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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 12 4105-4110
Copyright © 1997 by The Endocrine Society

Original Studies

Functional Differentiation of Placental Syncytiotrophoblasts during Baboon Pregnancy: Developmental Expression of Chorionic Somatomammotropin Messenger Ribonucleic Acid and Protein Levels1

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

Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction (B.M., E.D.A.), University of Maryland School of Medicine, Baltimore, Maryland 21201; and the Department of Physiology (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}ummc001.ummc.ab.umd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of the present study was to determine whether, in addition to the onset of chorionic somatomammotropin (CS) production previously shown to result from the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts, there is a further developmental increase in the capacity of syncytiotrophoblasts to produce CS with advancing stages of baboon pregnancy. Placentas were obtained from baboons in early (days 48–62), mid (days 97–110), and late (days 161–175) gestation (term = 184 days), and CS messenger ribonucleic acid (mRNA) and protein levels were determined in a syncytiotrophoblast-rich cell fraction isolated by Percoll gradient centrifugation. CS mRNA levels in syncytiotrophoblasts, expressed as a ratio of ß-actin, exhibited a progressive increase from early (0.04 ± 0.04 relative arbitrary units) to mid (2.37 ± 0.33; P < 0.001) to late (3.66 ± 0.39; P < 0.05) gestation. Levels of the 22-kDa CS protein were very low on days 48–55 (0.83 ± 0.09 arbitrary units), increased 10-fold (P < 0.001) on days 57–60 (8.11 ± 0.68), and increased (P < 0.001) to a maximum of 14.58 ± 0.58 near term. CS mRNA levels in whole placental villous tissue increased (P < 0.05) between early (0.89 ± 0.48) and mid (2.97 ± 0.47) gestation, then remained constant. CS protein exhibited a similar increase (P < 0.001) in villous tissue between early (2.32 ± 0.40) and mid (6.07 ± 0.24) gestation, then remained constant. The increase in mRNA and protein levels of CS in the placenta was accompanied by a progressive (P < 0.001) rise in serum CS. We conclude that in addition to the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts that has been well established to result in the onset of CS biosynthesis, villous syncytiotrophoblasts undergo functional/biochemical differentiation thereafter, manifested as an increase in the capacity for the synthesis of CS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FIRST half of human and nonhuman primate pregnancy is characterized by the proliferation and morphological differentiation of villous cytotrophoblasts into syncytiotrophoblasts (1, 2, 3). As a consequence of morphological differentiation, syncytiotrophoblasts synthesize peptide hormones, e.g. chorionic somatomammotropin (CS) (3, 4, 5, 6), as well as components of the steroid biosynthetic pathway, e.g. the P-450 cholesterol side-chain cleavage (scc) system (2, 6). CS is a single chain polypeptide hormone of 22 kDa molecular mass that is produced in progressively larger quantities within the placenta with advancing gestation (7, 8, 9, 10). The CS locus is comprised of at least three genes in the human (hCS-A, -B, and -L) (11, 12) and rhesus monkey (mCS-1, -2, and -3) (10). The hCS-A and -B (12, 13) and mCS-1 and -2 genes (10) encode identical proteins and exhibit 94% nucleotide and amino acid sequence homology with hCS-L and mCS-3, respectively. Although cAMP and other factors have the capacity to stimulate the expression of CS in trophoblasts in culture (14, 15, 16), the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts has been considered the principal mechanism by which the production of CS is initiated (4, 7, 17, 18). Consequently, it has generally been concluded that the progressive increases in CS in maternal serum (19), CS messenger ribonucleic acid (mRNA) and protein levels per g whole placental tissue (7, 10, 18), and CS mRNA in cultures of trophoblasts (20) simply reflected an increase in the number of syncytiotrophoblasts, and thus placental mass, and that maximal CS expression occurs with syncytial formation (7, 13, 18). In support of this concept, the content of CS mRNA per U syncytial mass determined by in situ hybridization was reported to be similar in the first and third trimesters of human pregnancy (4, 17).

In addition to the critical role that morphological differentiation has in the capacity of trophoblasts to produce hormones, we have recently shown an estrogen-dependent developmental increase in the expression of P-450scc (21, 22), low density lipoprotein (LDL) receptor (23), and LDL uptake (24, 25, 26), within a syncytiotrophoblast-rich cell fraction isolated from placentas in the latter two thirds of baboon pregnancy. We have proposed, therefore, that with advancing gestation syncytiotrophoblasts undergo a functional/biochemical differentiation process that is regulated by estrogen and that results in enhanced expression of key components of steroidogenesis.

The present study was conducted, therefore, to determine whether in addition to the onset of CS production clearly shown to result from the transformation of cytotrophoblasts into syncytiotrophoblasts, there is a further developmental increase in the capacity of syncytiotrophoblasts to produce CS as well as steroidogenic components with advancing baboon pregnancy. To examine this possibility, we determined CS mRNA and protein levels in a syncytiotrophoblast-rich cell fraction as well as in whole placental villous tissue obtained at early, mid, and late baboon gestation.


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

Female baboons (Papio anubis), weighing 13–15 kg, were obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX) and housed, maintained, and mated as previously described (27). Saphenous vein (4 mL) blood samples were obtained after brief sedation with an im injection of ketamine HCl at 1- to 2-day intervals between days 30–175 of gestation (length of gestation = 184 days), and serum was stored at -20 C. Animals were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol employed 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

Whole placentas were obtained from baboons not in labor at the time of pregnancy termination in early (days 48–62), mid (days 97–110), and late (days 161–175) gestation by elective cesarean section under halothane (1.0–1.5%)-nitrous oxide (0.5 L/min)-oxygen (2.0 L/min) anesthesia. Randomly selected sections of villous tissue were frozen in liquid nitrogen for subsequent RNA isolation and Western blot analysis or placed in Hanks’ Balanced Salt Solution (Life Technologies, Gaithersburg, MD) for trophoblast cell dispersion. A syncytiotrophoblast-enriched cell fraction was isolated by dispersion of villous tissue with 0.1% collagenase and 50% (24, 28) or 5–70% (2, 29) Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradient centrifugation. Human placental villous tissue was obtained after normal term spontaneous delivery.

Northern blot analysis

Total RNA was isolated by acid-guanidinium isothiocyanate-phenol-chloroform extraction (30). Five micrograms of polyadenylated [poly(A)+]-enriched RNA, purified by oligo(deoxythymidine)-cellulose chromatography (Pharmacia Biotech, Piscataway, NJ), was size-fractionated in 1% formaldehyde-agarose gel and transferred to nylon membrane (GeneScreen, DuPont-New England Nuclear, Boston, MA). Hybridization was performed for 23 h at 42 C, as previously described (23), using rhesus mCS-3 complementary DNA (cDNA), provided by Dr. Thaddeus Golos, Wisconsin Regional Primate Research Center, University of Wisconsin (Madison, WI), and human ß-actin cDNA (no. 65128, American Type Culture Collection, Rockville, MD). The mCS-3 cDNA was chosen because it hybridizes with mRNA that exhibits a progressive increase in expression in placental villi with advancing rhesus monkey gestation (10, 31). After hybridization with cDNAs labeled with [32P]deoxy-CTP (3000 Ci/mmol; Amersham, Arlington Heights, IL), membranes were washed using stringent conditions and exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY).

Western blot analysis

Syncytiotrophoblast cell fractions were solubilized in 0.5% Triton X-100, 25 mmol/L Tris, 250 mmol/L NaCl, and 5 mmol/L ethylenediamine tetraacetate, pH 7.5, containing 1 mmol/L phenylmethylsulfonylfluoride and 10 µg/mL leupeptin for 20 min on ice. Villous tissue was homogenized on ice in 6 vol 25 mmol/L Tris, 0.25 mol/L sucrose, 1 mmol/L ethylenediamine tetraacetate, and 1 mmol/L phenylmethylsulfonylfluoride, pH 7.4. Proteins (12 µg cells and 50 µg tissue) and serum samples (3 µL) were boiled for 2 min in SDS loading buffer, separated on 16% Tris-glycine gels (Novex, San Diego, CA) under nonreducing conditions at 140 V for 2 h (32), and transferred to nitrocellulose or polyvinylidene difluoride membranes at 4 C for 2 h under a constant current of 40 V using a Novex Blot Module. After washing and blocking the membrane for 2 h at room temperature in 3% BSA in TBST (50 mmol/L Tris, 0.9% NaCl, and 0.1% Tween-20), immobilized proteins were incubated with peroxidase-conjugated rabbit anti-hCS antibody (Dako Corp., Carpenteria, CA) for 1 h at room temperature in 0.78 µg/mL TBST. Chemiluminescence (ECL kit, Amersham) was visualized by exposure to x-ray film (Eastman Kodak).

Quantification of mRNA and protein

Scanning densitometry (Video Densitometer model 620, Bio-Rad, Richmond, CA) was performed to quantify the levels of mRNA and protein. The intensities of the bands were expressed as arbitrary units, and the levels of mRNA expression were standardized against ß-actin for each sample.

RIA of estradiol

Serum estradiol levels were determined by RIA using a solid phase 125I RIA (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). Intra- and interassay coefficients of variation were 6.2% and 7.3%, respectively.

Statistical analysis of data

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Syncytiotrophoblast CS mRNA and protein

We have shown previously by the extensive immunocytochemical localization of syncytiotrophoblast-specific pregnancy-specific ß1-glycoprotein and CS, that the placental cell fraction obtained by 50% Percoll is highly enriched for syncytiotrophoblasts (22, 28). Moreover, the relative purity of the cell fraction for syncytiotrophoblasts was similar at early, mid, and late baboon gestation (28).

Both human and baboon RNA from term placental trophoblast tissue hybridized with the mCS-3 cDNA to yield a single 0.9-kilobase mRNA transcript (Fig. 1Go). A representative Northern blot of CS mRNA expression in baboon syncytiotrophoblasts isolated by 50% Percoll centrifugation in early, mid, and late gestation is shown in Fig. 2AGo. Cumulative results for all animals are shown in Fig. 2BGo. CS mRNA levels (mean ± SE) in syncytiotrophoblasts were negligible early in gestation (0.04 ± 0.04 relative arbitrary units), then increased approximately 50-fold (P < 0.001) by midgestation (2.37 ± 0.33), and further increased (P < 0.05) in late gestation (3.66 ± 0.39).



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  		Figure 1. Northern blot of CS mRNA in baboon (B) and human (H) placental villous tissue. Poly(A)+-enriched RNA (5 µg) was hybridized with approximately 106 cpm/mL [32P]deoxy-CTP-labeled mCS-3 cDNA. The molecular size of transcript was determined from the migration pattern of a 0.24- to 9.5-kilobase RNA ladder. Autoradiogram exposure was for 20 min.

 


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  		Figure 2. A, Representative Northern blot of CS mRNA in baboon placental syncytiotrophoblasts. Poly(A+)-enriched RNA (5 µg) was obtained from syncytiotrophoblasts isolated by 50% Percoll gradient centrifugation at early (days 58–62; RNA from 5 animals pooled to yield 1 sample; lane 1), mid (days 97–110; RNA from 3 baboons pooled for sample in lane 2; samples in lanes 3 and 4 from individual baboons), and late (days 161–175; 3 individual baboons; lanes 5–7) gestation. Autoradiogram exposure was for 90 min. B, Cumulative results of the ratio of intensities of CS and ß-actin mRNA determined by autoradiographic densitometry in syncytiotrophoblasts. Placentas were obtained in early (3 samples pooled from 10 baboons), mid (7 samples pooled from 9 baboons), and late (8 samples pooled from 11 baboons) gestation. Each bar represents the mean ± SE and is expressed as relative arbitrary units. Values with different letter superscripts differ from P < 0.05 to P < 0.01 (by ANOVA and Newman-Keuls multiple comparison test).

 
The anti-hCS antibody recognized a 22-kDa CS protein in human and baboon placental trophoblast (Fig. 3Go). A representative Western blot of CS protein expression in syncytiotrophoblasts isolated by 5–70% Percoll gradient centrifugation is shown in Fig. 3AGo. Consistent with the results obtained for CS mRNA expression, levels of CS protein in syncytiotrophoblasts were very low on days 48–55 (0.83 ± 0.09), increased approximately 10-fold (P < 0.001) on days 57–60 (8.11 ± 0.68), and increased (P < 0.001) to a maximum of 14.58 ± 0.58 near term (Fig. 3BGo).



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  		Figure 3. A, Representative Western immunoblot of CS in baboon placental syncytiotrophoblasts. Proteins (12 µg) obtained from syncytiotrophoblasts isolated by 5–70% Percoll gradient centrifugation on days 48 (lane 1), 49 (lane 2), 54 (lane 3), 57 (lane 4), 59 (lane 5), 100 (lanes 6 and 7), and 170 (lane 8) of gestation were incubated with peroxidase-conjugated anti-hCS antibody, and chemiluminescence was visualized on x-ray film. The molecular mass of CS was determined relative to molecular mass standards (4–250 kDa) and was verified with placental hCS standard (lane 9). B, Cumulative results of CS protein levels determined by autoradiographic densitometry in placental syncytiotrophoblasts obtained from baboons in very early (days 48–55; seven samples from seven baboons), early (days 57–60; six samples from six baboons), mid (day 100; six samples from six baboons), and late (days 161–175; nine samples from nine baboons) gestation. Each bar represents the mean ± SE and is expressed as relative arbitrary units. Data points with different letter superscripts differ at P < 0.001.

 
Placental villous CS mRNA and protein

CS mRNA levels in whole placental villous tissue increased approximately 3-fold (P < 0.05) between early (0.89 ± 0.48) and mid (2.97 ± 0.47) gestation (Fig. 4Go), then plateaued and were maintained elevated through late gestation (3.83 ± 0.41). Consistent with CS mRNA expression, CS protein levels in villous tissue (Fig. 5Go) increased approximately 3-fold (P < 0.001) between early (2.32 ± 0.40) and mid (6.07 ± 0.24) gestation, then plateaued and remained constant through late gestation (5.78 ± 0.29).



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  		Figure 4. A, Representative Northern blot of CS mRNA in placental villous tissue during baboon pregnancy. Five micrograms of poly(A+)-enriched RNA were obtained from baboons in early (days 58–62; RNA from 5 animals pooled to yield 2 samples; lanes 1 and 2), mid (days 97–110; 4 individual baboons; lanes 3–6), and late (days 161–175; 5 individual baboons; lanes 7–11) gestation. Autoradiogram exposure was for 15 min. B, Cumulative results of the ratio of CS and ß-actin mRNA levels (mean ± SE) in placental villous tissue obtained from baboons in early (3 samples pooled from 10 baboons), mid (6 samples pooled from 8 baboons), and late (8 samples pooled from 11 baboons) gestation. Data points with different letter superscripts differ at P < 0.05.

 


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  		Figure 5. A, Representative Western immunoblot of CS in baboon placental villous tissue. Proteins (50 µg) from placental extracts were obtained from baboons in early (lanes 1–4), mid (lanes 5–8), and late (lanes 9–12) gestation (four animals for each gestational period). Placental hCS is shown in lanes 13 and 14. B, Cumulative results of CS protein levels (mean ± SE) in placental villous tissue obtained from baboons in early (n = 12), mid (n = 12), and late (n = 4) gestation. Data points with different letter superscripts differ at P < 0.001.

 
Serum CS

CS was not detectable by Western immunoblot in maternal serum obtained on days 40–50 of gestation (Fig. 6AGo). CS was first detectable at a very low level on day 60 of gestation (0.18 ± 0.07), then progressively increased (P < 0.001) during mid (3.14 ± 0.34) and late (5.57 ± 0.89) gestation.



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  		Figure 6. A, Representative Western immunoblot of serum CS during baboon pregnancy. Serum (3 µL) was obtained from baboons on days 45 (lanes 1–3), 60 (lanes 4–6), 100 (lanes 7–9), and 170 (lanes 10–12) of gestation (three baboons for each gestational period). The hCS standard is shown in lane 13. B, Cumulative results of CS protein levels (mean ± SE) in serum samples obtained from baboons in early (day 60; n = 17), mid (day 100; n = 12), and late (day 170; n = 6) gestation. Values with different letter superscripts are different at P < 0.001.

 
Placental weight progressively increased (P < 0.001) from early to mid to late gestation (Table 1Go). When expressed relative to placental weight, CS levels were approximately 7-fold (P < 0.01) greater at mid and late gestation than in early pregnancy, but were similar at mid and late gestation.


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  		Table 1. Maternal serum CS concentrations and placental weight in baboons

 
Serum estradiol

Maternal serum estradiol concentrations increased from a low of 0.13 ± 0.01 ng/mL on days 30–50 to 0.34 ± 0.06 ng/mL on days 51–59, then surged to 1.57 ± 0.12 ng/mL between days 60–80 of gestation. Estradiol then decreased before exhibiting a gradual and sustained increase throughout the second half of pregnancy, reaching peak values of 4–6 ng/mL near term.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study show for the first time in primate pregnancy that syncytiotrophoblasts isolated from the placenta in early, mid, and late baboon gestation exhibited a progressive developmental increase in the capacity to express CS mRNA and protein. It appears, therefore, that in addition to the initiation of CS expression resulting from the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts, clearly shown previously in humans and rhesus monkeys (4, 7, 10, 17, 18, 33, 34), after their formation, syncytiotrophoblasts display a further progressive increase in the capacity to synthesize CS with advancing primate pregnancy. The increase in CS expression by syncytiotrophoblasts was similar to that we recently reported for P-450scc (22) and LDL receptor (23) mRNA expression and LDL uptake (28) in baboon syncytiotrophoblasts. We propose, therefore, that in addition to the developmental increase in specific components of the progesterone biosynthetic pathway, the increase in CS mRNA and protein levels in syncytiotrophoblasts reflects a functional/biochemical differentiation of syncytiotrophoblasts after they have been formed from cytotrophoblasts. It is suggested that after their appearance, syncytiotrophoblasts undergo a functional change that engenders them with an enhanced capacity to produce protein and steroid hormones.

The marked rise in CS expression in whole placental villous tissue observed in this study with advancing gestation presumably reflected not only the extensive transformation of cytotrophoblasts into syncytiotrophoblasts, which occurs in the first half of pregnancy and has generally been considered the basis of the rise in CS in villous tissue, but also the functional differentiation of syncytiotrophoblasts. This is a very different concept from that held in the past, when it was concluded that the increase in CS expression per U whole villous tissue simply reflected the increase in the relative proportion of syncytiotrophoblasts (4, 17, 18).

Concomitantly with the developmental increase in CS mRNA and translated protein in the placenta, maternal serum CS concentrations rose to levels in late gestation about 30-fold higher than those in early baboon pregnancy. This is in agreement with previous findings in human (35, 36, 37) and monkey (38, 39, 40) pregnancy. As the relative increase in CS in placental villi was much less than that in the peripheral circulation, the progressive rise of this hormone in maternal serum has been generally concluded to reflect transformation of trophoblasts and increased placental mass (4, 37). However, based on the results of the present study, we propose that the progressive increase in serum CS levels reflects not only the latter aspects of development, but functional differentiation of placental trophoblasts as well, resulting in increased capacity of the syncytiotrophoblasts to produce CS.

The observation of a progressive increase in CS mRNA and protein levels in syncytiotrophoblasts with advancing baboon pregnancy contrasts with that obtained by in situ hybridization of CS in the human placenta (4). In the latter study it was concluded that the content of CS mRNA per U syncytial mass, estimated by the number of grains counted per syncytial nucleus, was constant in placentas obtained in the first and third trimesters. The reason(s) for these apparently conflicting results is unknown, although very different methodological procedures were employed in the two studies. The progressive increase in CS, determined by both Northern and Western blot in the present study, does not seem to reflect a difference in the qualitative nature of the syncytiotrophoblast cell preparation obtained in early, mid, and late gestation, because we have previously shown that the relative purity of this cell preparation appeared the same at each of these times in gestation (22, 28).

Considering the progressive increase in CS mRNA and protein levels in syncytiotrophoblasts in early, mid, and late baboon gestation, a comparable progressive rise in CS might have been anticipated in whole villous tissue throughout pregnancy. However, we have recently shown that although LDL receptor (23) and P-450scc (22) expression also progressively increased within syncytiotrophoblasts, the mRNA levels for these steroidogenic components in villous tissue remained constant or actually decreased with advancing gestation (23). We have suggested that the disproportionately large increase in the development of nonendocrine components of placental villous tissue, e.g. vascular tissue (41), that occurs in the second half of pregnancy may confound the measurement of syncytiotrophoblast endocrine function when assessing villous tissue. The increase in CS, expressed as a ratio of placental weight (Table 1Go) between early and midgestation, and the constant level thereafter are consistent with this possibility.

On the basis of several in vivo experimental approaches, we have previously demonstrated that estrogen regulates the functional differentiation of syncytiotrophoblasts and consequently the ontogenetic increase in expression of the LDL/P-450scc pathway in the second half of baboon pregnancy (24, 25, 26, 28, 42). Because the abrupt increase in syncytiotrophoblast CS expression on days 57–60 of baboon gestation (Fig. 3Go) was preceded by a rise in estrogen levels (Fig. 7Go), and there was a parallel rise in estrogen and CS thereafter, it is possible that CS formation within syncytiotrophoblasts is also dependent upon estrogen. Although further study is needed to determine this possibility, we propose that in the first half of gestation the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts enables the latter cells to produce CS, and that estrogen then acts upon syncytiotrophoblasts to regulate their biosynthetic capacity to form hormones.



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  		Figure 7. Maternal serum estradiol levels in baboons. Each data point represents the mean of three to eight baboons.

 
In conclusion, the present study shows a progressive developmental increase in CS mRNA and protein levels in syncytiotrophoblasts from early to mid to late baboon gestation. We suggest that in addition to the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts that has been well established to result in the onset of CS biosynthesis, villous syncytiotrophoblasts undergo functional/biochemical differentiation thereafter, manifested as an increase in the capacity for CS synthesis.


   Acknowledgments
 
The authors are grateful to Dr. Thaddeus Golos for his helpful advice and for generously providing the rhesus monkey CS-3 cDNA. The authors also appreciate the assistance of Mr. Jeffery S. Babischkin and Dr. William G. Zollers with the Northern blot analysis, and the secretarial assistance of Mrs. Wanda H. James.


   Footnotes
 
1 This work was supported by NIH Research Grant R01-HD-13294 and NIH National Research Scientist Award F32-HD-08075. Back

Received April 28, 1997.

Revised August 13, 1997.

Accepted August 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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