Prominent expression of xenobiotic efflux transporters in mouse extraembryonic fetal membranes compared with placenta

Abstract

Fetal exposure to xenobiotics can be restricted by transporters at the interface between maternal and fetal circulation. Previous work identified transporters in the placenta; however, less is known about the presence of these transporters in the fetal membranes (i.e., yolk sac and amniotic membranes). The purpose of this study was to quantify mRNA and protein expression of xenobiotic transporters in mouse placenta and fetal membranes during mid to late gestation. Concepti (placenta and fetal membranes, gestation day 11) or placenta and fetal membranes (gestation days 14 and 17) were collected from pregnant mice and analyzed for expression of multidrug resistance-associated proteins (Mrps), multidrug resistance proteins (Mdrs), multidrug and toxin extrusion proteins (Mates), breast cancer resistance protein (Bcrp), and organic anion-transporting polypeptides (Oatps). Maternal liver and kidneys were also collected at day 14 for mRNA and immunohistochemical analysis. mRNA expression of Mrp, Mdr, Bcrp, Mate-1, and Oatp isoforms was detected at day 11. The uptake carriers Oatp2a1, 3a1, 4a1, and 5a1 showed placenta-predominant expression. At days 14 and 17, fetal membranes expressed higher mRNA levels of the efflux transporters Mrp2 (7-fold), Mrp4 (5-fold), Mrp5 (3-fold), Mrp6 (12-fold), Bcrp (2-fold), and Mate-1 (7-fold) than placenta. Western blot analysis of Mrp2, Mrp4, Mrp6, and Bcrp confirmed higher expression in fetal membranes. Immunostaining revealed apical (Mrp2 and Bcrp) and basolateral (Mrp4, 5, and 6) cellular localization in epithelial cells of the yolk sac. In conclusion, xenobiotic transporters in the fetal membranes may provide an additional route to protect the fetus against endogenous chemicals and xenobiotics.

Figure 1. Tissue expression of uptake Oatp transporters

Total RNA was isolated from placenta, fetal membranes, maternal liver and kidney on day 14 of gestation. RNA was analyzed by bDNA assay for mRNA expression of Oatp1a5, 2a1, 2b1, 3a1, 4a1, and 5a1. The previous nomenclature for Oatp isoforms is presented in parentheses. The data are presented as mean relative light units (RLU) per 4 μg total RNA ± SE (n = 3).

Figure 2. Tissue expression of efflux Mrp, Mdr, Bcrp, and Mate transporters

Total RNA was isolated from placenta, fetal membranes, maternal liver, and kidney on day 14 of gestation. RNA was analyzed by bDNA assay for expression of Mrp1−6, Mdr1a, 1b, Bcrp, and Mate-1. The data are presented as mean relative light units (RLU) per 4 μg total RNA ± SE (n = 3).

Figure 3. Gestational expression of uptake Oatp transporters in placenta and fetal membranes

Total RNA was isolated from concepti (day 11) and placenta and fetal membranes (days 14 and 17). RNA was analyzed by bDNA assay for expression of Oatp1a5, 2a1, 2b1, 3a1, 4a1, and 5a1. The previous nomenclature for Oatp isoforms is presented in parentheses. The data are presented as mean relative light units (RLU) per 4 μg total RNA ± SE (n = 3−5). Asterisks (*) represent statistical differences (p<0.05) between placenta and fetal membranes. Daggers (†) represent statistical differences (p<0.05) compared to Day 14.

Figure 4. Gestational expression of efflux Mrp, Mdr, Bcrp, and Mate transporters in placenta and fetal membranes

Total RNA was isolated from concepti (day 11) and placenta and fetal membranes (days 14 and 17). RNA was analyzed by bDNA assay for expression of Mrp1−6, Mdr1a, Mdr1b, Mate-1, and Bcrp. The data are presented as mean relative light units (RLU) per 4 μg total RNA ± SE (n = 3−5). Asterisks (*) represent statistical differences (p<0.05) between placenta and fetal membranes. Daggers (†) represent statistical differences (p<0.05) compared to Day 14.

Figure 5. Protein expression of efflux transporters in placenta and fetal membranes

Western immunoblots were performed using homogenates (40 μg protein/well) from placenta and fetal membranes at 14 and 17 days of gestation. The data are presented as individual blots (A) and as mean trace density (O.D. x mm) ± S.E (n = 3) (B) for Mrp1, Mrp2, Mrp4, Mrp5, Mrp6, Bcrp, and Pgp. Blots were probed for β-actin staining to confirm equal protein loading. Due to differences in relative expression of β-actin between placenta and fetal membranes, transporter western blot data was not normalized to β-actin. Asterisks (*) represent statistical differences (p<0.05) between placenta and fetal membranes. Daggers (†) represent statistical differences (p<0.05) compared to Day 14.

Figure 6. Immunofluorescent detection of Mrp4, Mrp5, and Mrp6 transporters in fetal membranes

Indirect immunofluorescence against Mrp4, Mrp5, Mrp6 (green), and actin cytoskeleton (red) was conducted on day 14 fetal membrane cryosections. Images are shown at 40X (A, F, K) magnification. Cropped images were enlarged and provided as inserts (B, G, L). Actin staining is shown at high magnification (C, H, M). Sections were mounted in Prolong Gold containing DAPI for nuclear staining (blue). Maternal liver (D, I, N) and kidney (E, J, O) cryosections from gestational day 14 were stained for Mrp4, Mrp5, and Mrp6 proteins. Bar, 50 μm.

Figure 7. Immunofluorescent detection of Bcrp and Mrp2 transporters in fetal membranes

Indirect immunofluorescence against Bcrp, Mrp2 (green), and actin cytoskeleton (red) was conducted on day 14 fetal membrane cryosections. Images are shown at 40X (A, F) magnification. Cropped images were enlarged and provided as inserts (B, G). Actin staining is shown at high magnification (C, H). Sections were mounted in Prolong Gold containing DAPI for nuclear staining (blue). Maternal liver (D, I) and kidney (E, J) cryosections from gestational day 14 were stained for Bcrp and Mrp2 proteins. Bar, 50 μm.

Figure 8. Immunofluorescent co-localization analysis of Mrp2/Bcrp and Mrp2/Mrp4 proteins in fetal membranes

Double indirect immunofluorescence against Bcrp (green, A) and Mrp2 (red, B) proteins was performed on day 14 fetal membrane cryosections. Images A and B were merged to create image C demonstrating co-localization of Bcrp and Mrp2 at the apical surface. Double indirect immunofluorescence against Mrp4 (green, D) and Mrp2 (red, E) proteins was similarly performed on day 14 fetal membrane cryosections. Images D and E were merged to create image F demonstrating distinct localization of Mrp4 and Mrp2 to the basolateral and apical surfaces, respectively. 40X magnification; Bar, 50 μm.

Figure 9. Summary of xenobiotic transporter expression in mouse placenta and fetal membranes

The mouse placenta expresses a number of uptake (Oatp1a5, 2a1, 3a1, 4a1, 5a1) and efflux (Mrp1, Mrp5, Mdr/Pgp, Bcrp) transporters. Kalabis et al. demonstrated localization of Bcrp to the apical surface of the syncytiotrophoblasts in mouse placenta (Kalabis et al., 2007). Localization of Oatp, Mrp and Mdr transporters in mouse placenta is currently unknown. The amniotic membrane and inverted yolk sac enclose the fetus and are together known as the fetal membranes. Endoderm-derived epithelial cells of the yolk sac express transporters on the apical/maternal face (Mrp2 and Bcrp) and the basolateral/fetal face (Mrp4, 5, 6) (see insert). Mdr/Pgp, Mate-1, and Oatp1a5 transporters are also detected in the fetal membranes, although their cellular localization is unknown.