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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 9 4316-4323
Copyright © 2003 by The Endocrine Society

Functional Differentiation of the Placental Syncytiotrophoblast: Effect of Estrogen on Chorionic Somatomammotropin Expression during Early Primate Pregnancy

Biljana Musicki, Gerald J. Pepe and Eugene D. Albrecht

Departments of Obstetrics, Gynecology, and Reproductive Sciences and Physiology (B.M., E.D.A.), Center for Studies in Reproduction, The University of Maryland School of Medicine, Baltimore, Maryland 21201; and Department of Physiological Sciences (G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501

Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11-019, 655 West Baltimore Street, Baltimore, Maryland 21201. E-mail: ealbrech{at}umaryland.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen stimulates morphological and functional (i.e. steroidogenesis) differentiation of the primate placental trophoblast, and with advancing gestation there is an increase in estrogen and placental chorionic somatomammotropin (CS) mRNA and protein levels. To examine whether CS formation is regulated by estrogen, placental villous trophoblast CS was determined in baboons in which estradiol levels in uterine vein were increased 2- to 3-fold (P < 0.01) on d 60 of pregnancy (term = 184 d) by administration of aromatizable androstenedione on d 30–59 or estradiol benzoate on d 45–59 of gestation. Androstenedione and estradiol treatment resulted in a 75% decrease (P < 0.01) in placental whole villous CS-3 mRNA and CS protein levels, determined by Northern and Western blot analysis, on d 60, and a corresponding decrease in syncytiotrophoblast CS protein and maternal serum CS levels. In contrast, placental villous {Delta}5-3ß-hydroxysteroid dehydrogenase, 11ß-hydroxysteroid dehydrogenase-2, and P-450 aromatase protein levels were unaltered by androstenedione or estradiol treatment. Collectively, these results suggest that, in elevated levels, estrogen suppressed CS formation by villous syncytiotrophoblast during the first one third of primate pregnancy. Therefore, estrogen has very different and specific actions on steroid and peptide hormone biosynthesis within the placental trophoblast, which we propose are important in regulating placental function and promoting fetal-placental development in the primate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING HUMAN AND nonhuman primate pregnancy, cytotrophoblasts undergo morphological differentiation into the syncytiotrophoblast within the villous placenta (1, 2). As a result of this transformation process, the syncytiotrophoblast produces pregnancy-specific peptide hormones, such as placental lactogen/chorionic somatomammotropin (CS) (3, 4). CS is a member of the GH family, which consists of pituitary GH, placental GH variant, and three CS genes (5, 6). CS appears to have a role in regulating mammary development and lactogenesis, maternal intermediary metabolism, and fetal growth (7, 8, 9, 10); however, the regulation of placental CS formation is not well understood. Because there is a parallel increase in placental weight and placental CS formation during advancing human (11), rhesus monkey (12), and baboon (13) pregnancy, it has been suggested that CS is constitutively expressed. However, cAMP, prostaglandins, lipoproteins, and steroids regulate CS secretion in vitro by human and rhesus monkey trophoblast cells (6, 8, 14, 15, 16).

Using the baboon as a nonhuman primate model for the study of human pregnancy, we have recently shown that estrogen accelerated morphological differentiation of cytotrophoblasts into syncytiotrophoblast during the first half of pregnancy (17). We have also shown in the second half of baboon pregnancy that estrogen stimulated functional maturation of the syncytiotrophoblast, manifest as up-regulation of expression of low-density lipoprotein (LDL) receptor (18, 19, 20), P-450 cholesterol side-chain cleavage (P-450scc) enzyme (21), and the 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 and -2 enzymes, which regulate the switch in transplacental corticosteroid dynamics leading to maturation of the fetal pituitary adrenocortical axis (22, 23). In addition, the increase in estrogen levels and syncytiotrophoblast functional differentiation during advancing baboon pregnancy was associated with increased expression of CS mRNA and protein (13), and we proposed that CS formation results from both morphological differentiation of cytotrophoblasts into the syncytiotrophoblast and functional differentiation of the syncytiotrophoblast thereafter. It is possible, therefore, that estrogen regulates CS formation by the syncytiotrophoblast. To determine this possibility, in the present study we assessed whether placental trophoblast CS formation was altered by administration of aromatizable C19-steroid androstenedione or estradiol during early baboon pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female baboons (Papio anubis) weighing 12–15 kg were housed individually in large primate cages in air-conditioned rooms maintained at 22 C and fed monkey kibble and fresh fruit twice daily and water ad libitum. Females were paired with male baboons for 5 d at the time of ovulation as estimated by menstrual cycle history and the pattern of perineal turgescence. Day 1 of gestation was designated as 2 d preceding the onset of detumescence. Pregnant baboons were then either untreated, or treated with androstenedione (30 mg/d, sc in sesame oil) on d 30–59 of gestation, or with estradiol benzoate (250 µg/d, sc in sesame oil) on d 45–59 of gestation (length of gestation = 184 d). On d 60 (i.e. early) or d 100 (i.e. mid) of gestation baboons were anesthetized with halothane: nitrous oxide, blood samples (2 ml) obtained from maternal peripheral saphenous and left and right uterine veins and the fetus and placenta delivered by cesarean section.

Animals were cared for and used strictly in accordance with United States Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication no. 85-23, 1985). The experimental protocol used in the present study was approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.

Placental cell isolation

After removal of membranes and decidua, 8–10 sections of villous tissue randomly selected from various regions of the placenta were excised and frozen in liquid nitrogen for subsequent Northern and Western blot analysis of mRNA and protein in whole villous tissue. The entire remaining villous placenta was minced in Hanks’ balanced salt solution (Life Technologies, Gaithersburg, MD) and enzyme dispersed to obtain a syncytiotrophoblast-enriched fraction for Western blot analysis of protein. Villous tissue was dispersed in a shaking water bath at 37 C for 40 min with 0.1% collagenase (Type H Sigma Blend, Sigma Chemical Co, St. Louis, MO), 0.1% hyaluronidase (Type I-S, Sigma), and 0.01% deoxyribonuclease I (1680 Kunitz U/mg, Sigma), and applied to a 50% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradient to obtain a cell fraction that was comprised primarily (>90%) of CS-positive syncytiotrophoblast (24).

Northern blot analysis

Northern blot analysis of mRNA was performed essentially as described previously (25). Briefly, total RNA was isolated by 4 M guanidine: isothiocyanate homogenization, chloroform: isoamyl alcohol extraction (26) and cesium chloride gradient centrifugation. Polyadenylated [poly(A)+]-enriched RNA was purified from total RNA by oligo(deoxythymidine)-cellulose chromatography (Pharmacia), size-fractionated by 0.66 M formaldehyde-1% agarose gel electrophoresis and transferred to nylon membrane (Gene Screen, DuPont-NEN Life Science Products Corp., Boston, MA). Membranes were prehybridized for 16–24 h at 42 C in buffer containing 50% formamide, 0.1% polyvinyl pyrrolidone, 0.1% Ficoll, 2.5 x SSPE [0.375 M NaCl, 0.025 M NaH2 PO4-H2, 0.0025 M EDTA-N2 (pH 7.4)], 1% sodium dodecyl sulfate, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml). Poly (A)+ RNA (5 µg) was then hybridized for 23 h at 42 C with cDNAs for rhesus monkey mCS-3 (provided by Dr. Thaddeus Golos, Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, WI) or human ß-actin (no. 65128, American Type Culture Collection, Rockville, MD). mCS-3 is 94% homologous to mCS-1 and -2 in mRNA and deduced amino acid sequence, and although expressed in slightly lower level, mCS-3 was studied because it exhibited a developmental increase in placental expression with advancing rhesus monkey pregnancy compared with the other two genes (27). cDNAs were labeled with 50 µCi of [32P]deoxy-CTP (3000 Ci/mmol; Amersham Corp., Arlington Heights, IL) to a specific activity of approximately 109 dpm/µg DNA using the High-Prime DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). After hybridization membranes were washed in SSC (0.3 M NaCl, 0.03 M sodium citrate-2H2O) using stringent conditions and exposed at -80 C to Kodak X-AR film (Eastman Kodak, Rochester, NY).

Western immunoblot analysis

Proteins were analyzed by Western immunoblot as described previously (13). Placental whole villous tissue was homogenized on ice in 6 vol 25 mM Tris, 0.25 M sucrose, 1 mM ethylenediamine tetraacetate, and 1 mM phenylmethylsulfonylfluoride (pH 7.4). Syncytiotrophoblast fractions were solubilized for 20 min on ice in 0.5% Triton X-100, 25 mM Tris HCl, 0.25 M NaCl, and 5 mM ethylenediamine tetraacetate (pH 7.5), containing 1 mM phenylmethylsulfonylfluoride and 10 µg/ml leupeptin. Proteins (50 µg tissue and 12 µg cells) and serum samples (3 µl) were boiled for 2 min in sodium dodecyl sulfate loading buffer, separated on 16% Tris-glycine gels (Novex, San Diego, CA) under nonreducing conditions for 2 h (28) at 140 V, and transferred to nitrocellulose or polyvinylidene difluoride membranes for 2 h at 4 C under a constant current of 40 V using a Novex Blot Module. Membranes were washed and blocked for 2 h at room temperature in 3% BSA in TBST (50 mM Tris, 0.9% NaCl, and 0.1% Tween-20). Immobilized proteins were incubated for 1 h at room temperature with rabbit antibodies to human CS (recognizes all CS isoforms; Dako Corp., Carpinteria, CA), human 3ß-HSD (provided by Dr. J. Ian Mason, University of Edinburgh, Edinburgh, Scotland, UK), human 11ß-HSD-2 (provided by Professor Jonathan R. Seckl, University of Edinburgh), or human P-450 aromatase (provided by Dr. Evan Simpson, Prince Henry’s Institute of Medical Research, Monash Medical Center, Clayton, Victoria, Australia). Membranes were washed and incubated at room temperature for 1 h with donkey antirabbit IgG horseradish peroxidase-conjugated second antibody (Amersham). Chemiluminescence (ECL kit, Amersham) was visualized by exposure to x-ray film (Eastman Kodak).

Quantification of mRNA and protein

Protein and mRNA levels were quantitated by scanning densitometry (Video Densitometer model 620, Bio-Rad, Richmond, CA). Intensities of bands were expressed as arbitrary units, and mRNA levels were standardized against ß-actin for each sample.

Estradiol RIA

Serum estradiol levels were quantified by RIA using an automated chemiluminescent immunoassay system (Immulite, Diagnostic Products Corp., Los Angeles, CA), as described previously (29). Intra- and interassay coefficients of variation were 6.9% and 7.3%, respectively, and the minimal limit of detection was 12 pg/ml.

Statistical analysis of data

Data were expressed as the means ± SE. Statistical differences between groups were determined by ANOVA with post hoc comparisons of means by Newman-Keuls multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Placental and fetal body weights and serum estradiol levels

Mean (±SE) placental and fetal body weights in untreated baboons increased from 28.9 ± 1.6 g and 12.5 ± 0.7 g, respectively, on d 60 of gestation to values that were approximately 3-fold and 13-fold greater (P < 0.001) on d 100 (Table 1Go). Androstenedione or estradiol treatment had no significant effect on placental or fetal body weights at 60 d.


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  		TABLE 1. Placental and fetal body weights in androstenedione- or estradiol-treated baboons

 
Serum estradiol concentrations in maternal peripheral saphenous and uterine veins of untreated baboons increased (P < 0.01) from 0.17 ± 0.02 ng/ml and 0.47 ± 0.05 ng/ml, respectively, on d 60 to 1.82 ± 0.21 ng/ml and 6.70 ± 1.61 ng/ml, respectively, on d 100 of gestation (Fig. 1Go). Androstenedione administration increased (P < 0.01) serum estradiol levels by approximately 3-fold in saphenous (0.46 ± 0.09 ng/ml) and uterine (1.69 ± 0.33 ng/ml) veins on d 60 compared with untreated controls at this time of pregnancy (Fig. 1Go). Estradiol treatment increased (P < 0.01) estradiol concentrations in the peripheral (1.46 ± 0.41 ng/ml) and uterine (1.05 ± 0.25 ng/ml) veins by approximately 9-fold and 2-fold, respectively.



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  		FIG. 1. Serum estradiol concentrations in maternal peripheral saphenous ({square}) and uterine ({blacksquare}) veins of untreated baboons on d 60 (early, n = 7) and d 100 (mid, n = 4) of gestation (length of gestation = 184 d), and on d 60 in baboons treated with androstenedione ({Delta}4A, 30 mg/d, sc on d 30–59, n = 8) or estradiol benzoate (E2, 250 µg/d, sc on d 45–59, n = 5). Each bar represents mean ± SE. Système Internationale conversion factor for estradiol: l ng/ml = 4 nmol. *, Significantly greater (P < 0.01) than corresponding value in saphenous and uterine veins of untreated baboons on d 60.

 
Placental villous CS mRNA and protein

Baboon placenta hybridized with the mCS-3 cDNA to yield a single 0.9-kb mRNA transcript (Fig. 2Go). Placental villous CS-3 mRNA level, expressed as a ratio of ß-actin mRNA, increased (P < 0.01) between early and midgestation and was decreased (P < 0.05) on d 60 by androstenedione administration to a value (0.14 ± 0.08, relative arbitrary units) that was less than 20% of that observed in untreated baboons during early pregnancy (0.73 ± 0.12, Fig. 2Go). Estradiol treatment also suppressed (P < 0.01) CS-3 mRNA levels in whole villous tissue on d 60 of gestation (0.18 ± 0.09).



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  		FIG. 2. A, Northern blot of 0.9-kb CS-3 and 2.0-kb ß-actin mRNAs in baboon whole villous placenta. Five micrograms of poly(A+)-enriched RNA were obtained on d 60 (early, lanes 1–3, n = 3) and 100 (mid, lane 10, n = 1) of gestation from untreated baboons and on d 60 from baboons treated with androstenedione ({Delta}4A, lanes 4–6, n = 3), or estradiol (E2, lanes 7–9, n = 3) as detailed in the legend of Fig. 1Go. Autoradiogram exposure was 15 min. B, Ratio of CS-3 and ß-actin mRNA levels (means ± SE) expressed in relative arbitrary units in placental villous tissue obtained from baboons, the samples of which are shown in panel A, except at on d 100 where n = 4. Values with different letter superscripts differ at P < 0.05 to P < 0.01 (ANOVA and Newman-Keuls multiple comparisons test).

 
The 22-kDa CS protein determined by Western immunoblot in baboon placenta was verified with human placental CS standard, as shown previously (13). CS protein level in whole placental villous tissue from untreated baboons increased (P < 0.001) from 2.46 ± 0.53 (relative arbitrary units) on d 60 to 6.30 ± 0.21 on d 100 of gestation (Fig. 3Go). Androstenedione administration decreased (P < 0.01) CS protein level in placental villous tissue on d 60 to 0.74 ± 0.03, a value that was 30% of that observed in the untreated controls at this time in pregnancy. In estradiol-treated animals, placental villous CS protein was decreased (P < 0.01) to a level on d 60 (0.56 ± 0.19) that was 23% of that in untreated animals (Fig. 3Go).



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  		FIG. 3. A, Representative Western immunoblot of CS in whole placental villous tissue obtained on d 60 (lanes 1–4) and 100 (lanes 9–12) of gestation from untreated baboons, and on d 60 from animals treated with androstenedione (lanes 5 and 6) or estradiol benzoate (lanes 7 and 8). Placental protein (50 µg) and human placental CS standard (S) were incubated with peroxidase-conjugated human CS antibody and chemiluminescence visualized on x-ray film. B, Cumulative results of CS protein levels in placental villous tissue, determined by autoradiographic densitometry, obtained from untreated baboons on d 60 (early, n = 8) and 100 (mid, n = 8) of gestation, and on d 60 from androstenedione ({Delta}4A, n = 4) or estradiol benzoate (E2, n = 4)-treated baboons. Each bar represents the means ± SE expressed as relative arbitrary units. Data points with different letter superscripts differ at P < 0.01 to P < 0.001 (ANOVA and Newman-Keuls multiple comparisons test).

 
Placental syncytiotrophoblast and serum CS protein

Placental syncytiotrophoblast CS protein levels on d 60 of baboon gestation (9.59 ± 0.92) were decreased by androstenedione (5.25 ± 1.39) or estradiol (1.00 ± 0.17) treatment to values that were approximately 55% (P < 0.05) and 10% (P < 0.001), respectively, of that in untreated baboons(Fig. 4Go).



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  		FIG. 4. A, Western immunoblot of CS in isolated baboon placental syncytiotrophoblast. Protein (12 µg) from placental syncytiotrophoblast isolated by Percoll gradient centrifugation on d 60 from untreated baboons (lanes 1–5, n = 5) and baboons treated with androstenedione (lanes 6–7, n = 2) or estradiol (lanes 8–9, n = 2) as detailed in the legend of Fig. 1Go. B, Cumulative results of CS protein levels (mean ± SE) in placental syncytiotrophoblast obtained on d 60 from untreated baboons (n = 5) and animals treated with androstenedione ({Delta}4A, n = 4) or estradiol benzoate (E2, n = 4). Values with different letter superscripts differ at P < 0.05 to P < 0.001.

 
Peripheral serum CS protein levels increased (P < 0.001) from 0.18 ± 0.07 (arbitrary units) on d 60 to 3.14 ± 0.34 on d 100 of gestation in untreated baboons (Fig. 5Go). Administration of androstenedione or estradiol decreased peripheral serum CS levels on d 60 to nondetectable levels.



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  		FIG. 5. Serum CS protein levels (means ± SE, arbitrary units) determined by Western immunoblot in untreated baboons on d 60 (n = 17) and 100 (n = 12) of gestation and on d 60 from androstenedione ({Delta}4A, n = 8) or estradiol (E2, n = 8)-treated baboons. Values with different letter superscripts differ at P < 0.001.

 
Placental villous 3ß-HSD, 11ß-HSD-2, and P-450 aromatase proteins

In contrast to the inhibitory effects of estrogen on CS expression, 3ß-HSD protein levels in placental whole villous tissue (6.43 ± 0.55 arbitrary units, Fig. 6Go) or in syncytiotrophoblast (2.06 ± 0.43, Fig. 7Go) on d 60 of gestation were not significantly altered by androstenedione or estradiol treatment. Moreover, androstenedione or estradiol administration did not significantly change 11ß-HSD-2 protein levels in placental villous tissue early in pregnancy when compared with untreated baboons (5.98 ± 1.94, Fig. 8Go). Finally, P-450 aromatase protein levels in placental villous tissue on d 60 of gestation (6.06 ± 0.34) also were not significantly changed by treating baboons with androstenedione or estradiol early in pregnancy (Fig. 9Go).



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  		FIG. 6. A, Representative Western immunoblot of 3ß-HSD in whole placental villous tissue obtained on d 60 (lanes 1–4) and 100 (lanes 9–12) of gestation from untreated baboons, and on d 60 from baboons treated with androstenedione (lanes 5 and 6) or estradiol benzoate (lanes 7 and 8). B, Cumulative results of 3ß-HSD protein levels in placental villous tissue obtained from untreated baboons on d 60 (early, n = 8) and 100 (mid, n = 8) of gestation, and on d 60 from androstenedione ({Delta}4A, n = 4) or estradiol (E2, n = 4)-treated baboons. Each bar represents the means ± SE expressed in relative arbitrary units. There were no significant differences between the groups.

 


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  		FIG. 7. A, Representative Western immunoblot of 3ß-HSD in isolated baboon placental syncytiotrophoblast. Protein (12 µg) from placental syncytiotrophoblast isolated by Percoll gradient centrifugation on d 60 from untreated baboons (lanes 1 and 2) and baboons treated with androstenedione (lanes 3 and 4) or estradiol (lanes 5 and 6) as detailed in the legend of Fig. 1Go. B, Cumulative results of 3ß-HSD protein levels (mean ± SE) in placental syncytiotrophoblast obtained on d 60 from baboons untreated (n = 4) or treated with androstenedione ({Delta}4A, n = 4) or estradiol (E2, n = 4). There were no significant differences between the groups.

 


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  		FIG. 8. A, Representative Western immunoblot of 11ß-HSD-2 in whole placental villous tissue obtained on d 60 (lanes 1–4) and 100 (lanes 9–12) of gestation from untreated baboons, and on d 60 from animals treated with androstenedione (lanes 5 and 6) or estradiol benzoate (lanes 7 and 8). B, Cumulative results of 11ß-HSD-2 protein levels in villous tissue obtained from untreated baboons on d 60 (early, n = 8) and 100 (mid, n = 8) of gestation, and on d 60 from androstenedione ({Delta}4A, n = 4) or estradiol (E2, n = 4)-treated baboons. There were no significant differences between the groups.

 


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  		FIG. 9. A, Representative Western immunoblot of P-450 aromatase in whole placental villous tissue obtained on d 60 (lanes 1–4) and 100 (lanes 9–12) of gestation from untreated baboons, and on d 60 from baboons treated with androstenedione (lanes 5 and 6) or estradiol benzoate (lanes 7 and 8). B, Cumulative results of P-450 aromatase protein levels in villous tissue obtained from untreated baboons on d 60 (early, n = 8) and 100 (mid, n = 8) of gestation and on d 60 from androstenedione ({Delta}4A, n = 4) or estradiol (E2, n = 4)-treated baboons. There were no significant differences between the groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study show that elevating uterine vein serum estrogen concentrations by approximately 2- to 3-fold in early baboon pregnancy, by administration of aromatizable androstenedione or estradiol, resulted in a marked decrease in placental villous CS-3 mRNA and CS protein levels. The decline in whole villous CS expression in estrogen-treated baboons was associated with a corresponding decrease in CS protein levels in syncytiotrophoblast, the placental source of CS, and maternal serum into which placental CS is primarily secreted (30). The decrease in both placental CS mRNA and protein levels induced by estrogen, may reflect an increase in CS mRNA degradation and/or a decline in CS transcription within the syncytiotrophoblast. Collectively, these results suggest that in elevated levels estrogen suppressed CS formation by syncytiotrophoblast during the first one third of primate pregnancy.

We recently showed that androstenedione administration during early baboon pregnancy accelerated transformation of cytotrophoblasts into the syncytiotrophoblast (17), and although cytotrophoblast may produce some CS (1), it is generally considered that syncytiotrophoblast formation is paramount for CS biosynthesis (4, 31, 32). Therefore, the decrease in placental whole villous CS expression in estrogen-treated baboons of the current study is not the result of a decline in syncytiotrophoblast formation. The decline in CS protein levels in syncytiotrophoblast, as well as whole villous tissue, after exogenous estrogen is consistent with estrogen acting on syncytiotrophoblast CS formation.

In contrast to the inhibitory effect on CS, placental expression of 3ß-HSD, 11ß-HSD-2, and P-450 aromatase, enzymes expressed primarily by the syncytiotrophoblast (33, 34), and which may be regulated by hormones such as glucocorticosteroids (35), was unaltered by elevating estrogen early in pregnancy in baboons of the present study. Moreover, estrogen may up-regulate expression of vascular endothelial growth factor by the placental villous trophoblast during early baboon pregnancy (36). Therefore, estrogen appears to elicit very different and specific actions on biosynthesis of steroid and peptide hormones by the placental trophoblast during early primate pregnancy, which we propose are important in regulating placental function and promoting fetal-placental development in the primate.

In addition to morphological differentiation of cytotrophoblasts into the syncytiotrophoblast, a process that results in the onset of CS biosynthesis (32), after formation the syncytiotrophoblast exhibits increased capacity to synthesize CS (13), as well as LDL receptor (19, 25), and P-450scc (21), in association with the rise in estrogen that occurs with advancing baboon pregnancy (37). Moreover, inhibiting the action or production of estrogen in baboons suppressed placental receptor-mediated LDL uptake (18, 20) and P-450scc expression (21), effects reversed by exogenous estrogen. We have proposed, therefore, that once formed, the syncytiotrophoblast undergoes an estrogen-dependent functional or biochemical maturation that engenders it with an enhanced capacity to produce protein and steroid hormones (see Ref. 38 for review). However, the developmental increase in syncytiotrophoblast CS expression during the second half of baboon gestation coincides with endogenous estrogen levels that greatly exceed those attained on d 60 in animals of the current study by administration of androstenedione or estradiol that suppressed CS formation. Thus, there may be a developmental change in the regulation of placental CS formation by estrogen, whereby the syncytiotrophoblast loses or diminishes its responsivity to estrogen with respect to the inhibitory action on CS expression, but not to the stimulatory effects on expression of components of the steroidogenic pathway.

The physiological consequences of a selective inhibitory action of estrogen on placental CS formation early in pregnancy remain to be determined. However, members of the prolactin (PRL)/GH family, including CS, have the capacity to regulate angiogenesis. For example, intact GH stimulated vascular endothelial cell proliferation in vitro (39), whereas the 16-kDa N-terminal fragments of human PRL, CS, and GH inhibited angiogenesis in vitro (39, 40) and in vivo (41), by inducing endothelial cell apoptosis (42). The cathepsin enzymes that cleave PRL and related peptides are produced at the deciduo-placental interface (43), and smaller molecular weight fragments of these peptides appear in amniotic fluid and maternal serum (44), potentially providing a system in which CS and related peptides may regulate placental angiogenesis. Preliminary studies in our laboratory (Albrecht, E. D., V. A. Hildebrandt, and G. J. Pepe, unpublished observations) show that estrogen-stimulated expression of placental vascular endothelial growth factor and angiogenesis during early baboon pregnancy. It is possible, therefore, that the stimulatory effect of estrogen on placental neovascularization involves a decline in formation of CS fragments, which have the capacity to suppress angiogenesis. Indeed, we have shown that estrogen has an important role in promoting pregnancy maintenance (29).

In vitro studies show that various factors, including cAMP and high-density lipoprotein, stimulated CS production by the human and rhesus monkey trophoblast (8, 14, 15, 16, 43, 45). CS expression was enhanced by glucocorticoid, but not androstenedione or progesterone, in cultured rhesus monkey syncytiotrophoblast (15). Estrogen also increased CS formation when added to human trophoblast explants or cultures (46, 47), although this may in part result from the stimulatory action of these hormones on morphological differentiation of cytotrophoblasts into syncytiotrophoblast. In contrast, others have observed no effect of estrogen on trophoblast CS secretion in vitro (48) or plasma CS levels in women (49). The regulation of CS, therefore, appears multifaceted and whether the inhibitory effect of estrogen on placental trophoblast CS formation shown in baboons of the present study, reflected direct or indirect actions through these other endocrine pathways remains to be determined.

Serum estradiol levels were greater in the uterine than peripheral saphenous vein after androstenedione administration to baboons and also were greater in the uterine vein after androstenedione compared with estradiol treatment. This might be expected because, in contrast to estradiol, androstenedione is readily taken up by and aromatized within the primate placental trophoblast to estrogen, which is then selectively secreted into the maternal compartment via the uterine veins (33, 50). Nevertheless, both androstenedione and estradiol were effective in suppressing placental villous CS expression, although syncytiotrophoblast CS levels were lower in estradiol-treated than in androstenedione-treated baboon. The reason(s) for this difference is unknown, but as noted above did not reflect higher levels of estrogen within the placenta (i.e. uterine vein) after estradiol administration.

In summary, the present study shows that increasing estrogen by administration of androstenedione or estradiol in early baboon pregnancy suppressed CS mRNA and protein levels within the placental syncytiotrophoblast, whereas 3ß-HSD, 11ß-HSD-2, and P-450 aromatase expression was unaltered. Therefore, estrogen has very different and specific actions on steroid and peptide hormone biosynthesis within the placental trophoblast, which we propose are important in regulating placental function and promoting fetal-placental development in the primate.


   Acknowledgments
 
We are grateful to Dr. T. Golos for providing the rhesus monkey CS-3 cDNA, to Dr. J. I. Mason for the 3ß-HSD antibody, Dr. J. R. Seckl for the 11ß-HSD-2 antibody, and Dr. Evan Simpson for the P-450 aromatase antibody. We also sincerely appreciated the secretarial assistance of Mrs. Wanda H. James with the manuscript.


   Footnotes
 
This work was supported by NIH Research Grant R01 HD-13294.

Abbreviations: CS, Chorionic somatomammotropin; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; LDL, low-density lipoprotein; P-450scc, P-450 cholesterol side-chain cleavage; poly(A)+, polyadenylated (A)+; PRL, prolactin.

Received December 27, 2002.

Accepted May 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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