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a School of Anatomy and Human Biology and The Western Australian Institute for Medical Research, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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leptin, leptin receptor, placenta, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nulliparous albino Wistar rats aged between 9 and 12 wk were obtained from the Animal Resources Centre (Murdoch, Australia) and maintained under controlled conditions as previously described [16]. Rats were mated overnight, and the day on which spermatozoa were present in vaginal smear was designated gestational Day 1. Rats in this colony normally deliver on Day 23. All procedures involving animals were conducted only after approval by the Animal Ethics Committee of The University of Western Australia.
Experimental Glucocorticoid Manipulations
Increased glucocorticoid exposure was achieved both systemically by maternal dexamethasone treatment and locally within the placenta by treatment with carbenoxolone, an inhibitor of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), from Day 13 to Day 22 of pregnancy. Dexamethasone acetate was administered in drinking water (1 µg/ml) and carbenoxolone was administered twice daily (10 mg in 4% ethanol-saline, 0.1 ml s.c. injection at 0700 and 1730 h) over the same period. We have previously shown that these treatments reduce birthweight by 27% and 8%, respectively [17]. To reduce glucocorticoid exposure, mothers were treated with the 11ß-hydroxylase inhibitor, metyrapone, administered in physiological saline (in place of drinking water) at a concentration of 500 µg/ml; we have previously shown that this treatment enhances fetal and placental growth [16, 17].
Tissue Collection
On the day of tissue collection (Day 16 or Day 22), rats were anesthetized with halothane/nitrous oxide and uterine horns were exposed. Whole placentas were removed and immediately immersed in ice-cold MB Histochoice fixative (Amresco, Solon, OH) for 4 h, then processed for routine paraffin histology [22]. Additional placentas were dissected into the basal and labyrinth zones, snap frozen on liquid nitrogen, and stored at -80°C for subsequent Western blot and reverse-transcription polymerase chain reaction (RT-PCR) analyses.
Radioimmunoassay of Plasma Leptin
Blood samples were obtained at Day 22 of gestation from fetuses by decapitation (blood from four fetuses from each mother was pooled to provide sufficient volume), and maternal blood was collected from the dorsal aorta. The samples were collected into heparinized tubes, centrifuged at 11 000 rpm for 5 min, and plasma stored at -20°C until assayed. Plasma leptin concentrations were measured using a radioimmunoassay kit supplied by Linco Research (St. Charles, MO). The intra- and interassay coefficients of variation were 4.0% and 4.5%, respectively.
Immunocytochemical Analyses
Ob-R was immunolocalized in placental sections (5 µm) mounted on poly-L-lysine-coated slides (Sigma, St. Louis, MO) as previously described for localization of 11ß-HSD enzymes [22]. Briefly, endogenous peroxidase activity was blocked by incubation in 1% hydrogen peroxide in methanol for 10 min, and nonspecific binding was blocked by incubation with 1.5% BSA (Sigma) in PBS containing 0.1% Triton X. Sections were then incubated overnight at 4°C with primary antibody (diluted 1:50; K-20; Santa Cruz Biotechnology, Santa Cruz, CA) and then with biotinylated horse anti-goat secondary antibody (diluted 1:200; Vector Laboratories, Burlingame, CA) followed by Vectastain Elite ABC solution (Vector Laboratories). Positive signal was visualized using diaminobenzidene (DAB; Sigma), and sections were counterstained with Gill hematoxylin; no signal was evident after preincubation with blocking peptide (Santa Cruz Biotechnology) or in the absence of primary antibody. Because the primary antibody was generated against the common region of the Ob-R, it theoretically cross-reacts with all three major isoforms; by Western analysis, however, only the 120-kDa Ob-Rb isoform [23] was detected (see Results).
Western Blot Analysis
Western blot analysis was performed essentially as described by Shioda et al. [24]. Briefly, portions of basal and labyrinth tissue were homogenized (2 vol 10 mM Tris buffer containing 1.5 mM EDTA, 1 mM DTT, 1 mM PMSF, and 100 µg/ml trypsin inhibitor) and centrifuged at 105 000 x g for 30 min. Supernatant protein (100 µg) was resolved by SDS-PAGE (7% separating) and transferred to nitrocellulose membrane (Hybond C-Super, Amersham Pharmacia Biotech, Sydney, Australia). Membranes were incubated for 1 h in blocking solution containing 5% nonfat milk, then overnight at 4°C with Ob-R antibody (diluted 1:400, K-20; Santa Cruz Biotechnology). To identify immunoreactive bands, membranes were incubated with HRP-conjugated donkey anti-goat secondary antibody (diluted 1:5000; Chemicon International, Temecula, CA) and signals visualized using a chemiluminescence detection kit (SuperSignal Substrate, Pierce Chemical, Rockford, IL). Resultant autoradiographs were quantified by densitometry using Scion Image analysis software (Release beta 3b) as previously described [25].
Real-Time Quantitative RT-PCR Analysis
Total RNA from placental zones was isolated, reverse transcribed, and the resultant cDNA purified and quantified as previously described [8]. The cDNA primers to specific rat Ob-R isoforms [26] were positioned to span intron-exon junctions to distinguish cDNA from genomic DNA and were as follows: Ob-Ra, sense 5'-ATGAAGTGGCTTAGAATCCCTTCG-3', antisense 5'-TACTTCAAAGAGTGTCCGCTC-3'; Ob-Rb, sense 5'-ATGAAGTGGCTTAGAATCCCTTCG-3', antisense 5'-ATATCACTGATTCTGCATGCT-3'; Ob-Re, sense 5'-TTCCTGTGGGCAGAATCAGCACACACTGTT-3', antisense 5'-AAGCACAGTACACATACC-3' (GeneWorks, Adelaide, Australia). External standards were generated from regular PCR products (347, 375, and 305 base pairs, respectively, for Ob-Ra, Ob-Rb, and Ob-Re) and serial dilutions made in RNase-free water (1–107-fold dilutions).
Quantitative PCR and melting-curve analyses were performed in 10 µl reaction volumes in capillary tubes using the LightCycler system (Roche Diagnostics, Indianapolis, IN) as previously described for Ob-Rb [8]. Primers (as above) and MgCl2 concentrations were optimized as follows: Ob-Ra, primers 0.3 µM, MgCl2 3 mM; Ob-Rb, primers 0.3 µM, MgCl2 3 mM; Ob-Re, primers 0.4 µM, MgCl2 2 mM. The PCR cycling conditions included an initial denaturation at 95°C for 10 min followed by 55 cycles at 95°C for 15 sec; 54°C for 5 sec (Ob-Re 50°C), and 72°C for 16 sec. Melting-curve analysis showed a single PCR product for each Ob-R isoform, and this was confirmed by gel electrophoresis (data not shown). Fluorescence values were analyzed and a standard curve constructed using the LightCycler software. The intra- and interassay coefficients of variation, respectively, were as follows: Ob-Ra, 0.5% and 0.3%; Ob-Rb, 0.5% and 2.0%; Ob-Re, 0.2% and 0.3%.
Statistical Analysis
All data are expressed as the mean ± SEM, where a minimum of three animals was used for each experimental variable and each litter represented an n of one. Variation in Ob-R protein and mRNA expression related to placental zone, stage of pregnancy, or treatment was assessed by one- or two-way ANOVAs as appropriate. The effects of glucocorticoid manipulations on maternal and fetal plasma leptin were assessed by one-way ANOVAs. Where the F-test for the ANOVA reached statistical significance (P < 0.05), differences among specific means were assessed by least significant difference (LSD) tests [27].
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RESULTS |
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Expression of mRNAs encoding Ob-Ra, Ob-Rb, and Ob-Re were detected in both the basal and labyrinth zones of the rat placenta at Days 16 and 22 of pregnancy (Fig. 1). Expression of Ob-Ra and Ob-Re mRNAs both varied with gestational age (P < 0.05) and placental zone (P < 0.02), and in each case, there was significant interaction between these sources of variation (P < 0.05, two-way ANOVA). Specifically, the major effects were that both Ob-Ra and Ob-Re mRNA expression increased (P < 0.01) in the labyrinth zone from Day 16 to Day 22 (by 4.8- and 1.9-fold, respectively), whereas basal zone expression of both isoforms remained relatively stable. Expression of Ob-Rb appeared slightly higher in labyrinth zone at both stages of pregnancy, but this just failed to reach statistical significance (P = 0.07, two-way ANOVA). Regardless of stage of pregnancy or placental zone, expression of Ob-Re clearly exceeded that of Ob-Ra and Ob-Rb (by 3.7- to 10.7-fold).
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Western analysis of Ob-R showed a clear immunoreactive signal at the expected size of Ob-Rb (120 kDa [23]) in both basal and labyrinth zones at Days 16 and 22 (Fig. 2). Generally, expression of Ob-Rb protein was consistent with Ob-Rb mRNA expression, although Ob-Rb protein did fall significantly (36%, P < 0.05) in basal zone from Day 16 to Day 22. A second immunoreactive band was observed at 50 kDa in all samples, but this did not vary with either gestational age or placental zone (data not shown).
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Immunocytochemical localization of the Ob-R confirmed its presence in both the basal and labyrinth zones at Days 16 and 22 (Fig. 3). Specifically, immunostaining was apparent in trophoblast tissue at both stages of pregnancy and within the adventitial layer of fetal blood vessels in the placental labyrinth zone, particularly at Day 22 (Fig. 3b).
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Effects of Glucocorticoid Manipulations on Placental Ob-R Expression
Fetal weight in untreated animals (5.34 ± 0.17 g) was significantly greater than that in animals treated with dexamethasone (3.59 ± 0.17 g; P < 0.001) or carbenoxolone (4.62 ± 0.11 g; P < 0.05). In contrast, fetal weight appeared to be elevated after treatment with metyrapone (5.77 ± 0.20 g), although this just failed to reach statistical significance (difference = 0.43 g; LSD0.05 = 0.49 g). Placental expression of Ob-Ra, Ob-Rb, and Ob-Re mRNAs each varied with glucocorticoid manipulation and with placental zone (each P < 0.01, separate two-way ANOVAs). Specifically, Ob-Rb expression in the labyrinth zone declined by 32% (P < 0.05) after dexamethasone treatment but increased by 39% following suppression of endogenous glucocorticoid synthesis by metyrapone (P < 0.05; Fig. 4). Similar effects were observed for Ob-Rb expression in the basal zone, but differences reached statistical significance (P < 0.05) only between the metyrapone and the dexamethasone or carbenoxolone groups. Metyrapone treatment also markedly increased (P < 0.01) placental mRNA expression of Ob-Ra (basal, 2.2-fold increase; labyrinth, 1.9-fold) and Ob-Re (basal, 3.5-fold; labyrinth, 2.3-fold), but dexamethasone and carbenoxolone were without effect on expression of these isoforms.
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Placental expression of the 120-kDa Ob-Rb protein also varied significantly with treatment (P < 0.01), and these effects were generally consistent with those observed for Ob-Rb mRNA (compare Figs. 4 and 5). Specifically, dexamethasone reduced Ob-Rb protein in the basal zone by 46% (P < 0.02, Fig. 5), and carbenoxolone reduced Ob-Rb expression relative to the metyrapone group (37%, P < 0.05) but not the control group (26%, not significant). A similar trend was observed for Ob-Rb protein expression in the labyrinth zone among the four groups, but this did not reach statistical significance. The 50-kDa signal was again seen in all samples, but this did not vary significantly with either treatment or placental zone (data not shown).
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Effects of Glucocorticoid Manipulations on Maternal> and Fetal Plasma Leptin
Treatment of pregnant mothers with dexamethasone resulted in a 2.7-fold increase (P < 0.01) in maternal plasma leptin but an 82% decrease (P < 0.001) in fetal plasma leptin (Fig. 6). Carbenoxolone also reduced fetal plasma leptin (52%, P < 0.01) but had no effect on maternal leptin, whereas metyrapone had no effect on either maternal or fetal plasma leptin concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placental expression of Ob-Rb, the only isoform with full signal transduction capability [11], was readily detected by RT-PCR and Western analysis, particularly in the labyrinth zone, which also displayed the highest Ob-R immunolocalization. This placental region is the site of maternal-fetal exchange and undergoes considerable growth (more than threefold increase in weight [28]) over the period of gestation studied. Quantitative RT-PCR analysis showed that Ob-Ra and Ob-Re were also expressed in the two placental zones, and at Day 22, expression of both isoforms clearly exceeded that of Ob-Rb in the labyrinth zone. Interestingly, Western analysis showed a strong immunoreactive signal at 50 kDa, as previously reported for other tissues [29, 30]. This molecular weight species appears too small to be either Ob-Ra or Ob-Re, and Hill et al. [29] have suggested that it may be a breakdown product of the Ob-Re isoform. Regardless of its precise identity, there was no evidence of any change in expression of this 50-kDa species with gestational age, placental zone, or glucocorticoid manipulation.
Immunolocalization of Ob-R to trophoblast cells suggests that leptin is likely to exert direct effects on placental function. Given the highly proliferative state of the placenta over the last week of pregnancy and the proposed role of leptin as a fetal growth factor [3, 5–7], leptin may exert a mitogenic effect on trophoblast, similar to its role in the skin [31]. Indeed, our observation that dexamethasone suppresses both placental growth [16] and Ob-Rb expression further supports this contention. Moreover, immunolocalization of Ob-R to placental blood vessels is consistent with the proposed role of leptin as an angiogenic factor [32], which may be particularly important during the rapid and marked growth of the labyrinth zone during late pregnancy.
The Ob-Ra isoform is thought to promote the transport of leptin across physiological barriers, a process that occurs preferentially from the apical to basal surface of cells in vitro [33]. Such a role for Ob-Ra is supported by its relatively high expression in choroid plexus and microvessels of the brain [34]. Because the labyrinth zone of the rat placenta is the site of feto-maternal exchange, its relatively high expression of Ob-Ra, especially at Day 22 of pregnancy, is consistent with a role for this isoform in the transplacental passage of maternal leptin to the fetus. Consistent with this proposal, we have recently observed an increase in the passage of 125I-leptin from maternal to fetal blood between Days 16 and 22 of rat pregnancy (unpublished results). This transport of maternal leptin across the placenta is likely to be of particular importance in rodents because, unlike the human placenta [35], that of rodents appears to synthesize little if any de novo leptin [36, 37].
Placental expression of mRNA encoding the soluble isoform Ob-Re was clearly the highest among the three isoforms, particularly in the labyrinth zone, and this expression is consistent with our recent observation that plasma leptin-binding activity increases progressively until late pregnancy [8]. Thus, the rat appears similar to the mouse in that placental Ob-Re is secreted into the maternal circulation and thereby increases plasma leptin-binding activity [9]. Further studies are required to establish the role, if any, of placental Ob-Re within the fetal circulation.
The inhibition of placental Ob-Rb expression (both mRNA and protein) following treatment with dexamethasone is consistent with previous reports suggesting that glucocorticoids act as counterregulatory hormones to leptin action [18, 19]. The fall in Ob-Rb expression would be expected to limit any growth-promoting effects of leptin in the placenta and thus may contribute to glucocorticoid suppression of placental growth. The inhibitory effect of glucocorticoids on Ob-Rb expression appears to operate across the full physiological range of concentrations because inhibition of endogenous glucocorticoid synthesis by metyrapone enhanced expression of mRNA encoding Ob-Rb as well as those for Ob-Ra and Ob-Re. This further suggests that placental expression of all three Ob-R isoforms is tonically suppressed by basal levels of endogenous glucocorticoids, highlighting the need for local glucocorticoid levels to be tightly regulated within the placenta. We have previously demonstrated that this regulation is mediated by zone-specific expression of the 11ß-HSD enzymes in the placenta [28].
Dexamethasone treatment also increased plasma leptin concentrations in the maternal compartment but had the opposite effect on plasma leptin in the fetus, similar to recent observations by Sugden et al. [21]. The increase in maternal leptin following dexamethasone treatment likely reflects stimulation of maternal adipocyte leptin production as previously reported [19, 20], but the reasons for reduced plasma leptin levels in the fetus are unclear. One explanation is that maternal dexamethasone treatment compromised transplacental passage of maternal leptin to the fetus. Consistent with this explanation, we have recently observed that maternal dexamethasone treatment reduces the transplacental passage of 125I-leptin from the mother to the fetus (unpublished results). Interestingly, despite this reduction in placental leptin transport, the present work shows that expression of Ob-Ra, the isoform normally associated with transport of leptin, is unaffected by dexamethasone. Recent work in the rodent brain indicates that Ob-Rc may also be an important player in leptin transport [38], and so it would be of interest to assess whether this isoform is also expressed in the placenta and if its expression is reduced by glucocorticoids. In addition, glucocorticoid-induced changes in nonspecific mediators of maternal-fetal leptin transport, such as placental blood flow and placental surface area, could also account for the apparent reduction in placental leptin transport.
In conclusion, this study shows that the three major isoforms of the leptin receptor, Ob-Ra, Ob-Rb, and Ob-Re, are all expressed in the rat placenta, each with distinct spatial and temporal patterns. Manipulation of placental glucocorticoid exposure markedly altered expression of all Ob-R isoforms, consistent with a positive role for leptin on fetal and placental growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Brendan Waddell, School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. FAX: 61 8 9380 1051; bwaddell{at}anhb.uwa.edu.au
Received: 14 March 2002.
First decision: 10 April 2002.
Accepted: 15 May 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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