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BOR - Papers in Press, published online ahead of print May 25, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042242
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BIOLOGY OF REPRODUCTION 73, 591–597 (2005)
DOI: 10.1095/biolreprod.105.042242
© 2005 by the Society for the Study of Reproduction, Inc.

Multidrug Resistance Phosphoglycoprotein (ABCB1) in the Mouse Placenta: Fetal Protection1

Grazyna M. Kalabis 3, Alice Kostaki 3, Marcus H. Andrews 3, Sophie Petropoulos 3, William Gibb 6, and Stephen G. Matthews 2,3,4, 5  

Departments of Physiology,3 Obstetrics and Gynaecology,4 Medicine,5 University of Toronto, Toronto, Ontario, Canada M5S 1A8 Departments of Obstetrics and Gynaecology and Cellular and Molecular Medicine,6 University of Ottawa, Ottawa, Ontario, Canada K1H 8M5


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The multidrug resistance phosphoglycoprotein ATP-binding cassette subfamily B (ABCB1) actively extrudes a range of structurally and functionally diverse xenobiotics as well as glucocorticoids. ABCB1 is present in many cancer cell types as well as in normal tissues. Although it has been localized within the mouse placenta, virtually nothing is known about its regulation. In the mouse, two genes, Abcb1a and Abcb1b, encode ABCB1. We hypothesized that there are changes in placental Abcb1a and Abcb1b gene expression and ABCB1 protein levels during pregnancy. Using in situ hybridization, we demonstrated that Abcb1b mRNA is the predominant placental isoform and that there are profound gestational changes in the expression of both Abcb1a and Abcb1b mRNA. Placentas from pregnant mice were analyzed between Embryonic Days (E) 9.5 and 19 (term ~ 19.5d). Abcb1b mRNA was detected in invading trophoblast cells by E9.5, peaked within the placental labyrinth at E12.5, and then progressively decreased toward term (P < 0.0001). Abcb1a mRNA, although lower than that of Abcb1b at midgestation, paralleled changes in Abcb1b mRNA. Changes in Abcb1 mRNA were reflected by a significant decrease in ABCB1 protein (P < 0.05). A strong correlation existed between placental Abcb1b mRNA and maternal progesterone concentrations, indicating a potential role of progesterone in regulation of placental Abcb1b mRNA. In conclusion, there are dramatic decreases in Abcb1a and Abcb1b mRNA and in ABCB1 at the maternal-fetal interface over the second half of gestation, suggesting that the fetus may become increasingly susceptible to the influences of xenobiotics and natural steroids in the maternal circulation.

pregnancy, early development, placenta, placental transport, syncytiotrophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The multidrug resistance phosphoglycoprotein ATP-binding cassette subfamily B (ABCB1) belongs to a superfamily of ATP-dependent transporters originally discovered in tumor cells [1], and has been shown to be involved in the development of tumor resistance to chemotherapeutic agents. ABCB1 actively transports a wide range of other structurally diverse hydrophobic and amphipathic substrates, including antiretroviral HIV drugs (e.g., saquinavir), herbicides and pesticides (e.g., ivermectin), cardiac glycosides (e.g., digoxin), analgesics (e.g., morphine), and epileptic drugs (e.g., phenytoin), as well as synthetic and endogenous steroids (e.g., dexamethasone, cortisol) [2–8].

More recently, ABCB1 has been identified in a number of normal tissues, including the intestines, kidney, liver, and adrenal, where it plays an important role in limiting absorption and/or facilitating excretion of a wide range of substrates [9–11]. ABCB1 has also been detected in tissues with barrier functions, such as the endothelial cells of the blood-brain and blood-testes barriers, as well as the placental syncytiotrophoblasts [12–14]. It has been shown that ABCB1 in these tissues is responsible for preventing transfer of substrates into the brain and testis as well as transplacental transfer from mother to fetus [3, 8].

In the human, ABCB1 is encoded by a single Abcb1 gene, whereas in the mouse, two closely located genes, Abcb1a and Abcb1b, encode ABCB1A and ABCB1B isoforms of the protein [4, 15]. The two mouse isoforms of ABCB1 exhibit specific but overlapping substrate specificity and organ distribution [10, 16]. Together, the distribution and function of ABCB1A and ABCB1B is similar to that of human ABCB1 encoded by the single human Abcb1 gene [15].

The placenta serves a critical function in the exchange of nutrients, hormones, and other molecules essential for the maintenance of pregnancy and normal fetal development. It has also been implicated in the protection of the developing fetus from potentially detrimental environmental xenobiotics. Although early studies failed to detect ABCB1 in the placenta [11], others have described ABCB1 expression in the placental trophoblast [9, 12, 17]. Mice in which both Abcb1a and Abcb1b had been knocked out exhibited dramatically elevated placental penetration of digoxin, saquinavir, and paclitaxel [8] and increased sensitivity to avermectin-induced teratogenicity [17]. This suggests that placental ABCB1 is involved in limiting transplacental transfer of teratogens and drugs from mother to fetus, effectively reducing fetal susceptibility to these factors.

Despite these important findings, the developmental expression of Abcb1a and Abcb1b in the placenta through gestation and their contribution to fetal protection remains largely unknown. Studies of ABCB1 regulation in other tissues [18, 19] and cell lines [20–22] indicate that Abcb1 genes can be positively regulated by progesterone. The latter decreases rapidly in the maternal circulation in late gestation [23, 24]. In the present study, we hypothesized that 1) both Abcb1a and Abcb1b are expressed in the placental trophoblasts and that levels of mRNA and protein change with the progression of gestation; if progesterone positively regulates placental Abcb1 gene expression, a reduction in expression would be predicted; and 2) Abcb1a and Abcb1b gene expression will be differentially expressed in the placenta throughout gestation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Female FVB mice (Taconic, Germantown, NY) were mated in our colony. Two Abcb1a and Abcb1b double knockout mice (Taconic) were bred to act as negative controls. Pregnancy was defined after presence of vaginal plug and designated as Embryonic Day (E) 0.5 (average gestation period ~ 19.5 days). Mice were killed by cervical dislocation at E9.5, E12.5, E15.5, E18.5, and E19. Trunk blood was collected from the dam immediately following cervical dislocation, and plasma was separated and frozen. These studies were performed using protocols approved by the Animal Care Committee at the University of Toronto and in accordance with the Canadian Council for Animal Care.

The relative expression of placental Abcb1a and Abcb1b was evaluated in tissue derived from midgestation (E9.5) to near term (E19). At E9.5, the entire yolk sac (the fetus and the placenta) was carefully excised to exclude surrounding uterine tissue. For E12.5 through E19, the placenta was separated from the fetus and hemisected such that the coronal cut traversed fetal to maternal surfaces. Tissues were rapidly frozen (–80°C). A minimum of six dams were used per gestational age (average litter size = 8).

In Situ Hybridization

The method for in situ hybridization has been described previously [25]. Briefly, placental cryosections (10µm) were mounted onto poly-L–-lysine coated slides, dried, and fixed in paraformaldehyde (4%). Antisense oligonucleotide probes were labeled using terminal deoxynucleotidyl transferase (Gibco, Burlington, ON, Canada) and [35S]-dATP (1300 Ci/ mmol; Perkin Elmer, Woodbridge, ON, Canada) to a specific activity of 1.0 x 109 cpm/mg. Labeled probes in hybridization buffer (200 µL) were applied to slides at a concentration of 1.0 x 105 cpm/ml. Separate oligonucleotide (45mer) antisense probe complimentary to bases 2348–2392 of mouse Abcb1a (GenBank accession no. NM011076) and bases 295–339 of Abcb1b mRNA (GenBank accession no. NM011075) were designed to differentiate between Abcb1a and Abcb1b mRNA. Slides were incubated overnight in a moistened chamber (42.5°C). After washing in 1x SSC (20 min at 23°C, then 35 min at 55°C), slides were rinsed, dehydrated in ethanol, dried, and exposed to autoradiographic film (Biomax MR, Kodak, Perkin Elmer) for 21 days. Films were developed using an automatic processor. Specificity of the probes was determined using a sense probe.

The relative expression (relative optical density [ROD]) of Abcb1a and Abcb1b mRNA was determined using a computerized image analysis system (MCID; Imaging Research, St Catherine's, ON, Canada). Several placentas per litter, with a minimum of six litters per gestational age, were analyzed, and a litter mean calculated. All tissue sections were processed simultaneously to allow for direct comparison between gestational groups. 14C-labeled standards were used to ensure that image analysis was undertaken within the linear range of the autoradiographic film, as described previously [25]. The Abcb1a and Abcb1b mRNA expression was further visualized using high-resolution silver grain emulsion autoradiography. Following in situ hybridization, the slides were dipped into photographic emulsion (LM; Amersham Pharmacia) and allowed to expose for 8 wk.

Immunohistochemistry

Frozen cryosections mounted onto poly-L-lysine coated slides were postfixed in acetone (10 min) and dried. Fixed sections were incubated in hydrogen peroxide solution (0.03% in PBS, pH 7.5, 30 min) and then rinsed in PBS (5 min). Immunohistochemistry was performed using the anti-rabbit Vectastain Elite ABC Kit (Vector Laboratories). Briefly, sections were incubated in blocking serum at room temperature (1 h) and then rinsed in PBS. Sections were incubated overnight at 4°C with H-241 rabbit polyclonal primary antibody specific for ABCB1 protein (1:50 dilution, sc-8313; Santa Cruz Biotechnology). ABCB1 was visualized using diaminobenzidine, and sections were dehydrated and counterstained with hematoxylin. The specificity of the antibody was confirmed by incubation of tissues known to contain discrete expression of ABCB1 (mouse brain, liver, kidney, adrenal gland, intestines; data not shown). Negative controls included incubation with preimmune serum as well as analysis in Abcb1a and Abcb1b knockout animals.

Western Blot Analysis

ABCB1 protein expression from E12.5 to E19 was assessed by Western blotting, as described previously [26]. The yolk sac derived at E9.5 was not analyzed for ABCB1 because of difficulty in accurately isolating placenta from the embryo. Briefly, frozen placenta was homogenized in ice-cold RIPA buffer, centrifuged (4°C, 10 000 x g, 10 min) and the resulting supernatant recentrifuged. The supernatant was assayed for total protein using the method described by Bradford [27]. Laemmli sample buffer (15 µl; Sigma-Aldrich, Oakville, Canada) was added to each sample (50 µg protein) and the protein was denatured (5 min at 95°C). The samples were subjected to SDS-PAGE electrophoresis (8% resolving polyacrylamide gel) and transferred electrophoretically to a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, Canada). Nitrocellulose membranes were blocked overnight (4°C) in skim milk (5% wt/vol of PBS with Tween 20 [PBS-T]). Membranes were washed with PBS-T and then incubated with C219 mouse monoclonal antibody (1:200 dilution, 1 h at 23°C; BP1199; ID Labs, London, ON, Canada) in 5% skim milk in PBS-T. The membranes were then washed in PBS-T and incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:7000 dilution, 1 h, 23°C; NEN, Boston, MA) followed by Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer). The bands were visualized by exposure to Kodak Blue X-OMAT film (Perkin Elmer) for 1 min. Membranes were stripped in Restore Western Blot Stripping buffer (30 min, 23°C; Pierce, MJS Bioynx, Mississauga, ON, Canada) and reanalyzed for ß subunit of G protein (Gß) (1:3000 dilution, rabbit polyclonal sc-8261; Santa Cruz Biotechnology). The relative optical density of the bands was measured using computerized image analysis and were standardized against the Gß signal. All Western analysis was performed at least two times for each placenta, data were pooled, and a mean ROD for ABCB1 value was derived for each gestational age. There are no antibodies currently available that differentiate between the ABCB1A and ABCB1B isoforms; thus, Western analysis represents the levels of both isoforms. The specificity of the antibody that detects the 170-kDa band was confirmed by preabsorption of the primary antibody with excess protein epitope (custom-synthesized by Sigma-Aldrich). The latter was designed on the basis of previous preabsorption studies [28].

Progesterone Assay

Maternal plasma progesterone concentrations were determined by radioimmunoassay using a commercially available kit (ICN, Costa Mesa, CA). For each pregnancy, maternal plasma progesterone concentrations were correlated with placental Abcb1a and Abcb1b mRNA levels.

Statistical Analysis

Group data are presented as means ± SEM and were statistically analyzed using multiple ANOVA followed by the Duncan method of post hoc comparison (Statistica; Statsoft Inc., Tulsa, OK). The correlation between maternal progesterone and Abcb1a and Abcb1b mRNA levels was also determined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Although both Abcb1a and Abcb1b mRNA are expressed in the mouse placenta, Abcb1b mRNA was the predominant isoform. This was especially evident at E9.5 through E15.5 (Figs. 1 and 2). Levels of Abcb1b mRNA were high at midgestation, with the highest level observed at E12.5. However, by E15.5 there was a significant decline (P < 0.05) in Abcb1b mRNA expression, as well as a reduction of Abcb1a mRNA. This pattern continued with the progression of gestation such that by E18.5 both Abcb1a and Abcb1b mRNA were dramatically reduced; this was particularly striking for Abcb1b mRNA (P < 0.001). Levels remained low at E19 and were comparable to those observed in the Abcb1a- and Abcb1b-deficient mice (Fig. 1).



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  		FIG. 1. Representative images of Abcb1a (A, C, E, and G) and Abcb1b mRNA (B, D, F, and H) expression in the mouse placenta at Embryonic Day (E) 9.5 (A and B), E12.5 (C and D), E15.5 (E and F) and E18.5 (G and H). Abcb1b mRNA expression in Abcb1a- and Abcb1b-deficient mice is shown in K. In situ hybridization using a sense control probe is shown in L. Note that at E9.5 the images show the whole conceptus, including the developing placenta and the fetus within the yolk sac. Representative high-resolution silver grain emulsion radiographs of placental Abcb1b mRNA within the interface are shown for E9.5 (I) and E12.5 (J). White arrows point to placental-fetal interface. The black arrow points to the labyrinth within the placenta. Bar = 1 mm (A) and 50 µm (I). Dec, Decidua. Exposure time: film, 21 days; emulsions, 8 wk



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  		FIG. 2. Placental Abcb1a (A) and Abcb1b (B) mRNA levels at Embryonic Day (E) 9.5 (n = 7 dams), E12.5 (n = 6), E15.5 (n = 6), E18.5 (n = 7) and E19 (n = 6). Bar represents mean ± SEM relative optical density (ROD). *P < 0.05, **P < 0.001 vs. E12.5. Note the difference in the y-axis scale

At E9.5, expression of Abcb1a and Abcb1b mRNA was selectively localized within a thin layer of cells at the placental-fetal interface (Fig. 1, A and B). This represents an area of chorionic stem cells that will differentiate into trophoblast cells of the labyrinth. Silver emulsion autoradiographs confirm that the signal was concentrated within select cells of this placental-fetal interface (Fig. 1I). At E12.5, the area of expression dramatically expanded (Fig. 1, C and D), consistent with the pronounced development of the placental labyrinth between E9.5 and E12.5 [29, 30]. This specific localization of the signal within the labyrinth was further illustrated by silver emulsion autoradiography (Fig. 1J); once again expression of Abcb1b mRNA was significantly more pronounced than for Abcb1a mRNA (Fig. 1, C and D). Differences between the two isoforms remained pronounced at E15.5 (Fig. 1, E and F), but levels decreased significantly at E18.5 (Fig. 1, G and H) to those observed in Abcb1a- and Abcb1b-deficient mice (Fig. 1K).

The pattern of ABCB1 protein expression throughout gestation was consistent with that described for mRNA. After testing various antibodies in a number of ABCB1-positive tissues, we found that the H-241 rabbit polyclonal antibody provided optimal staining under our experimental conditions. At E9.5, the protein is shown to be specifically localized within cells at the placental-fetal interface (Fig. 3, A and B). With progression of gestation, at E12.5 and E15.5 (Fig. 3, C–E), localization of ABCB1 became restricted to the luminal membranes of the syncytial trophoblast layer (Fig. 3D) facing the maternal circulation. There was a dramatic decrease in the intensity of staining at the end of gestation (Fig. 3G), again consistent with both the pattern and intensity of declining mRNA expression. There was no visible staining in the placental labyrinth of ABCB1 deficient mice (Fig. 3H).



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  		FIG. 3. Representative images of placental ABCB1 protein following immunohistochemical staining using H-241 polyclonal antibody at Embryonic Day (E) 9.5 (A and B), E12.5 (C and D), E15.5 (E) and E18.5 (G). Images of control placenta incubated with preimmune serum (F) and placenta from Abcb1a- and Abcb1b-deficient mice (H). T, Trophoblast stem cells within the chorion; Dec, decidua; L, labyrinth; FC, fetal capillary; GC, giant cells. Black arrow points to the syncytial trophoblast cell layer; arrowhead points to the labyrinth-decidua interface. Bar = 50µm (A) and 17µm (B)

The level of ABCB1 protein expression was quantified through Western immunoblotting using C219 mouse monoclonal antibody previously reported to specifically recognize ABCB1 [31, 32]. We confirmed the specificity of this antibody through preincubation with the peptide epitope and prevention of binding at 170 kDa (Fig. 4A). As identified with in situ hybridization and immunohistochemistry, there was a significant decline in ABCB1 protein expression from midgestation to the end of gestation (Fig. 4).



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  		FIG. 4. A) Example of Western blot of placental ABCB1 and Gß protein levels at Embryonic Day (E) 12.5 (n = 6), E15.5 (n = 6), E18.5 (n = 6) and E19 (n = 5) using C219 antibody. Pre, Preabsorption of C219 prevented antibody binding. B) Relative levels of ABCB1 expressed as a ratio to Gß at each of the gestational levels. Each bar represents mean ± SEM ABCB1/Gß ratio. *P <0.05 vs. E12.5

The mean maternal plasma progesterone concentration was highest at midgestation (E9.5) but began to fall at E15.5 with a significant decline at E18.5 (P < 0.0001; Fig. 5). There was a highly significant positive correlation between Abcb1b mRNA expression and maternal plasma progesterone concentration (F1,101 = 54.5; P < 0.0001; r = 0.5921). The correlation between maternal progesterone concentrations and Abcb1a mRNA was not statistically significant.



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  		FIG. 5. Maternal plasma progesterone levels at Embryonic Day (E) 9.5 (n = 7 dams), E12.5 (n = 6), E15.5 (n = 6), E18.5 (n = 7) and E19 (n = 6). Each bar represents the mean ± SEM expressed as ng/ml. *P < 0.05, **P < 0.001 vs. E9.5


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to have determined the development of the placental ABCB1 system in the mouse. Throughout gestation the placenta plays a critical role in regulating the exchange of various substances between the maternal and fetal circulation, and placental ABCB1 has been shown to be an integral component of this barrier. We have demonstrated that placental expression of Abcb1a and Abcb1b mRNA and associated ABCB1 protein dramatically decreases from midgestation to the end of gestation. Although both Abcb1a and Abcb1b are expressed, the predominant isoform expressed is Abcb1b mRNA. The highest expression of Abcb1a and Abcb1b mRNA, and of ABCB1 protein, occurs at E12.5; expression begins to decline at E15.5 such that by E18.5 there is a highly significant decrease in both Abcb1a and Abcb1b expression. This corresponds to decreasing levels of progesterone within the maternal plasma.

A limited number of studies have attempted to define the functional importance of ABCB1 within the placenta. These have focused on one time point and have provided somewhat contradictory results [8, 17, 33, 34]. The present study is the first to determine the developmental profile of Abcb1a and Abcb1b mRNA and ABCB1 expression in the placenta throughout gestation in the mouse. Studies using mice in which Abcb1a, or Abcb1a and Abcb1b, are absent have suggested that the fetuses of these animals are more susceptible to the adverse effects of xenobiotics [8], including teratogens [17]. In the human, ABCB1 has been localized to the luminal membrane of the placental syncytiotrophoblast cells [12–14, 35]. The mouse placenta is trichorial, consisting of three layers of trophoblasts [36]. The syncytiotrophoblast layer II, which is the continuous syncytial layer in contact with maternal blood, shows staining for ABCB1 (Fig. 3D).

Embryonic Day 9.5 represents the stage in mouse placentation during which the trophoblast stem cells in the chorion begin to differentiate into trophoblasts within the labyrinth, the site of exchange between the maternal and fetal circulations [29, 30]. Using immunohistochemistry and in situ hybridization followed by high resolution silver grain emulsion autoradiography, we have demonstrated that it is within this region that Abcb1a and Abcb1b mRNA, as well as ABCB1, are localized at high levels. At E9.5, this region represents the primary interface between mother and fetus. As gestation progresses to E12.5, the levels of Abcb1a and Abcb1b mRNA, as well as ABCB1, are elevated. There is also expansion of the area expressing Abcb1a and Abcb1b mRNA, and this is consistent with the structural and functional development of the labyrinth. At this stage, the expression of the Abcb1a and Abcb1b mRNA and ABCB1 protein is localized to the syncytialized layer of trophoblast cells in the labyrinth. At E15.5 there is a significant decline in both Abcb1b mRNA and ABCB1 expression, though levels remain relatively high. The high levels of Abcb1b mRNA and ABCB1 at this time corroborate a previous study that demonstrated a significant difference in fetal transfer of ABCB1 substrates (saquinavir, digoxin, and paclitaxel) between mice deficient in Abcb1a and Abcb1b and their wild-type counterparts at E15 [8].

The dramatic decrease in both Abcb1a and Abcb1b mRNA between E15.5 and E18.5 is paralleled by a decrease in ABCB1. This decline is consistent with studies that demonstrated little difference in the transplacental transfer of digoxin between Abcb1b-deficient and wild-type mice at E17 [33]. The dramatic decline in Abcb1 mRNA isoforms that we have identified during late gestation in the mouse placenta parallels recent findings that Abcb1 mRNA and ABCB1 protein expression in the human placenta decreases at term (Sun et al., unpublished results).

The finding that ABCB1B is the predominant isoform within the mouse placenta is consistent with previous suggestions that this isoform predominates in tissues with endocrine function [7, 37, 38]. Indeed, high levels of ABCB1B have been demonstrated in tissues such as adrenal gland and gravid uterus, where it has been shown to be regulated, at least in part, by progesterone [20]. Although ABCB1B predominates in the placenta, ABCB1A isoform may also play a role in transplacental transfer of xenobiotic agents. Utilizing the CF-1 mouse, in which a subgroup carries a natural mutation of exon 23 in the Abcb1a gene, Lankas and colleagues demonstrated increased teratogenic effects of avermectin in mice carrying the mutation compared to normal CF-1 mice [17]. Evidence indicates that there are subtle differences in substrate affinity and the rate of substrate efflux between ABCB1A and ABCB1B [15]. The functional significance of such differences in isoform expression and activity within the placenta remains to be determined.

The significant reduction in ABCB1 that we have identified in the mouse placenta and in the human placenta (Sun et al., unpublished results), combined with the functional studies indicating that placental ABCB1 actively prevents transplacental transfer of clinically important drugs, has tremendous implications for both maternal and infant health. Importantly, recent studies interrogating the importance of ABCB1 expression at the blood-brain barrier have determined that endogenous (cortisol and corticosterone) and exogenous (dexamethasone) glucocorticoids are also substrates for ABCB1 [5, 39–42]. Transfer of endogenous and exogenous glucocorticoids into the adult brain is significantly elevated in Abcb1a knockout, and in Abcb1a and Abcb1b double knockout mice. In the case of cortisol and corticosterone, there is a 4.6- and 2-fold elevation in transfer into the brain in Abcb1a and Abcb1b double knockout mice [40]. Data would suggest that the protein encoded by Abcb1b, but not Abcb1a, can transport corticosterone, although both can transport cortisol. This clearly has implications for the mouse placenta, in which we have shown Abcb1b to predominate. Maternal plasma concentrations of corticosterone are several times higher than those in the fetal circulation, and it is important for the fetus to be protected from high concentrations of glucocorticoids [43]. It has been elegantly shown that placental 11ß-hydroxysteroid dehydrogenase (11ß-HSD) Type 2 plays an important role in preventing maternal glucocorticoids from entering the fetus [44] and decreases significantly at term [45, 46]. It is likely that placental ABCB1 also plays an important role in excluding maternal glucocorticoid from the fetus. Such a role is also likely in the human where cortisol is the primary circulating glucocorticoid. The decrease in placental Abcb1a and Abcb1b toward the end of gestation may be important in facilitating transplacental transport of glucocorticoids that are known to be necessary for normal fetal development and birth. The relative importance of ABCB1 and 11ß-HSD Type 2 enzyme in regulation of transplacental transfer of endogenous glucocorticoids in the mouse and human, and the potential ramification of parallel decreases toward the end of gestation in terms of fetal development, warrants further investigation.

There are many circumstances (cancer, HIV infection, preterm labor) during pregnancy that require the administration of drugs to either the mother or the fetus, and establishing the benefit to risk ratio for both mother and fetus is often difficult. For example, women at risk of premature delivery are treated with synthetic glucocorticoids (dexamethasone or betamethasone) to mature the fetal lungs. Because of the difficulty in diagnosing preterm labor and the effectiveness of the treatment, approximately 7% of all pregnant women will receive at least one course of synthetic glucocorticoids between 24 and 34 wk of gestation [47]. ABCB1 is effective at preventing synthetic glucocorticoids entering the syncytiotrophoblast and the fetus [48]. Therefore, it is highly likely, given our findings in mouse and human pregnancy, that placental ABCB1 actively inhibits transplacental transfer of a number of drugs, and that the degree of fetal protection will depend on the timing of administration.

In the present study, we have shown a significant correlation between maternal plasma progesterone concentration and placental Abcb1b mRNA, but not Abcb1a mRNA, levels. Progesterone has been shown to interact with, but not be transported by, ABCB1 [5, 22]. In vivo [18] and in vitro [21, 49] studies suggest that it may play a role in the regulation of ABCB1 expression. Progesterone has been shown to increase Abcb1b mRNA expression in the uterus of ovariectomized mice [20]. Further, studies have demonstrated that the promoter region of the Abcb1b gene is responsive to the A form of the progesterone receptor [21]. Our studies are consistent with the possibility that progesterone maintains placental Abcb1b mRNA, though further studies are required to investigate the association. Little is currently known about regulatory control of expression of ABCB1 in normal tissues, and it is quite possible that placental Abcb1a and Abcb1b are independently regulated.

Virtually nothing is known with regard to expression of ABCB1 in the developing fetus. It is possible that the expression of ABCB1 increases in fetal tissues in late gestation, conferring protection at a local rather than a placental level. Further, other fetal protective mechanisms, such as the cytochrome P-450 enzyme system and Phase II enzymatic pathways of xenobiotic metabolism, which are known to increase in late gestation [50], may take over the role of fetal protection. Finally, it is also possible that another recently discovered placental efflux transporter, breast cancer-resistant protein (ABCG2), becomes important in late gestation. This transporter has overlapping substrate specificity to ABCB1 and has recently been identified in the human and mouse placenta [51].

In conclusion, our present studies indicate that ABCB1 is expressed at high levels in the syncytial trophoblasts at early stages of placental formation in the mouse and that the Abcb1b mRNA isoform predominates. There is a significant reduction in both placental Abcb1a and Abcb1b mRNA in late gestation, and this is consistent with our recent findings in the human placenta. These dramatic decreases in the expression of Abcb1a and Abcb1b and associated proteins at the primary maternal-fetal interface may indicate that the fetus becomes increasingly susceptible to the influences of toxic substances and therapeutic agents, as well as to a number of natural steroids present in the maternal circulation. Further functional studies are clearly warranted. In light of the large number of substances that are actively excluded from the fetus by ABCB1, understanding its function and regulation may aid in the design of both maternal and fetal therapeutic strategies in pregnancy.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. John Kingdom, Yong Lu, and Meihua Sun for their expert suggestions and advice with immunohistochemistry. We wish to thank Sonja Banjanin and Elaine Setiawan for their technical assistance.


   FOOTNOTES
 
1 Supported by the Canadian Institutes of Health Research (FRN-57746 to S.G.M. and W.G.). Back

2 Correspondence: Stephen G. Matthews, Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. FAX: 416 978 4940; stephen.matthews{at}utoronto.ca Back

Received: 30 March 2005.

First decision: 26 April 2005.

Accepted: 25 May 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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