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a Department of Obstetrics, Gynecology and Reproductive Sciences, Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201
b Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23501
ABSTRACT
The present study was conducted to determine the developmental expression of placental insulin-like growth factor (IGF)-II, IGF-binding protein (IGFBP)-1 and -2, and IGF-II receptor mRNA expression during baboon pregnancy and whether estrogen, the levels of which increase with advancing pregnancy, regulates placental trophoblast IGF-II mRNA expression. Levels of the IGF-II 6.1-kilobase (kb) and 4.9-kb mRNA transcripts determined by Northern blot analysis progressively increased three- to fourfold in placental syncytiotrophoblast and whole-villous tissue between early (Day 60), mid (Day 100), and late (Day 170) baboon gestation (term = 184 days). In contrast, syncytiotrophoblast IGFBP-1 and -2 mRNA levels decreased, and IGF-II receptor mRNA expression remained relatively constant, with advancing baboon pregnancy. Placental cytotrophoblast IGF-II mRNA levels determined by competitive reverse transcription-polymerase chain reaction on Day 54 of gestation were increased (P < 0.05) almost twofold at 18 h after acute administration of estradiol to baboons, whereas long-term estrogen treatment had no effect. We propose that these changes in trophoblast IGF expression would provide a mechanism for enhancing net bioavailability and bioreactivity of IGF-II locally to promote the growth and development of the placenta and, consequently, of the fetus during primate pregnancy.
estradiol, growth factors, placenta, pregnancy, syncytiotrophoblast
INTRODUCTION
The placenta undergoes growth as well as morphological and functional changes with advancing primate pregnancy to promote fetal development. Placental growth results from the proliferation of stem cell-like villous cytotrophoblasts and their morphological differentiation and recruitment into the syncytiotrophoblast [1]. Using the baboon as a nonhuman primate model to study human fetal-placental development, we have recently shown that placental villous cytotrophoblast transformation into syncytiotrophoblast was accelerated early during gestation in baboons in which estrogen levels were increased by administration of aromatizable androstenedione [2]. Estrogen also stimulated morphological differentiation of human cytotrophoblasts into syncytiotrophoblast in vitro [3]. We have also shown that, after formation, the syncytiotrophoblast undergoes an estrogen-dependent functional differentiation, manifesting as enhanced expression of the low-density lipoprotein receptor and P450 cholesterol side-chain components of the progesterone biosynthetic pathway [4, 5] and the 11ß-hydroxysteroid dehydrogenase enzymes, which govern transplacental corticosteroid metabolism and maturation of the fetal pituitary-adrenocortical axis [6, 7].
Although the primate placenta expresses the estrogen receptor [8], it remains to be determined whether estrogen stimulates trophoblast morphological and functional differentiation directly or indirectly via other factors. Peptide growth factors, such as insulin-like growth factor (IGF)-II, have a major role in placental and, consequently, fetal growth and development [9, 10]. For example, a decrease in placental and fetal growth occurred in transgenic mice with reduced or absent IGF-II gene expression [11], and umbilical serum IGF-I and -II concentrations were positively correlated with human birth weight and gestational age [12, 13]. Moreover, the mRNA and protein for IGF-II are expressed in human placental villous cytotrophoblasts and syncytiotrophoblast [14–17]. Human placental explants [18] and baboon placental trophoblast incubates [19] produced greater quantities of IGF-II than of IGF-I.
The IGF-binding proteins (IGFBPs) transport IGF-I and -II to promote their bioavailability and biologic actions [9]. The human placenta expresses certain IGFBPs [16, 20], and human umbilical serum concentrations of IGFBP-1 and -2 were inversely correlated with serum levels of unbound IGF-I and -II and with birth weight [13]. The actions of IGF-I and -II are mediated via the IGF type I and mannose-6-phosphate/type II receptors [9]. The mRNA for IGF-II receptor is expressed in human placental cytotrophoblasts [15] and syncytiotrophoblast [21].
Estrogen has a central role in primate fetal-placental development, and IGFs are important to placental growth and development. However, it is not known whether IGF-II and its binding proteins and receptors are developmentally regulated in the primate placenta, or whether estrogen regulates their expression. To determine these possibilities, mRNA levels for IGF-II, IGFBP-1 and -2, and the IGF-II receptor were determined in syncytiotrophoblast and whole-villous tissue obtained from the placenta at early, mid, and late baboon pregnancy, and IGF-II mRNA expression was determined in villous cytotrophoblasts after both short- and long-term estradiol administration in early baboon pregnancy.
MATERIALS AND METHODS
Animals
Female baboons (Papio anubis), weighing 13–18 kg, were housed individually in aluminum-stainless steel, large-primate cages and received high-protein monkey chow (Purina Mills, Inc., St. Louis, MO) and fresh fruit twice daily, vitamins daily, and water ad libitum. Females were paired with male baboons for up to 5 days at the expected time of ovulation as determined by menstrual cycle history and turgescence of external sex skin. Placentas were obtained for the developmental study of IGF-II, IGFBP, and IGF-II receptor mRNA levels by Northern blot analysis from untreated baboons at early (Day 60, n = 11), mid (Day 100, n = 10), and late (Day 170, n = 12) gestation (term = 184 days) via cesarean section under halothane/nitrous oxide/oxygen anesthesia. Placentas for cytotrophoblast IGF-II mRNA analysis by competitive reverse transcription-polymerase chain reaction (RT-PCR) were also obtained by cesarean section on Day 54 from baboons 6 h (n = 3) or 18 h (n = 3) after administration via an antecubital vein of 500 µg of free 17ß-estradiol in 1.0 ml of 5% (v/v) ethanol:saline or 18 h after administration of saline vehicle alone (n = 6). Additional baboons were treated s.c. with either 250 µg/day of estradiol benzoate in 1.0 ml of sesame oil on Days 45–59 (n = 5), 30 mg/day of androstenedione in 1.0 ml of sesame oil on Days 30–59 (n = 2), or sesame oil vehicle only (n = 7), with placentas being removed by cesarean section on Day 60 for quantification of cytotrophoblast mRNA levels by competitive RT-PCR. Serum estradiol concentrations were determined by automated chemiluminescent immunoassay (Immulite; Diagnostic Products Corp., Los Angeles, CA), as described previously [2], performed on blood samples (2 ml) obtained from a uterine vein immediately before removing the fetus and placenta. Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication 85-23, 1985). 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 Dispersion
Placental and fetal body weights were determined, and placentas were immediately placed on ice and rinsed thoroughly in saline. Sections (
5 mm3) of whole-villous tissue obtained in areas not involving calcification or infarction were snap-frozen and stored in liquid nitrogen for subsequent mRNA analysis by Northern blot. Additional villous tissue was harvested and placed in ice-cold Hanks balanced salt solution (HBSS; Life Technologies, Inc., Gaithersburg, MD), and a syncytiotrophoblast-rich fraction was obtained for the developmental study of mRNA levels by Northern blot analysis as previously described [22]. Briefly, tissue was minced in calcium- and magnesium-free HBSS and cells dispersed in HBSS containing 0.1% collagenase (type 1A, 420 U/mg; Sigma, St. Louis, MO), 0.1% hyaluronidase (type I-S, 300 U/mg; Sigma), 0.01% deoxyribonuclease (DNase-I, 2000 Kunitz U/mg; Sigma), 1.0% lipoprotein-free fetal bovine serum (Life Technologies), 0.025% soybean trypsin inhibitor (type I-S; Sigma), and 4 mM NaHCO3 (pH 7.4) at 37°C. Placental cells were separated from erythrocytes on a preformed, 50% Percoll (Pharmacia Biotech, Piscataway, NJ) gradient, and purified syncytiotrophoblast was washed and then resuspended in HBSS. As shown previously [22, 23], syncytiotrophoblast was the principal tissue present in the cell fraction obtained by collagenase dispersion and 50% Percoll gradient centrifugation at early, mid, and late baboon gestation, as determined by extensive immunocytochemical reactivity with pregnancy-specific B1-glycoprotein and placental lactogen. A cytotrophoblast-enriched fraction for assay of mRNA levels by competitive RT-PCR was obtained from the placentas of estrogen-treated baboons by trypsin dispersion and 5–70% Percoll gradient centrifugation as described previously by our own and other laboratories [24, 25].
Northern Blot Analysis of mRNA
Preparation of RNA Placental syncytiotrophoblast, cytotrophoblasts, and whole-villous tissue were homogenized in 4 M guanidine isothiocyanate, and RNA was extracted with chloroform:isoamyl alcohol (24:1 [v/v]). The aqueous layer was forced through a 23-gauge needle, layered onto a 5.7 M cesium chloride gradient, and centrifuged at 174 000 x g for 21 h at 20°C. The RNA was resuspended in 0.3 M sodium acetate, precipitated with ethanol, dissolved in diethyl pyrocarbonate-treated water, and integrity-assessed by evaluation of 18S and 28S ribosomal RNA. The poly(A)+-enriched RNA was prepared by centrifugation of 500–750 µg of total RNA over columns of oligo(deoxythymidine) cellulose (Pharmacia Biotech). The RNA from placentas was pooled in certain cases, particularly at early gestation, when as many as three individual placentas were utilized to yield one sample.
Northern blot analysis
Approximately 5 µg of poly(A)+-enriched RNA were denatured in 50% formamide, 2.2 M formaldehyde, and 20 mM 3-[N-morpholino] propane sulfonic acid (MOPS; pH 7.0) and size-fractioned by electrophoresis in 1.0% agarose gel containing 0.6 M formaldehyde and 20 mM MOPS (pH 7.0). The RNA was transferred overnight by capillary action in 10x SSC (1.5 M NaCl and 0.15 M sodium citrate-2H2O [pH 7.0]) onto a nylon membrane (Gene Screen; Dupont-New England Nuclear Corp., Boston, MA). The RNA-containing membranes were ultraviolet (UV) cross-linked, baked in a vacuum oven at 80°C for 2 h, and prehybridized in buffer containing 50% formamide, 0.1% polyvinylpyrrolidone, 0.1% BSA, 0.1% Ficoll, 2.5x SSPE (0.375 M NaC1, 0.025 M NaH2PO4-H2O, and 0.0025 M EDTA), 1.0% SDS, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml) for 24 h at 42°C before the addition of probe. The human cDNAs for IGF-II (no. 57482) and ß-actin (no. 65128) were obtained from American Type Culture Collection (Rockville, MD). The cDNA for human IGFBP-1 was provided by the Collaborations Program of Genetech (South San Francisco, CA). The human IGFBP-2 cDNA was provided by Dr. Shunichi Shimasaka (Whittier Institute for Diabetes and Endocrinology, La Jolla, CA), and the human IGF-II receptor cDNA was provided by Dr. William Sly (St. Louis University Medical Center, St. Louis, MO). The cDNAs were labeled with 50 µCi of 
-32P-dCTP (3000 Ci/mmol; Amersham Corp., Arlington Heights, IL) to a specific activity of approximately 109 dpm/µg of DNA using the Random-Primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Hybridization was performed in fresh buffer at 42°C for 21–24 h with approximately 106 cpm/ml of 32P-cDNA probe. After hybridization, the membrane was washed twice at room temperature for 5 min in 2x SSC, then at 65°C for 7–20 min (depending on the probe utilized) in 2x SSC and 1% SDS. Membranes were exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) at -80°C. After exposure, membranes were stripped before rehybridization.
Quantification of mRNA Intensities of blots were analyzed by densitometric autoradiographic scanning using a Model 620 Video Densitometer and 1-D Analyst II software (Bio-Rad, Richmond, CA). The relative intensities of the mRNA transcripts for IGF-II, IGFBPs, and IGF-II receptor were related to those of ß-actin to determine specific effects on expression.
Competitive RT-PCR of IGF-II mRNA
The mRNA levels for IGF-II were quantified by the competitive RT-PCR assay established by Riedy et al. [26] as modified in our laboratory [4].
Primer sequence The IGF-II oligonucleotide primers were synthesized by Life Technologies, selected from the human IGF-II cDNA sequence [27], and flanked a portion of the sequence spanning two exons and overlapping an intron. The primers were as follows: primer 1, downstream, 5'-TGA ACG CCT CGA GCT CCT TGG CGA GCT GCT TCC AGG TGT CAT ATT-3' (position 469–445 linked to 384–365); primer 2, upstream, 5'-AAT TTA ATA CGA CTC ACT ATA GGG AGC AAG CCG TGT GAG CCG TCG-3' (position T7 polymerase sequence; the italicized sequences are linked to 187–206); primer 3, downstream, 5'-GCC TCG AGC TCC TTG GCG AG-3' (position 464–445); and primer 4, upstream, 5'-GCA AGC CGT GTG AGC CGT CG-3' (position 187–206).
Construction of internal standard RNA The IGF-II competitive reference standard (CRS) was prepared according to the method of Riedy et al. [26] using RT-PCR to generate the cDNA template and transcription with T7 polymerase. Total RNA (3 µg) from baboon placenta was reverse-transcribed at 42°C for 60 min in a reaction mixture (20 µl) containing 1 mM each of deoxy(d)-ATP, dCTP, dGTP, and dTTP (Promega Corp., Madison, WI); 1 mM dithiothreitol; 200 U of SUPERSCRIPT RNase H-RT (Life Technologies); 40 U of RNAguard (Pharmacia Biotech); 50 mM Tris-HCl; 75 mM KCl; 3.0 mM MgCl2; and 250 ng of random primers (Life Technologies). After 60 min, the RT mixture was incubated at 70°C for 15 min and then cooled to 4°C, after which 5 µl were added to a PCR mixture (45 µl) containing 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 10 mM Tris-HCl; 1.5 mM MgCl2; 50 mM KCl; 1.25 U of cloned thermus aquaticus DNA polymerase (Amplitaq; Perkin-Elmer Corp., Norwalk, CT); and 20 pmol each of primers 1 and 2. The PCR was performed in a programmable thermal cycler (MJ Research, Inc., Cambridge, MA), and the sample was amplified in 22 or 25 sequential cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min. After the last cycle, the sample was incubated for an additional 5 min at 72°C. An aliquot of the PCR reaction was fractionated by electrophoresis in a 2% agarose gel and visualized in ethidium bromide. The amplified product (248 base pairs [bp]) contained a sequence for T7 polymerase and a 60-bp designated deletion as compared to the target (wild-type) mRNA strand. The PCR product was gel-purified using the QIAEX II gel extraction kit (Qiagen, Valencia, CA). The IGF-II CRS was synthesized from 150 ng of gel-purified template using MEGAscript T7 In Vitro Transcription Kit (Ambion, Inc., Austin, TX).
Competitive RT-PCR A constant amount of total RNA (250 ng) from villous cytotrophoblast was added to the RT mixture containing threefold serial dilutions of the IGF-II CRS (1350–50 attomoles). After completion of the RT, 20 pmol each of primers 3 and 4 were added for the PCR. Negative controls in which either RNA or RT was omitted from the reaction were performed to test for contaminating DNA. The PCR products (278-bp target and 218-bp CRS) were fractionated by electrophoresis, visualized with a UV transilluminator, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA).
Quantification of IGF-II Photographs (negative image) of the amplified products were analyzed by autoradiography using the Model 620 Video Densitometer and 1-D Analyst II software. Intensity was represented as the relative area under each sample band. A correction factor [28] was used to account for the differences in size of the target and CRS cDNAs. The logarithm (log) of the ratio of CRS to target area was plotted as a function of the concentration of IGF-II CRS added to each PCR reaction. The concentration of IGF-II mRNA was determined in which the ratio of CRS to target area was equal to 1 (i.e., equivalence point).
Statistical Analysis
The ANOVA and Newman-Keuls multiple-comparisons test were employed to assess the effects of advancing gestation or estrogen treatment on mRNA levels. Student t-test was utilized to determine differences in IGF-II mRNA transcripts between syncytiotrophoblast and villous tissue.
RESULTS
Placental and Fetal Body Weights and Serum Estradiol
Placental and fetal body weights (means ± SEM) increased (P < 0.01) from 25.9 ± 1.4 and 11.9 ± 0.8 g, respectively, on Day 60 of gestation to 169.0 ± 7.1 and 768.2 ± 27.1 g, respectively, on Day 170 (Table 1). Mean (± SEM) serum estradiol levels in the uterine vein at the time of cesarean section increased from 0.47 ± 0.05 ng/ml on Day 60 of gestation to 8.85 ± 0.81 ng/ml on Day 170 (Table 1).
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Developmental Expression of Placental IGF-II, IGFBP-1 and -2, and IGF-II Receptor mRNA
The mRNA expression was determined in three different sets of placental tissue obtained during early, mid, and late baboon gestation. Representative Northern blots from one set and cumulative results of the three tissue sets are presented in each figure. In both the syncytiotrophoblast (Fig. 1A) and whole-villous tissue (Fig. 2A), major IGF-II mRNA transcripts of 6.1 and 4.9 kilobases (kb) were expressed in baboons. In the syncytiotrophoblast fraction, levels of the IGF-II 6.1-kb (Fig. 1B) and 4.9-kb (not shown) mRNA transcripts, expressed as a ratio of ß-actin, increased, although not significantly, by approximately three- to fourfold, from 0.63 ± 0.40 and 0.62 ± 0.36 (relative arbitrary units), respectively, on Day 60 to 2.72 ± 0.69 and 2.01 ± 0.50, respectively, on Day 170. In whole-placental villous tissue, levels of the IGF-II 6.1-kb (Fig. 2B) and 4.9-kb (not shown) mRNA transcripts, corrected for ß-actin, also exhibited a progressive increase between early (0.40 ± 0.08 and 1.80 ± 0.78, respectively) and late (1.31 ± 0.24 and 3.78 ± 0.70, respectively) gestation. Mean (± SEM) ratio of the IGF-II 6.1-kb and IGF-II 4.9-kb mRNA transcript was always greater (P < 0.0001) in the syncytiotrophoblast fraction (1.32 ± 0.09) (Fig. 1A) than in whole-placental villous tissue (0.32 ± 0.03) (Fig. 2A). However, the ratio of the two IGF-II mRNA transcripts was similar at early, mid, and late gestation within each tissue fraction.
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In contrast to the developmental increase in IGF-II, syncytiotrophoblast IGFBP-1 (P < 0.05 to P < 0.001) (Fig. 3) and IGFBP-2 (Fig. 4) mRNA levels exhibited a progressive decrease between early (5.73 ± 1.0 and 2.16 ± 1.11, respectively) and late (0.26 ± 0.09 and 0.63 ± 0.12, respectively) gestation. The IGFBP-1 and -2 mRNA levels in whole-villous tissue (data not shown) also appeared to decline, but not significantly, between Day 60 (3.09 ± 1.81 and 2.69 ± 2.20, respectively) and Day 170 (1.74 ± 0.78 and 1.24 ± 0.20, respectively) of baboon pregnancy.
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The IGF-II receptor mRNA levels in the syncytiotrophoblast (Fig. 5A) and villous tissue (Fig. 5B) as well as IGF type 1 receptor mRNA levels in the placenta (data not shown) were not significantly different at early, mid, and late baboon gestation.
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Effect of Estrogen on Placental IGF-II mRNA Expression
Placental and fetal body weights were not altered by acute or chronic administration of estrogen (Table 1). Maternal and uterine venous serum estradiol levels on Day 54 of gestation were increased (P < 0.01) by approximately five- and fourfold at 6 and 18 h, respectively, after a bolus injection of estradiol (Table 1).
Figure 6 illustrates a representative quantitative analysis of IGF-II mRNA levels by competitive RT-PCR in baboon placental villous cytotrophoblasts obtained from untreated and estrogen-treated (18 h) baboons. The expected 278-bp IGF-II target and 218-bp IGF-II CRS products generated by PCR are shown in Figure 6A. No PCR product appeared when RNA or RT was omitted from the reaction (data not shown). The slopes of the log of CRS to target areas plotted as a function of increasing amounts of CRS were similar for RNA obtained from untreated and estrogen-treated baboons (Fig. 6B), indicating no difference in amplification frequency.
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Placental villous cytotrophoblast IGF-II mRNA levels, as determined by competitive RT-PCR, were similar in baboons on Day 54 of gestation 6 h after administration of estradiol (1554 ± 298 attomoles/µg of total RNA) (Fig. 7) and saline vehicle (1588 ± 206 attomoles/µg of total RNA). However, placental cytotrophoblast IGF-II mRNA levels were increased (P < 0.05) almost twofold at 18 h after estrogen administration (2941 ± 412 attomoles/µg of total RNA) compared to saline controls (Fig. 7).
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Uterine venous serum estradiol concentrations on Day 60 of gestation were increased approximately threefold by chronic administration of estrogen (1.36 ± 0.29 ng/ml) (Table 1). In contrast to the acute effect of estrogen, however, cytotrophoblast IGF-II mRNA levels were similar on Day 60 in untreated baboons and in animals treated long-term with estradiol or androstenedione (Table 2).
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DISCUSSION
The present study shows a developmental increase in IGF-II mRNA levels in placental syncytiotrophoblast and whole-villous tissue with advancing baboon gestation. In contrast, placental syncytiotrophoblast IGFBP-1 and -2 mRNA levels progressively decreased between early and late baboon pregnancy, whereas IGF-II receptor expression was not significantly altered. Consistent with these results, IGF-II mRNA and protein have been localized by immunocytochemistry and in situ hybridization within the syncytiotrophoblast of the rhesus monkey [29] and human [15, 16] placenta, and we have previously shown that baboon syncytiotrophoblast incubates synthesized and/or secreted significant quantities of IGF-II [19]. Because the IGF-II receptor is also expressed in the trophoblast, locally produced IGF-II could affect the growth of the trophoblast and villous mesenchyme locally. Moreover, binding of IGFs to the binding proteins may decrease their availability to bind to receptor [9, 10], providing a further mechanism for regulating placental-fetal growth. Indeed, the decrease in placental and fetal growth in IGF-II null transgenic mice [11], IGF-II overexpression in patients with Beckwith-Wiedemann overgrowth syndrome [30], IGF-II promotion of mitosis in human placental trophoblasts in culture [15], and the positive correlation of IGF-II levels and human birth weight [12, 13] demonstrate the importance of IGF-II, acting in an autocrine/paracrine fashion, in promoting placental and fetal growth. Collectively, based on the results of the present and previously published studies, we suggest that the developmental increase in IGF-II and decrease in IGFBPs in the syncytiotrophoblast in baboons of the present study would potentially enhance the net bioreactivity of IGF-II, thereby promoting growth and development of the placenta during primate pregnancy.
The present study also shows that IGF-II mRNA was expressed in baboon placental villous cytotrophoblasts, which is consistent with the localization of IGF-II mRNA and/or protein in human placental cytotrophoblasts [15–17]. Moreover, the present results show that cytotrophoblast IGF-II mRNA levels were increased shortly after acute administration of estradiol to baboons early during gestation, when estrogen levels are typically low. Estrogen also accelerated morphological differentiation of villous cytotrophoblasts into syncytiotrophoblast in vivo in baboons [2] and in vitro in human placental cultures [3], and it stimulated functional differentiation of the syncytiotrophoblast during baboon pregnancy [5]. Estrogen also increased IGF-II mRNA levels and cell proliferation in vivo in the uterine myometrium of the rhesus monkey [31], and IGF-II promoted morphological differentiation and growth of the trophoblast [18]. Collectively, based on our results in the primate placenta, we further propose that estrogen's action in promoting placental cytotrophoblast morphological differentiation and syncytiotrophoblast functional differentiation may be modulated by IGF-II.
Although cytotrophoblast IGF-II mRNA expression in baboons was acutely up-regulated by estrogen, cytotrophoblast IGF-II mRNA levels were not changed by long-term administration of estradiol or aromatizable androstenedione. Moreover, the decrease in placental IGF-II protein formation induced in baboons by fetectomy was not reversed by long-term administration of estradiol or androstenedione [19]. This apparent discrepancy between acute and chronic estrogen treatment could reflect potential changes in the IGF-II mRNA turnover rate in primate placenta, which would not be detected by the analysis of steady-state mRNA. Because only mRNA levels were determined in baboons of the present study, analysis of IGF-II protein levels will need to be quantified to definitively determine the role of estrogen in placental IGF-II biosynthesis. Regardless of the difference in IGF-II mRNA expression after acute and chronic estrogen treatment, placental and fetal body weights were not changed by increasing estrogen levels during early baboon pregnancy. Obviously, the regulation of placental IGF production and fetal-placental growth is complex, involving steroid hormones, peptide growth factors, and other factors.
It is generally accepted that IGFBP-1 is expressed primarily by the decidua in early human [17], baboon [32, 33], and rhesus monkey [34] pregnancy. However, using poly(A)+-enriched RNA and Northern blot analysis, the present study shows that the baboon syncytiotrophoblast and whole-villous tissue also express IGFBP-1 and -2 mRNA, particularly during early pregnancy. These latter observations are consistent with the localization of IGFBP-1 [20] and IGFBP-2 [16] proteins by immunocytochemistry in the human syncytiotrophoblast. We suggest, therefore, that the gestation-dependent increase in IGF-II combined with the decline in IGFBP expression within the trophoblast would provide a mechanism for promoting IGF-II action locally in the placental villi.
In summary, the present study shows a progressive increase in IGF-II and decrease in IGFB-1 and -2 mRNA levels in placental syncytiotrophoblast with advancing baboon pregnancy. Moreover, placental villous cytotrophoblast IGF-II mRNA expression was acutely up-regulated by administration of estrogen during early baboon pregnancy. We suggest that these changes in trophoblast IGF-II and IGFBP expression would provide a mechanism for enhancing net bioavailability and bioreactivity of IGF-II locally to promote the growth and development of the placenta and, consequently, of the fetus during advancing primate pregnancy.
ACKNOWLEDGMENTS
The authors appreciate the secretarial assistance of Mrs. Wanda James with the computer graphics and word processing of this manuscript.
FOOTNOTES
First decision: 13 March 2001.
1 Supported by NIH Research Grant R01 HD-13294. 
2 Correspondence: Eugene D. Albrecht, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11-019, 655 West Baltimore St., Baltimore, MD 21201. FAX: 410 706 5747; ealbrech{at}umaryland.edu
3 Current address: Ivy Animal Health, Inc., Overland Park, KS 66214.
Accepted: May 31, 2001.
Received: February 12, 2001.
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5-3ß-hydroxysteroid dehydrogenase isomerase messenger ribonucleic acids during baboon pregnancy. Placenta 1996; 17:595-602[CrossRef][Medline]This article has been cited by other articles:
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)- and estradiol (•)-treated baboons were plotted as a function of CRS added to each PCR reaction. Lines were constructed by linear regression analysis and IGF-II mRNA levels determined from the equivalence points (i.e., intersection of vertical with regression lines)