard developer and fixer, and the sections were then counterstained with hematoxylin-eosin.
Immunohistochemistry. Immunohistochemistry on 5-µm sections was performed using standard technique with secondary antibody, ABC reagent, and DAB-Tris-peroxidase kits obtained from Vector (Burlington). Sections were counterstained with Contrast Red (GIBCO BRL, Burlington) or 1% methyl green, washed, dehydrated, and mounted. Assessments of staining intensity were determined in a blinded fashion (no knowledge of the genotype). The reproducibility of the results was confirmed independently on at least three separate litters.
For immunohistochemistry of CaSR and PTH/PTHrP receptor, sections were treated to reduce cross-linking and unmask the epitopes. After the deparaffinization and rehydration steps, sections were incubated in 4% paraformaldehyde for 15 min, then in 10 µg/ml of proteinase K (GIBCO BRL) in PBS at 37°C for 15 min, and then in 4% paraformaldehyde again for 15 min to stop the reaction. After this, the standard immunohistochemical technique was resumed with administration of the blocking serum. For each primary antibody, the appropriate concentration and incubation conditions were determined empirically. Concentrations ranged from 1:250 to 1:1,000, with optimal staining generally observed at 1:500. The specific details of PTHrP immunohistochemistry have been previously described (36). For remaining primary antibodies, incubations were at room temperature for 1-2 h, except for anti-VDR and anti-CaSR antibodies, which were applied at 4°C overnight. The CaSR and PTH/PTHrP receptor antibodies were diluted in 1% bovine serum albumin in PBS; the PTHrP antibody was diluted in PBS containing 5% newborn bovine serum (GIBCO BRL); all other primary antibodies were diluted in PBS containing 2% blocking serum.Controls. There were several levels of controls for the immunohistochemistry and in situ hybridization. Pthrp-null, Pthr1-null, Casr-null, Vdr-null, and Ct-null placentas were used to confirm the specificity of the respective cDNAs and antibodies. All comparisons between wt and null placentas were made between placentas that had been obtained from within the same litter (i.e., siblings) and that had been fixed, embedded, sectioned, and treated together. For immunohistochemistry, adjacent control sections also had the primary antibody omitted. For in situ hybridization, sense and anti-sense riboprobes made from the same cDNA were applied on adjacent sections; also, different antisense probes (i.e., different cDNAs) were studied on adjacent sections in the same experiments. Because of the practicalities of space limitations, it is not possible to show all controls in the figures.
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RESULTS |
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For consistency with the literature, the layers of the IPYS will be referred to as the columnar layer and the parietal layer. The yolk sac proper (i.e., the bulk of the yolk sac, which is outside the placenta) will be referred to as the extraplacental yolk sac and its layers as the visceral layer and the parietal layer. The columnar layer of the IPYS is contiguous with the visceral layer of the extraplacental yolk sac, and both parietal layers are also contiguous.
Calbindin-D9k and calbindin-D28k.
Calbindin-D9k mRNA was intensely expressed in the columnar
cells of the IPYS, with little or no signal detected in the parietal cells (Fig. 2, a and
c). The signal was typically so intense that the columnar
cells were blackened and obliterated by silver grains on the
bright-field image, but silver grain deposition was not readily
apparent in the remainder of the placenta (Fig. 2a).
However, on the dark-field images, calbindin-D9k mRNA was
clearly present at a much lower intensity in the labyrinthine
trophoblasts than in the visceral layer of the extraplacental yolk sac
(Fig. 2c). Immunohistochemistry demonstrated that
calbindin-D9k was present in both columnar and parietal
IPYS cells, although more intensely on the columnar side (Fig.
2g).
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Ca2+-ATPase.
Examination of Ca2+-ATPase mRNA and protein revealed an
expression pattern similar to that of calbindin-D9k. By in
situ hybridization, the mRNA signal was most intense in the columnar
cells of the IPYS, resulting in a blackened bright-field image (Fig.
3a),
whereas the less intense expression in the labyrinthine trophoblasts
and visceral layer of the extraplacental yolk sac was apparent only on
the corresponding dark-field image (Fig. 3b).
Immunohistochemistry determined that both the columnar and parietal
cells of the IPYS express Ca2+-ATPase, although the
staining is more intense on the columnar side (concordant with the mRNA
signal intensity) (Fig. 3, c and d).
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PTH/PTHrP receptor. PTH/PTHrP receptor mRNA was most intensely expressed in the parietal cells of the IPYS overlying Reichert's membrane, with reduced mRNA signal intensity evident in the columnar cells of the IPYS (Fig. 3, e, f, and g). Labyrinthine trophoblasts and the visceral layer of the extraplacental yolk sac showed no detectable PTH/PTHrP receptor mRNA in bright- or dark-field images. Immunohistochemistry with anti-PTH/PTHrP receptor antibody showed a similar pattern of immunoreactivity confined to the parietal layer of the IPYS, in addition to adjacent epithelial cells of maternal blood vessels (not shown). In control experiments using Pthr1-null placentas, the PTH/PTHrP receptor mRNA and protein were absent (data not shown).
PTHrP. PTHrP mRNA was present throughout the placenta, including the IPYS, trophoblasts, and visceral layer of the extraplacental yolk sac, but the signal was most intense in the parietal cells of the IPYS (Fig. 3, h and i). The PTHrP mRNA was absent in sections obtained from Pthrp-null placentas (Fig. 3j). When anti-PTHrP antibody was used, the peptide was found to be diffusely expressed in all three trophoblast types, the IPYS, and the visceral layer of the extraplacental yolk sac. The highest intensity of PTHrP immunoreactivity was observed in the spongiotrophoblasts and IPYS. Within the IPYS, the columnar cells had the highest intensity of staining, with less apparent staining in the parietal cells of the IPYS (Fig. 3k). With use of sections obtained from Pthrp-null placentas, the specificity of the anti-PTHrP antibody was confirmed (Fig. 3l).
CaSR. CaSR mRNA was detectable by in situ hybridization only in the IPYS cells and not in the trophoblasts or the extraplacental yolk sac. Although more intense on the parietal side, CaSR mRNA could be detected on the columnar side of the IPYS as well (Fig. 3, m and n). Placentas obtained from Casr-null mice could not be used as controls for in situ hybridization, because a truncated (mutant) CaSR mRNA is transcribed and is detected by Northern blot and in situ hybridization (unpublished data). By immunohistochemistry, with a monoclonal antibody directed against a region of the CaSR that has been deleted in the Casr-null mice, the CaSR was found to be expressed in both layers of the IPYS and in the surrounding trophoblasts of wt placenta (Fig. 3o) and absent in placentas obtained from Casr-null mice (Fig. 3p).
VDR. By use of riboprobes generated from a cDNA for the murine VDR, a low level of mRNA signal was detected in wt placenta that was not strikingly different from the background signal observed in Vdr-null placentas (not shown). This finding indicated that VDR mRNA must be present at a low level, if present at all. With use of antibody to VDR, and when wt were compared with Vdr-null placentas, nuclear staining for VDR was detected in many columnar cells of the IPYS and in a few parietal cells (Fig. 3, q and r). No apparent VDR immunoreactivity could be detected in trophoblasts or the extraplacental yolk sac.
Calcitonin.
Calcitonin mRNA was found to be diffusely expressed throughout the
placenta, including the IPYS and the labyrinthine trophoblasts (Fig.
4, a and
b). No apparent signal was detected in the extraplacental yolk sac. No calcitonin mRNA was detected in Ct-null
placentas (Fig. 4, c and d), confirming the
specificity of the signal detected in wt placenta. Calcitonin
immunoreactivity was present at low levels diffusely in the
labyrinthine trophoblasts and in both layers of the IPYS of wt placenta
(Fig. 4e), with no immunoreactivity detected in
Ct-null placenta (Fig. 4f).
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Calcitonin receptor. Antibody to the calcitonin receptor revealed modest but diffuse staining in the IPYS and labyrinthine trophoblasts, with the most intense staining in the visceral and parietal layers of the extraplacental yolk sac (Fig. 4, g and h).
57-kDa calbindin. The expression of 57-kDa calbindin was most apparent in spongiotrophoblasts and giant trophoblasts at the periphery and base of the placenta and in the visceral layer of the extraplacental yolk sac (Fig. 4, i and j). There was comparatively less intense mRNA signal visualized in the labyrinthine trophoblasts, and no definite mRNA was detected in either layer of the IPYS.
-Fetoprotein, nodal, placental lactogen, and proliferin.
As a guide to the relative expression of calciotropic factors in
the IPYS, we also examined the expression of other placental markers,
including
-fetoprotein (a yolk sac marker, Fig. 4, k and
l), nodal (a spongiotrophoblast marker, not shown),
placental lactogen, and proliferin (markers of giant trophoblasts, not
shown). None of these factors was expressed in the IPYS, as determined by in situ hybridization using specific riboprobes.
-Fetoprotein was
intensely expressed in the visceral layer of the extraplacental yolk
sac, but immediately upon entry into the placenta, the corresponding columnar cells of the IPYS did not express
-fetoprotein (Fig. 4,
k and l). Nodal was expressed in
spongiotrophoblasts, and placental lactogen and proliferin were
expressed in giant trophoblasts, and none of these appeared to be
present in the IPYS (data not shown).
Pthrp-null and Pthr1-null placentas.
Previously, Pthrp-null fetuses had been shown to have
reduced placental calcium transfer compared with their littermates, whereas Pthr1-null placentas had been shown to have
increased placental calcium transfer (35). Placentas of wt
and Pthrp-null siblings were examined to determine whether
reduced placental calcium transfer was associated with any abnormality
in structure or in gene or protein expression. Pthr1-null
placentas were similarly examined to determine whether increased
placental calcium transfer was accompanied by any changes in placental
structure or in gene or protein expression. The absence of PTHrP mRNA
and protein in Pthrp-null placentas has already been
demonstrated (Fig. 3, j and l). The IPYS was far
less often present in sections obtained from Pthrp-null
placentas, but it was readily and abundantly present in wt and
Pthr1-null placentas. In Pthrp-null sections that
did have IPYS, calbindin-D9k mRNA signal intensity was
sharply reduced in the Pthrp-null placentas, such that it
was not apparent in the bright-field image (Fig. 2, b vs.
a) and was apparent only in the dark-field image (Fig. 2,
d vs. c). The expression of the corresponding
protein was also reduced in intensity, as judged by
immunohistochemistry by use of anti-calbindin-D9k antibody (Fig. 2, f and h vs. e and
g). The reduction in calbindin-D9k was specific,
because adjacent sections from the same placentas showed normal
intensity of expression of Ca2+-ATPase, the PTH/PTHrP
receptor, -fetoprotein, 57-kDa calbindin, and other calcitropic
mRNAs (data not shown). Examination of placentas from the
Pthr1-null mice showed no abnormality of
calbindin-D9k or any other calcitropic gene expression,
apart from loss of the PTH/PTHrP receptor (data not shown).
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DISCUSSION |
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This study has comprehensively examined calcium-binding proteins,
calcitropic hormones, and their receptors in the murine placenta during
late gestation in the interval of rapid calcium exchange. We have used
the semi-quantitative technique of mRNA in situ hybridization, aided by
the use of specific antibodies for immunohistochemistry where
available, and by the use of placentas obtained from specific gene
knockout models. The null placentas have enabled us to more rigorously
test the specificity of the mRNAs and proteins that were detected. We
have determined that many of these factors are expressed more intensely
in the IPYS than in the trophoblasts (summarized in Table
1), a finding that may indicate the
importance of the IPYS in maternal-fetal calcium transfer. This led us
to hypothesize that abnormalities in calcitropic gene expression would
be present in placentas in which placental calcium transfer is
downregulated and upregulated. We tested this hypothesis by examining
placentas obtained from Pthrp-null fetuses (in which
placental calcium transfer is downregulated) and Pthr1-null fetuses (in which placental calcium transfer is upregulated). We found
that Pthrp-null placentas have less IPYS and reduced expression of calbindin-D9k mRNA and protein in the IPYS;
these findings are not present in Pthr1-null placentas.
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These results indicate that the columnar cells of the IPYS have the most intense expression (mRNA and protein) of Ca2+-ATPase and calbindin D9k, two of the components thought to be necessary for maternal-fetal calcium transfer. In addition, the IPYS expresses other calcitropic hormones and receptors more intensely than in the surrounding trophoblasts, including PTHrP adjacent to the PTH/PTHrP receptor, CaSR, and VDR; the IPYS also expresses calcitonin and the calcitonin receptor. The localization of PTHrP to the placenta is particularly relevant, because this peptide regulates placental calcium transfer in mice and sheep (1, 10, 35, 54). Expression of CaSR by the IPYS confers the capability of calcium sensing, which in turn might be required to regulate the synthesis and release of some of the hormones expressed in the IPYS and the rate of calcium transfer. This hypothesis is supported by our previous observation that Casr-null fetuses have a reduced rate of placental calcium transfer (33). The functional importance of IPYS expression of VDR, calcitonin, and the calcitonin receptor is less certain, given the published evidence that calcitriol and calcitonin may not be required for placental calcium transfer or fetal calcium homeostasis (reviewed in Ref. 34).
We found that the IPYS did not express 57-kDa calbindin,
-fetoprotein, nodal, proliferin, or placental lactogen. 57-kDa
calbindin is homologous to calreticulin, a protein present in the
endoplasmic reticulum, and its relevance to calcium transfer is less
certain. Downregulation of its expression with antisense technology
reduced calcium uptake (a first step in calcium transport) in a
choriocarcinoma cell line (21). A midmolecular fragment of
PTHrP (but not PTH or amino-terminal PTHrP) upregulated the expression
of 57-kDa calbindin in placental organ cultures and a choriocarcinoma
cell line (20). However, we observed no reduction in the
expression of 57-kDa calbindin in the Pthrp-null placenta,
suggesting that the PTHrP midmolecule may not be an important
physiological regulator of 57-kDa calbindin expression in mice. That
the IPYS does not express
-fetoprotein is a further indication that
the IPYS cells function differently than the yolk sac cells from which
they are derived. Similarly, the IPYS does not express genes that are
characteristic of specific trophoblast cell types, including nodal
(expressed in spongiotrophoblasts), proliferin, and placental lactogen
(expressed in giant trophoblasts). The specific markers of the IPYS
appear to be calcium-regulating proteins and receptors, as well as
platelet-derived growth factor receptor-
.
The observation that fewer sections of Pthrp-null placenta have IPYS suggests that the absence of PTHrP may impair formation or growth of the IPYS. Furthermore, because the IPYS appeared to be present in normal amounts in Pthr1-null sections, any action of PTHrP to regulate IPYS formation must be independent of the PTH/PTHrP receptor. Placental calcium transfer is regulated by the PTHrP midmolecule, independent of the PTH/PTHrP receptor (1, 10, 35, 54); the development and expansion of the IPYS may similarly require the PTHrP midmolecule. The reduced rate of placental calcium transfer in Pthrp-null fetuses may be partly due to the decreased volume of IPYS.
Apart from calbindin-D9k, the placental localization of calcium-binding proteins and calcitropic hormones has not been systematically studied in other species. However, it is evident that the localization of calbindin-D9k differs significantly among species, and this may indicate that calcium transfer occurs at different sites of the placenta within different species. In human and other primate placentas, calbindin-D9k is highly expressed in trophoblasts. As noted in this report and the published literature, in rodent placentas it is not the trophoblasts (which make up the bulk of the placenta) but the IPYS cells that express the highest levels (mRNA and protein) of calbindin-D9K during the time frame of rapid placental calcium transfer (4, 7, 13, 14). In the epitheliochorial placentas of sheep, cows, and goats, calbindin-D9k is mainly concentrated not in the trophoblasts of the placentome (which has the greater surface area for maternal-fetal exchange) but in a smaller structure, the interplacentomal trophoblast cells (i.e., the flat intercotyledonary trophoblasts) (43, 44, 52). Thus the hemochorial placentas of rodents and the epitheliochorial placentas of ruminants have concentrated the expression of calbindin-D9k in small structures within the placenta, as opposed to the trophoblasts, which make up the bulk of the placenta.
In both rodents and ruminants compared with humans, the amount of calcium transferred to the fetus from the mother is proportionately much greater, and the time frame in which the transfer can be accomplished is much shorter. A human fetus typically accumulates 21 g of calcium by term, requiring an average daily transfer of 200 mg calcium from the mother in the third trimester (16). A fetal rat accretes ~12 mg of calcium in the last 5 days of gestation, which requires that the mother provide 24 mg of calcium per day to a litter of 10 fetuses (11). A fetal lamb accretes 75 g of calcium by term, which requires that the mother provide 6 g of calcium per day to her typically twin lambs in late pregnancy (19). Rodents and ruminants may have developed these specialized areas within the placenta in order for calcium transfer to be facilitated.
The question remains as to how the IPYS might be involved in maternal-fetal calcium transfer, i.e., by what route(s) can the IPYS effect calcium transfer? The answer to this question may be apparent by considering the anatomic localization of the IPYS and the process of eggshell calcification in birds and (especially) egg-laying mammals.
First, as noted in this report, the IPYS abundantly expresses many of
the proteins and receptors that might be required for calcium transfer
to occur. Second, the IPYS is positioned between thin-walled fetal
vessels and maternal blood spaces at the fetal pole of the placenta.
Calcium and other substances might transfer from maternal to fetal
vessels directly across IPYS and the sinus of Duval. Third, the IPYS
communicates with the yolk sac cavity and, through the parietal yolk
sac layer that overlies the uterine decidua, it communicates with the
uterine epithelium that is not in contact with the placenta itself
(Fig. 1B). Calcium and other substances excreted or secreted
by uterine epithelium may, therefore, be transported by the parietal
yolk sac layer into the yolk sac cavity and thence to the IPYS.
Exuberant calcium secretion by uterine epithelial cells is a
fundamental step in the process of eggshell calcification in birds and
egg-laying mammals. Figure 5
schematically depicts these two postulated routes by which calcium may
reach the fetal circulation via the IPYS.
|
Thus the IPYS may have evolved to permit more rapid transfer of calcium
(and possibly other nutrients or minerals) by utilizing all of the
uterine lumen to secrete calcium into the yolk sac (and thence to the
IPYS and fetal circulation), instead of limiting maternal-fetal
exchange to the uterine-placental interface. As well, the IPYS may
provide a direct short-circuit between fetal and maternal circulations
across the sinus of Duval within the rodent placenta. Bruns et al.
(6) have previously proposed that the process of eggshell
calcification (calcium secretion by uterine epithelial cells) suggests
that one function of the IPYS is to take calcium from the uterine
epithelium and transfer it to the fetus. Therefore, it may be that
rodents have adapted a strategy of egg-laying animals to maximally
increase the surface area through which maternal-fetal calcium transfer
can occur in late gestation. If this hypothesis is correct, fetuses
that completely lack IPYS [platelet-derived growth factor
receptor--null fetuses (45)] should have reduced
placental calcium transfer and impaired skeletal mineralization near term.
In conclusion, our observation of intense expression of calcitropic
genes, calbindin-D9k, and Ca2+-ATPase
("calcium transfer machinery") in the IPYS of murine placenta implies function, and we speculate that one role of the IPYS is to
transfer calcium to the fetus. The concentrated expression of
calcitropic factors within the IPYS may enable calcium transfer to be
locally regulated (e.g., calcium sensing by the CaSR and stimulation of
active maternal-fetal calcium transfer by PTHrP), and the growth and
physical size of the IPYS may be controlled by some of these same
factors (e.g., PTHrP and platelet-derived growth factor receptor-).
The reduction in placental calcium transfer rate in
Pthrp-null fetuses may be a consequence of loss of the
tropic effect of PTHrP on growth of the IPYS and the local effect of
PTHrP to stimulate placental calcium transfer. Similarly, the reduced
placental calcium transfer of Casr-null fetuses may be the
result of the loss of calcium sensing within the IPYS. However, further
study is needed to confirm the role of the IPYS and to determine how
much calcium is transferred via the IPYS as opposed to trophoblasts. It
is evident that the IPYS is not simply a nonfunctional remnant of the
primitive yolk sac but is a functional structure whose importance
remains to be fully elucidated.
![]() |
ACKNOWLEDGEMENTS |
---|
Acknowledgment is made to Dr. Marie Demay for the Vdr-null mice, Dr. Jon Seidman for the Casr-null mice, and Dr. Robert F. Gagel for the Ct-null mice; Dr. Jane Moseley for critical review of the manuscript; Drs. John Harnett and Henry M. Kronenberg for their support and guidance; and Judy Foote for additional technical support.
![]() |
FOOTNOTES |
---|
This work was supported by a Scholarship award (MSH 35674) and an operating grant (MT-15439) (both to C. S. Kovacs) from the Medical Research Council of Canada (now Canadian Institutes of Health Research). Additional research support (to C. S. Kovacs) was obtained from the Medical Research Foundation, the Research and Development Committee, the Faculty of Medicine, and the Discipline of Medicine, all at Memorial University of Newfoundland.
Address for reprint requests and other correspondence: C. S. Kovacs, Faculty of MedicineEndocrinology, Memorial Univ. of Newfoundland, 300 Prince Philip Drive, St. John's, NF A1B 3V6, Canada
(E-mail: ckovacs{at}mun.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00369.2001
Received 13 August 2001; accepted in final form 14 November 2001.
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