Regulation of expression and localisation of the Na+/H+ exchanger (NHE) 3 and the NHE regulatory factor 2 in baboon placental syncytiotrophoblast by oestrogen.

Our understanding of the regulation of the expression of the sodium hydrogen exchangers (NHE) and their regulatory factors (NHERF), which play important roles in fetal-placental homeostasis, is incomplete. We previously showed that the expression and localisation of NHE3 and NHERF2 in the juxtanuclear compartment of the placental syncytiotrophoblast were markedly decreased between mid and late baboon pregnancy. In the current study, immunocytochemical fluorescence localisation and level of NHE3/NHE1 and NHERF1/NHERF2 proteins were determined in late gestation in baboons untreated or treated throughout the second half of gestation with an aromatase inhibitor CGS 20267 alone (reduced oestrogen levels by >95%) or with oestradiol to determine whether oestrogen regulated antiporter developmental expression. The immunocytochemical expression of NHE3 and NHERF2 in the juxtanuclear compartment was minimal in baboons untreated or treated with CGS 20267 plus oestradiol (i.e. oestrogen-replete) but extensive in oestrogen-suppressed animals. Moreover, the abundant expression of NHERF2 in fetal vascular endothelium of oestrogen-replete baboons was decreased in oestrogen-suppressed animals. In contrast, expression and localisation of NHE1 and NHERF1 in the placental syncytiotrophoblast were not altered by oestrogen deprivation in baboons. Based on our current and previous findings, we propose that oestrogen plays an important role in regulating localisation and expression of components of the NHE system within and consequently development and function of the primate placental syncytiotrophoblast.

Estrogen regulates 11 beta-hydroxysteroid dehydrogenase-1 and -2 localization in placental syncytiotrophoblast in the second half of primate pregnancy.

We recently demonstrated that the 11 beta-hydroxysteroid dehydrogenase enzymes catalyzing cortisol-cortisone reduction (11 beta-hydroxysteroid dehydrogenase-1) and oxidation (11 beta-hydroxysteroid dehydrogenase-2) are located in different regions of the baboon and human placental syncytiotrophoblast. Moreover, there was a 2-fold increase in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes contiguous with the basal membrane (BMm) between mid and late baboon gestation. Our laboratories have also shown that estrogen regulates syncytiotrophoblast functional differentiation. Therefore, the current study determined whether the change in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in the BMm was regulated by estrogen. Placentas were obtained on d 165 of gestation (term = d 184) from baboons that were untreated or were treated daily beginning on d 100 with the aromatase inhibitor CGS 20267, which reduced uterine and maternal serum E2 by more than 95% or with CGS 20267 plus E2 benzoate. Western blot analyses and immunofluorescence confirmed that in untreated controls the expression of 11 beta-hydroxysteroid dehydrogenase-1 was abundant in the microvillus membranes and considerably less in the BMm. In contrast, expression of 11 beta-hydroxysteroid dehydrogenase-2 was abundant in more internal regions of the syncytiotrophoblast, including the BMm, but was not detected in the microvillus membranes. The 11 beta-hydroxysteroid dehydrogenase-2 protein level was significantly decreased in the BMm of placentas from estrogen-suppressed baboons, resulting in a 2-fold decrease in the ratio of these enzymes in membranes juxta the fetal blood, and these changes were partially restored by CGS 20267 and E2. In contrast, estrogen had no effect on the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in whole villous homogenate or the micro-villus membranes. Collectively, these results indicate that estrogen regulates the developmental increase in the ratio of 11 beta-hydroxysteroid dehydrogenase-2 to 11 beta-hydroxysteroid dehydrogenase-1 in syncytiotrophoblast membranes juxta fetal blood, providing the subcellular architectural mechanism responsible for the previously demonstrated estrogen-dependent switch in transplacental glucocorticoid metabolism that regulates maturation of the primate fetal pituitary-adrenocortical axis.

Expression of the mRNAs and Proteins for the Na(+)/H(+) exchangers and their regulatory factors in baboon and human placental syncytiotrophoblast.

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na(+)/H(+) exchangers (e.g. Na(+)/H(+) exchanger-1 and -3) and their regulatory proteins, Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein play a major role in regulating Na(+)/H(+) exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na(+) and H(+) between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na(+)/H(+) exchanger-1 and -3 were compartmentalized and associated with expression of Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na(+)/H(+) exchanger-1 and 190-bp Na(+)/H(+) exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na(+)/H(+) exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na(+)/H(+) exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na(+)/H(+) exchanger-1 and -3 was confirmed by immunocytochemistry. The Na(+)/H(+) exchanger-3 regulatory protein, Na(+)/H(+) exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na(+)/H(+) exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na(+)/H(+) exchanger-1 and -3 and their regulatory factors and that Na(+)/H(+) exchanger-1 and Na(+)/H(+) exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na(+)/H(+) exchanger-3 and Na(+)/H(+) exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na(+)/H(+) exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na(+)/H(+) exchange between the placenta and the maternal and fetal circulations.

Localization and developmental regulation of 11beta-hydroxysteroid dehydrogenase-1 and -2 in the baboon syncytiotrophoblast.

We previously showed that the messenger RNA and protein levels of the 11ss-hydroxysteroid dehydrogenase (11betaHSD) enzymes catalyzing glucocorticoid reduction (11betaHSD-1) and oxidation (11betaHSD-2) increased with advancing baboon gestation and concluded that the estrogen-regulated change in placental cortisol metabolism from reduction at midgestation to oxidation near term is not simply the result of a change in the relative concentrations of these two enzymes. Therefore, in the current study we determined whether 11betaHSD-1 and -2 are located in different regions of the baboon and human syncytiotrophoblast and whether there is a developmental change in their localization with advancing baboon gestation. Western blot analyses, immunofluorescence, and electron microscopic immunocytochemistry indicated that 11betaHSD-1 expression was abundant in microvillus membranes (MVM) juxta the maternal circulation, and their levels are significantly lower, but detectable, in more internal regions of the syncytiotrophoblast, including membranes contiguous with the basal membrane (BM(m)) facing the fetal vasculature in both the human and baboon. In contrast, in both species 11betaHSD-2 expression was limited in the MVM and extensive throughout the remainder of the syncytiotrophoblast, including the BM(m). In the baboon, the relative mean (+/-SE) concentrations (arbitrary densitometric units per microgram protein) of 11betaHSD-1 in the MVM were similar at mid (i.e. day 100; 38,859 +/- 3,484; n = 3) and late (i.e. day 180; 43,561 +/- 1,784; n = 3) gestation (term = day 184) and exceeded (P < 0.01) respective values for 11betaHSD-2 by approximately 16-fold. In contrast, levels of 11betaHSD-1 in the BM(m) declined (P < 0.05) by approximately 50% between mid (7,099 +/- 758) and late (4,013 +/- 738) gestation, whereas levels of 11betaHSD-2 in this fraction increased. Thus, the ratio of 11betaHSD-2 to 11betaHSD-1 in the BM(m) at midgestation (1.22 +/- 0.10) was increased (P < 0.05) 2-fold in late gestation (2.66 +/- 0.05). Collectively, these findings indicate that the 11betaHSD-1 and -2 enzymes are localized to different membrane fractions of the baboon and human placental syncytiotrophoblast. Moreover, we propose that the developmental increase in the ratio of 11betaHSD-2 to 11betaHSD-1 in membranes facing fetal blood near term is consistent with and perhaps the subcellular mechanism responsible for the previously demonstrated switch in transplacental glucocorticoid metabolism from reduction at midgestation to oxidation late in gestation and appears to be responsible for the activation/maturation of the fetal pituitary-adrenocortical axis.

Expression of the 11beta-hydroxysteroid dehydrogenase types 1 and 2 proteins in human and baboon placental syncytiotrophoblast.

We have shown that the placenta, via metabolism of maternal cortisol and cortisone by the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes types 1 and 2 in the syncytiotrophoblast, regulates the maturation of the fetal pituitary adrenocortical axis in the baboon. Because the timing and regulation of fetal adrenal development by fetal ACTH in the human seem to parallel that in the baboon, we propose that the placental 11beta-HSD-1 and -2 system also has a role in regulating the development of the fetal pituitary adrenocortical axis during human pregnancy. However, although the human placenta has been shown to express the 11beta-HSD-2, it remains to be determined unequivocally whether 11beta-HSD-1 protein is present in the human placental syncytiotrophoblast. To answer this question, enriched fractions of syncytiotrophoblast were prepared from human and baboon term placentae and proteins probed with polyclonal antibodies directed to amino acids 22-36 or 66-77 of human 11beta-HSD-1. The 11beta-HSD-1 was detected by Western blot analysis as a 32-kDa protein in human and baboon syncytiotrophoblast and as a 34-kDa protein in adult baboon liver. Localization of the 11beta-HSD-1 to the syncytiotrophoblast was confirmed by immunocytochemistry following antigen retrieval. These results show that both human and baboon placental syncytiotrophoblast expressed the 11beta-HSD-1, as well as the 11beta-HSD-2, proteins. Because 11beta-HSD-1 can function as a reductase, the expression of 11beta-HSD-1 in human syncytiotrophoblast would be consistent with the ability of this tissue to convert cortisone to cortisol and provide a means by which transplacental transport of cortisol could regulate the fetal pituitary adrenocortical axis in the human, as recently shown experimentally in the non-human primate baboon model.

Inhibition of fetal adrenal adrenocorticotropin receptor messenger ribonucleic acid expression by betamethasone administration to the baboon fetus in late gestation.

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3beta-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 microl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 +/- 0.07 arbitrary densitometric units) than in untreated controls (0.63 +/- 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of beta-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 +/- 0.02) than in untreated controls (0.84 +/- 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (microm) of the definitive zone (183 +/- 14 vs. 128 +/- 7; determined by immunohistochemical expression of 3beta-HSD), as well as the levels of the mRNA and protein for 3beta-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17alpha-hydroxylase-C(17,20) lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3beta-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta.

Cortisol-cortisone interconversion is catalyzed by the NADP/NADPH-dependent oxido-reductase, 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) and the NAD-dependent oxidase, 11betaHSD-2. Because of the importance of placental corticosteroid metabolism in dictating the amount of cortisol arriving in the fetus to regulate fetal pituitary-adrenocortical function, the present study determined whether there was a developmental change in the expression of 11betaHSD-1 and/or -2 in placental syncytiotrophoblast, the site of maternal:fetal exchange. A syncytiotrophoblast-enriched (>95%) cell fraction was isolated from baboon placentas obtained at early (day 60), mid (day 100), and late (day 165) gestation (term = day 184), and 11betaHSD-1 and -2 messenger RNA (mRNA) and protein levels were determined by Northern and Western blots. The levels (mean +/- SE) of the single 1.6-kilobase (kb) mRNA for 11betaHSD-1, expressed as a ratio to beta-actin, increased (P < 0.05) between early (0.36 +/- 0.16; n = 4) and mid (0.95 +/- 0.21; n = 11) gestation and further increased (P < 0.05) by late gestation (1.82 +/- 0.29; n = 13). Similarly, the levels of the single 1.9-kb mRNA for 11betaHSD-2 in late gestation (2.46 +/- 0.35; n = 8) were greater (P < 0.05) than respective values at mid (1.36 +/- 0.22; n = 8) and early (0.64; n = 2) gestation. The levels of 11betaHSD-1 (arbitrary densitometric units), detected as a dominant band of 34 kDa, were greater (P < 0.05) in late gestation (2.6 +/- 0.2; n = 4) than at early (1.2 +/- 0.1; n = 4) or mid (1.9 +/- 0.3; n = 4) gestation. In contrast, 11betaHSD-2 was not detected by Western blot in syncytiotrophoblast isolated by collagenase dispersion. However, immunocytochemistry revealed that 11betaHSD-2 was present in and localized to the syncytiotrophoblast layer of the baboon placenta and that expression in late gestation (n = 4) appeared to exceed that in placentas of early (n = 4) and mid (n = 4) gestation. These results indicate that both 11betaHSD-1 and 11betaHSD-2 were expressed in syncytiotrophoblasts of the baboon placenta and that the mRNA and protein levels of these two 11betaHSD enzymes increased with advancing gestation. However, because 11betaHSD-2 was not detected in syncytiotrophoblast isolated by collagenase dispersion, we suggest that the 11betaHSD-1 and -2 reside in different membrane fractions of the syncytiotrophoblast. Consequently, the estrogen-regulated change in transplacental cortisol metabolism with advancing gestation may result in a developmental change in the expression and location of the two 11betaHSD enzymes controlling cortisol-cortisone metabolism and transfer into the fetus, resulting in activation of the fetal pituitary adrenocortical system.

Interconversion of cortisol and cortisone in the baboon placenta at midgestation: expression of 11beta-hydroxysteroid dehydrogenase type 1 messenger RNA.

At midgestation in the baboon, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalysed interconversion of cortisol (F) and cortisone (E) during transuterine passage favors the formation of F. Because the site(s) of oxidation/reduction of F and E are not clear, the present study compared F-E interconversion in placenta, decidua and chorion in vitro. In addition, because the reduction of E to F is catalysed only by the 11beta-HSD-1, we also determined whether the mRNA for this enzyme was expressed in baboon placenta, including placental syncytiotrophoblast cells; the site of fetal-maternal exchange. Placentas were obtained on day 100 of gestation (term = day 184) and villous tissue, decidua and chorion isolated, minced in HBSS and incubated (300-400 mg) in duplicate for 0.1-24 h in Medium 199 containing 10% fetal bovine serum and [3H]F and [14C]E. Radiolabelled F and E were purified from incubates and the percentage conversion of F to E and E to F was calculated. In decidua, mean (+/- SE; n = 3) conversion of E to F (69 +/- 2%) was greater than oxidation of F to E (26 +/- 2%). Conversion of E to F in placenta (50 +/- 1%) and chorion (39 +/- 9%) was also extensive and greater than or equal to that for the oxidation of F to E (39 +/- 4% and 32 +/- 4%, respectively). The apparent ratio of 11beta-HSD reductive/oxidative activity was maintained when respective tissues from four baboons were incubated for 18 h with or without 4.4 microM excess radioinert substrate. Expression of 11beta-HSD-1 mRNA was determined by Northern blot by hybridization of poly (A) + -enriched RNA from tissues obtained at midgestation with [32P]labelled human 11beta-HSD-1 cDNA. This cDNA hybridized to a single mRNA species of 1.6 kb in decidua, whole placental villous tissue and syncytiotrophoblast cells, but not to RNA isolated from fetal baboon kidney. The results of the present study demonstrate that at midgestation in the baboon, 11beta-HSD activity in intact placenta and decidua in vitro favored the formation of F from E, presumably catalysed by the 11beta-HSD-1 enzyme protein, the mRNA for which is present in placental syncytiotrophoblasts. Moreover, based on overall mass and extensive vascularity, we suggest that the net formation of F from E during transuterine passage in vivo at this stage of gestation results from the 11beta-HSD-1 in the syncytiotrophoblast cells of the baboon placenta.