help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Henson, M. C.
Right arrow Articles by Swan, K. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Henson, M. C.
Right arrow Articles by Swan, K. F.
Endocrinology Vol. 138, No. 4 1385-1391
Copyright © 1997 by The Endocrine Society

ARTICLES

Effects of Reduced Maternal Lipoprotein-Cholesterol Availability on Placental Progesterone Biosynthesis in the Baboon1

Michael C. Henson, Sheila J. Greene, Brett C. Reggio, Wenliang Shi and Kenneth F. Swan

Departments of Obstetrics/Gynecology (M.C.H., S.J.G., B.C.R., W.S., K.F.S.) and Physiology (M.C.H., W.S.), Tulane University School of Medicine, New Orleans, Louisiana 70112-2699; and Tulane Regional Primate Research Center (M.C.H.), Covington, Louisiana 70433-8915

Address all correspondence and requests for reprints to: Michael C. Henson, Ph.D., Department of Obstetrics/Gynecology SL-11, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Maternal low density lipoprotein (LDL) is the principal source of cholesterol substrate for progesterone biosynthesis in the primate placental syncytiotrophoblast. The relationship of LDL-cholesterol availability and other potential cholesterol-yielding pathways to placental progesterone production have not, however, been demonstrated in vivo in a nonhuman primate. Therefore, maternal peripheral lipoprotein-cholesterol and progesterone concentrations were determined in blood samples obtained by venipuncture, from day 72 until day 100, from pregnant baboons (Papio sp) that were either untreated (n = 4) or treated (n = 3) with the inhibitor of hepatic lipoprotein production, 4-aminopyrazolo [3–4-d]pyrimidine (4-APP, 10 mg/kg BW) on days 98–99 of pregnancy (term = 184 days). Although LDL-cholesterol and progesterone levels remained unchanged in untreated animals, LDL-cholesterol concentrations were 9-fold lower (P < 0.005) in baboons receiving 4-APP than in untreated baboons 2 days following initial administration. Commensurate progesterone levels were 3.5-fold lower (P < 0.03) in 4-APP-treated baboons than in untreated baboons. RT-PCR was used to approximate relative changes in transcription of messengers RNAs (mRNAs) for selected cholesterol-sensitive pathways in placental tissue collected on day 100. Thus, expression of mRNAs for LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase appeared enhanced, whereas acyl-coenzyme A:cholesterol acyl transferase (ACAT) mRNA was diminished in syncytiotrophoblast-enriched cell fractions as a result of 4-APP administration. No relative differences in mRNAs were apparent in whole placental villous tissue, however, as a result of 4-APP treatment.

In summary, this experiment demonstrates a significant decline in progesterone production elicited by maternal LDL-cholesterol withdrawal, and attests to the efficacy of 4-APP administration during baboon pregnancy. These results also suggest a commensurate regulation of cholesterol-sensitive pathways in primate syncytiotrophoblast. However, no relative differences were apparent in mRNA levels for LDL receptor, HMG-CoA and ACAT in whole placental villous tissue as a result of LDL-cholesterol withdrawal, which may suggest potential disparities in the mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. those in proliferative nonendocrine placental constituents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE, a steroid hormone produced first during gestation in the ovary and later in the placenta, is vital to the maintenance of primate pregnancy (1). Cholesterol, the necessary substrate for progesterone production by both the ovarian corpus luteum (2) and the placental syncytiotrophoblast (3, 4), is derived primarily from low density lipoprotein (LDL) supplied by the maternal peripheral circulation (5). Thus, LDL is bound to specific receptors on the plasma membrane and is internalized by endocytosis. Resultant endocytic vesicles then fuse with lysosomes, where the protein portion of LDL is hydrolyzed to free amino acids, and stored cholesterol esters are hydrolyzed by an acid lipase. Unesterified cholesterol can then fuel steroidogenesis or be reesterified for storage through the action of the enzyme acyl-coenzyme A:cholesterol acyl transferase (ACAT), which is activated in the presence of surplus cholesterol. Also when cholesterol concentrations are high, the LDL receptor gene is not transcribed into messenger RNA (mRNA), thereby decreasing the number of membrane-bound receptors through direct feedback inhibition. Further, 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase) is inhibited, suppressing the de novo synthesis of cholesterol from acetate. Conversely, in times of intracellular cholesterol shortage, stores are deesterified via the cholesterol ester hydrolase enzyme system. Available substrate can be converted to pregnenolone via P-450 cholesterol side-chain cleavage enzyme, then to progesterone by {Delta}5-3ß-hydroxysteroid dehydrogenase.

It has previously been observed that pregnant women deprived of optimal LDL-cholesterol availability (6) maintained rates of progesterone biosynthesis adequate for pregnancy maintenance, and it has been proposed, therefore, that enzyme systems regulating alternate cholesterol-yielding pathways in the placenta may be sufficient to support a significant, albeit reduced, level of progesterone production (7). We have determined that estrogen regulates LDL uptake in the placental syncytiotrophoblast of the baboon (Papio sp, Refs. 8–10) as it does in other tissues of various species (11, 12, 13), and subsequently reported that as maternal peripheral estrogens levels rise with advancing gestation in the baboon, LDL-cholesterol uptake by the syncytiotrophoblast increases commensurately (14). However, although the LDL receptor of the baboon syncytiotrophoblast appears exquisitely sensitive to the influence of estrogen, we have also observed that progesterone continued to be secreted in amounts adequate for conceptus maintenance when placental LDL uptake was severely inhibited due to estrogen deprivation (9, 10). In a related study, Albrecht and co-workers (15) recently determined that LDL receptor mRNA concentrations in syncytiotrophoblast-enriched fractions were significantly greater at term than at early or mid gestation, although mRNA levels in unprocessed whole villous tissue remained unchanged with advancing baboon pregnancy. In view of this evidence in both the human and nonhuman primate, therefore, we hypothesize that the primate syncytiotrophoblast may potentially derive significant amounts of the cholesterol necessary for limited progesterone production from sources secondary to the preferred LDL receptor pathway, and that mechanisms responsible for cholesterol homeostasis in the steroidogenically active syncytiotrophoblast may be regulated divergently from those in proliferative nonendocrine constituents of the placenta.

Pathways identified as potential sources of cholesterol substrate have been investigated in rodents via treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP), an adenine analog that inhibits the hepatic secretion of lipoproteins (16). However, analogous studies have not been reported in primates, which are dependent upon placental, rather than luteal, progesterone production throughout most of gestation. Thus, in this initial study, we determined LDL-cholesterol and progesterone concentrations in maternal serum, as well as relative abundances of LDL receptor, HMG-CoA reductase, and ACAT mRNAs in placental tissue from both untreated and 4-APP-treated baboons. Our objectives were: 1) to determine the efficacy of 4-APP as an inhibitor of lipoprotein-cholesterol production in the pregnant baboon; 2) to quantitate the short-term effect of LDL-cholesterol withdrawal on placental progesterone production, as measured in the maternal peripheral circulation; 3) to demonstrate the preliminary effect(s) of reduced lipoprotein-cholesterol availability on the relative abundances of placental LDL receptor, HMG-CoA reductase, and ACAT mRNAs, as determined by RT-PCR; and 4) to investigate the potential for differences in the relative expression of these mRNAs in unprocessed villous tissue vs. a purified syncytiotrophoblast-enriched cell fraction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female baboons (Papio sp), weighing 13–17 kg, were individually housed in stainless steel cages at the Tulane Regional Primate Research Center, essentially as previously described (8, 9, 10, 14). A 12-h light, 12-h dark photoperiod was maintained in air-conditioned rooms, and animals received a primate maintenance ration (Teklad Monkey Chow, Harlan/Teklad, Bartonsville, IL) with fresh fruit daily and water ad libitum. Females were quartered with males for mating in indoor/outdoor runs for 5–6 days coinciding with the estimated time of ovulation, as determined by menstrual cycle records and visible turgescence of external sex skin. Blood samples were obtained from pregnant baboons at 2-day intervals from day 72 until day 100 of gestation (term = 184 days). This extended period of sample collection was deemed necessary to establish reliable baseline profiles for maternal peripheral progesterone and lipoprotein-cholesterol. Thus, at 0800–0900 h, a 6-ml blood sample was withdrawn from an antecubital vein via 21-gauge needle after brief restraint and sedation with an im injection of ketamine HCl (10–15 mg/kg BW, Ketalar, Fort Dodge Pharmaceuticals, Fort Dodge, IA). Three baboons were administered 10 mg/kg BW 4-APP (Sigma Chemical Co., St. Louis, MO), po in fruit, on days 98 and 99 of gestation, whereas four baboons remained untreated and received fruit without 4-APP. Animals were maintained and used strictly in accordance with United States Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals (NIH Publication 86–23). The experimental protocol employed in the present study was approved by the Institutional Animal Care and Use Committee of the Tulane Regional Primate Research Center.

Placental collection and preparation of a syncytiotrophoblast-enriched fraction
Baboon placental tissue was obtained via cesarean section on day 100 of gestation under isofluorane anesthesia, following sedation with ketamine HCl/atropine. Samples of placental villous tissue were taken immediately and flash-frozen in liquid nitrogen for storage at -80 C. The remainder of placental tissue was chilled on ice, and cells were dispersed as we have described previously (8, 9, 10, 14). Briefly, villous tissue was minced in calcium and magnesium-free Hank’s Balanced Salt Solution (HBSS; GIBCO, Grand Island, NY) and cells dispersed in HBSS containing 0.1% collagenase (type 1A; 420 U/mg; Sigma), 0.1% hyaluronidase (type I-S; 300 U/mg; Sigma), 0.01% deoxyribonuclease (DNase-I; 2000 Kunitz U/mg; Sigma) 1.0% lipoprotein-free FBS (GIBCO), 0.025% soybean trypsin inhibitor (Sigma) and 4 mM NaHCO3, pH 7.4, at 37 C. Cells suspended in HBSS were layered on preformed 50% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) gradients and an enriched cell fraction sequestered via centrifugation (800 x g, 10 min). We have previously reported that placental cells isolated by this method are primarily syncytiotrophoblasts, as determined via immunohistochemistry (14).

Poly (A)+ RNA extraction
mRNA was isolated from both placental villous tissue and syncytiotrophoblast-enriched cell fractions via the PolyATtract System 1000 (Promega, Madison, WI). According to the manufacturers protocol and in reference to Chirgwin et al. (17), up to 0.125 g of cells or tissue was homogenized in 1 ml of GTC extraction buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.1, 2% ß-mercaptoethanol). Two milliliters dilution buffer (6 x SSC, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.25% SDS, 1% ß-mercaptoethanol) were preheated to 70 C, added to the homogenate, and mixed by inversion. Biotinylated oligo(dT) probe, which binds to the polyA tail of mRNAs, was then added in an amount determined by tissue mass and incubated (5 min, 70 C). The homogenate was centrifugally cleared of debris and Streptavidin MagneSphere Paramagnetic Particles (SA-PMP; Promega) added. Within this suspension, SA-PMPs bind the biotinylated oligo(dT)/mRNA complex. This mixture was then subjected to a magnetic field for capture of SA-PMP complexes, which were then resuspended in nuclease-free water. Following dissociation in water, purified mRNA was precipitated with 0.1 vol 3 M sodium acetate and 1 vol isopropanol, resuspended in diethylpyrocarbonate-treated water, quantitated spectrophotometrically, and stored at -80 C.

RT-PCR
Oligonucleotide primers were synthesized (Tulane Molecular Biology Consortium, Department of Biochemistry, Tulane Medical School) according to the published sequences for ACAT (18, 19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (20, 21), HMG-CoA reductase (20, 22), and LDL receptor (20, 23). PCR primer sequences and product sizes are listed in Table 1Go. Sequences for oligonucleotide primers are approximately 50% GC content and are designed to be RNA specific by spanning the junction of two exons. Complementary DNAs (cDNAs) were synthesized using the SuperScript Preamplification System (GIBCO/BRL, Gaithersburg, MD). Briefly, 0.1 µg mRNA was diluted to 11 µl with diethylpyrocarbonate water. One microliter random hexamer was added to the mRNA, and the mixture incubated (70 C, 10 min) and then placed on ice. Following centrifugation, kit reagents are added to a final concentration of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol and 0.5 mM deoxynucleotide triphosphate. The mixture was incubated (25 C, 5 min) and 1 µl SuperScript II RT (200 U) added. The reaction was incubated (42 C, 50 min) and then terminated (70 C, 15 min). One microliter RNase H was added, and the mixture incubated (37 C, 20 min). PCR proceeded according to the GeneAmp PCR Reagent kit protocol (Perkin-Elmer/Cetus, Norwalk, CT). Briefly, 3.5 µl cDNA was placed in an oil-free thermal cycler tube with kit reagents to a final concentration of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% wt/vol gelatin, 200 µM deoxynucleotide triphosphates, 0.8 µM of each primer, and AmpliTaq DNA Polymerase (1.25 U). PCR was performed (TEMP-TRONIC Thermocycler; Barnstead/Thermolyne, Dubuque, IA) with denaturation at 94 C/90 sec, annealing at 58 C/60 sec, and extension at 72 C/60 sec, for 30 cycles. PCR products were visualized in 2% agarose gels containing ethidium bromide. PCR reactions were accompanied by controls, including: 1) GAPDH as a positive cDNA synthesis control; 2) RNA that had not been transcribed into cDNA as a genomic DNA contamination control; and 3) sterile water blanks as a reagent control.


View this table:
[in this window]
[in a new window]
  		Table 1. Sequences of human ACAT, GAPDH, HMG-CoA reductase, and LDL receptor oligonucleotide primers used for RT-PCR analyses

 
Quantitation of serum lipoproteins
Serum concentrations of LDL- and high density lipoprotein (HDL)-cholesterol were determined according to the method of Noma et al. (24), as adapted for use in our laboratory (14). Briefly, serum (0.2 ml) was first treated with 150 mg sodium heparin (Sigma), 7.35 g CaCl2, and 475 mg NiCl2 to isolate HDL, and then with 500 mg Amberlite anion exchange resin (Sigma) to isolate HDL plus LDL. Two milliliters of O-pthalaldehyde solution (50 mg/100 ml glacial acetic acid; Sigma) were added to both lipoprotein precipitates and cholesterol standards, and following addition of 1.0 ml H2SO4, were incubated (room temperature, 10 min). Absorbance was determined at 550 nm. Serum LDL concentrations were calculated by subtraction of cholesterol concentrations in HDL fractions from concentrations in HDL plus LDL fractions. The interassay coefficient of variation (n = 5 assays) for an LDL-cholesterol standard was 10.2% and for HDL-cholesterol was 11.9%. The intraassay coefficient of variation (n = 3 replicates) was 1.7% for LDL-cholesterol and 2.3% for HDL-cholesterol.

Quantitation of serum progesterone
Serum samples were stored at -20 C until progesterone concentrations were determined by RIA, as we have previously described (14, 25), using Coat-A-Count solid-phase [125I]RIA, according to the directions of the manufacturer (Diagnostic Products Corp., Los Angeles, CA). Inter- and intraassay coefficients of variation for progesterone RIA were 7.4% (n = 4) and 7.0% (n = 3), respectively, with a minimal detection limit of 30 pg/ml.

Statistical analysis
Student’s t test, using the TTEST procedure of the Statistical Analysis System (26), was employed to assess the effects of 4-APP treatment on measured parameters. A significant difference between means was understood to exist when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
4-APP (10 mg/kg BW) elicited a 63% decline in maternal lipoprotein-cholesterol concentrations (combined LDL + HDL) within 24 h in pregnant baboons (Fig. 1AGo). A second administration resulted in a mean lipoprotein-cholesterol concentration 82% lower than that measured before 4-APP. Lipoprotein-cholesterol concentrations in untreated pregnant baboons showed neither a significant increase or decline throughout the same gestational period. LDL- and HDL-cholesterol fractions in untreated baboons also showed no significant fluctuations over the period corresponding to that of 4-APP treatment. LDL- and HDL-cholesterol fractions in 4-APP-treated animals, however, decreased 84% and 74%, respectively, 48 h following initial administration of the drug. Commensurately, maternal peripheral progesterone levels remained unchanged in untreated baboons, but declined 74% in animals that received 4-APP for 2 days (Fig. 1BGo). The effects of 4-APP on maternal LDL-cholesterol and progesterone concentrations are summarized in Fig. 2Go. Thus, maternal LDL-cholesterol levels were approximately 9-fold lower (P < 0.005) in pregnant baboons that received 4-APP (4.5 ± 2.3 mg/dl, mean ± SEM) than in untreated baboons (41.2 ± 6.0 mg/dl), whereas concomitant progesterone concentrations were 3.5-fold lower (P < 0.03) in 4-APP-treated (9.5 ± 3.8 ng/ml) than in untreated (33.9 ± 6.1 ng/ml) pregnant baboons.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Maternal serum lipoprotein-cholesterol (A) and progesterone (B) concentrations between days 72–100 of gestation in untreated pregnant baboons ({circ}, n = 4) and in pregnant baboons that received 10 mg/kg BW 4-APP on days 98 and 99 (•, n = 3). Experiments were terminated upon cesarean section (C-SEC) on day 100 of gestation. Values are means ± SEM.

 


View larger version (18K):
[in this window]
[in a new window]
  		Figure 2. Maternal serum LDL-cholesterol and P4 concentrations on day 100 of gestation in untreated baboons (n = 4) and in baboons that received 10 mg/kg BW 4-APP on days 98 and 99 (n = 3). Values are means ± SEM. Common lowercase letters indicate significant differences between means (a = P < 0.005, b = P < 0.03).

 
Relative amounts of mRNAs for ACAT, HMG-CoA reductase, LDL receptor, and GAPDH were compared via semiquantitative RT-PCR in whole placental villous tissue and purified syncytiotrophoblast-enriched fractions. An identical amount of mRNA (0.1 µg) was used in all RT-PCR reactions, and PCR products from genes of interest were compared with the PCR product for a housekeeping gene, GAPDH. Results are illustrated in Fig. 3Go, A-C. In all panels, lane 1 is a DNA ladder, lanes 2–5 represent placental tissue from an untreated baboon, and lanes 6–9 from a baboon that received 10 mg/kg BW 4-APP on days 98–99 of gestation. Also in all panels, lanes 2 and 3 and 6 and 7 are whole villous tissue and lanes 4 and 5 and 8 and 9 are purified syncytiotrophoblast-enriched cell fractions. Even-numbered lanes represent the gene of interest (LDL receptor, HMG-CoA reductase, ACAT), and odd-numbered lanes represent GAPDH. In Fig. 3AGo, LDL receptor mRNA was detected in relatively equal abundance in whole villous tissue from both untreated (lane 2) and 4-APP-treated (lane 6) baboons. The abundance of LDL receptor mRNA in the syncytiotrophoblast-enriched placental cell fraction from the 4-APP-treated animal (lane 8), however, appeared substantially enhanced over the untreated baboon (lane 4). In Fig. 3BGo, HMG-CoA reductase expression was similar in whole villous tissue taken from untreated (lane 2) and 4-APP-treated (lane 6) baboons. However, as with LDL receptor, HMG-CoA reductase mRNA in the syncytiotrophoblast-enriched fraction appears up-regulated in the 4-APP-treated (lane 8) vs. untreated (lane 4) baboons. In panel Fig. 3CGo, ACAT mRNA abundance was relatively unchanged in untreated (lane 2) and 4-APP treated (lane 6) whole villous tissue, as seen with LDL-receptor and HMG-CoA reductase. However, ACAT mRNA appeared substantially diminished in the syncytiotrophoblast-enriched fraction, as a result of 4-APP treatment (lane 8) vs. untreated (lane 4). The potential effects of 4-APP treatment on the relative abundances of mRNAs are summarized in Table 2Go. Thus, the results suggested an up-regulation of LDL receptor and HMG-CoA reductase mRNAs and a comensurate down-regulation of ACAT mRNA in a syncytiotrophoblast-enriched fraction as a result of cholesterol withdrawal in 4-APP-treated animals. GAPDH transcripts remained relatively unchanged from those of untreated controls in both unprocessed placental villous tissue and in purified syncytiotrophoblast cells as a consequence of 4-APP administration.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 3. RT-PCR of mRNA in untreated (lanes 2–5) and 4-APP-treated (lanes 6–9) baboons. A, Lanes 2 and 6 are LDL receptor (258 bp), and lanes 3 and 7 are GAPDH (240 bp) in samples of gross villous tissue. Lanes 4 and 8 are LDL receptor, and lanes 5 and 9 are GAPDH in syncytiotrophoblast-enriched fractions. B, Lanes 2 and 6 are HMG-CoA reductase (246 bp), and lanes 3 and 7 are GAPDH (240 bp) in samples of gross villous tissue. Lanes 4 and 8 are HMG-CoA reductase, and lanes 5 and 9 are GAPDH in syncytiotrophoblast-enriched fractions. C, Lanes 2 and 6 are ACAT (1664 bp), and lanes 3 and 7 are GAPDH (240 bp) in samples of gross villous tissue. Lanes 4 and 8 are ACAT, and lanes 5 and 9 are GAPDH in syncytiotrophoblast-enriched fractions. Lane 1 in each panel (A, B, and C) is a 1-kilobase DNA ladder.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Summary of effects of 4-APP treatment on LDL receptor, HMG-CoA reductase, ACAT, and GAPDH mRNAs in baboon placental whole villous tissue and syncytiotrophoblast-enriched cell fractions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LDL-cholesterol, manufactured in the liver and present in abundance in the maternal peripheral circulation, is the preferred substrate for placental progesterone biosynthesis in both the human and nonhuman primate. However, the potential for significant substrate contributions by nonlipoprotein cholesterol sources to placental steroidogenesis has been noted in pregnant women afflicted with either homozygous familial hypobetalipoproteinemia or phenotypic abetalipoproteinemia (6). Although limited by the amount of LDL-cholesterol available as precursor, placental progesterone biosynthesis in these women, although diminished, was maintained at a rate sufficient for the successful maintenance of pregnancy, suggesting that intracellular de novo cholesterol synthesis may have been enhanced to provide the necessary precursor. Such coordinated regulation of the LDL receptor and HMG-CoA reductase is well-known in cultured cells (5) and has also been demonstrated in vivo (27). Thus, hepatic mRNA levels for the LDL receptor and HMG-CoA reductase declined in mice fed a cholesterol-enriched diet, demonstrating a coordinated down-regulation necessary for the preservation of normal intracellular cholesterol homeostasis. Similarly, in humans administered mevinolin, an HMG-CoA reductase inhibitor, peripheral plasma LDL concentrations declined by means of enhanced receptor-mediated lipoprotein clearance (28, 29). In respect to steroidogenic tissues, administration of lipoprotein-cholesterol diminished HMG-CoA reductase activity in human choriocarcinoma cells (30) and demonstrated that in the absence of LDL, such cells secrete approximately 50% of the progesterone normally produced when cocultured with LDL-cholesterol. This result strongly suggests that de novo cholesterol synthesis and/or deesterification of intracellular stores constituted a viable source(s) of substrate for progesterone biosynthesis in the absence of LDL (31, 32). Indeed, the capacity for de novo cholesterol production by normal human trophoblast may be similar, as it was reported that only two-thirds of the placental progesterone produced in women infused with radiolabeled cholesterol could be attributed to lipoprotein-cholesterol substrate derived from the maternal circulation (3, 33). The remaining one-third of the cholesterol used by the syncytiotrophoblast for progesterone biosynthesis may have been synthesized intracellularly via HMG-CoA reductase and/or mobilized from esterfied stores. The possibility that the human placenta is capable of producing significant amounts of cholesterol in the absence of preferred LDL-bound substrate has also been addressed in syncytiotrophoblast cultures (34). Thus, the presence of lipoprotein-cholesterol inhibited the incorporation of acetate into cholesterol, although sufficient cholesterol was mobilized from esterified stores and/or synthesized de novo in the absence of LDL, to support a level of progesterone production that was approximately 50% of that measured when LDL was present in cell medium. HMG-CoA reductase, ACAT, and cholesterol ester hydrolase activities, measured radioenzymatically, were adequate to support this rate of production and is in agreement with others who have reported that activities of enzymes mediating de novo cholesterol biosynthesis (35) and cholesterol esterification/deesterification (36) are significant in normal human placental trophoblast. Coordinated regulation of these secondary cholesterol-yielding mechanisms with the preferred LDL-cholesterol pathway had not been demonstrated in vivo in a nonhuman primate model, before the current study.

Mechanisms regulating cholesterol homeostasis have been studied in the absence of lipoprotein-derived cholesterol in rodents, however, via administration of 4-APP. PMS/human CG-primed, superovulated, immature rats were treated with 4-APP (25 mg/kg BW, ip) daily for 3 days, and resultant plasma cholesterol concentrations were 5-fold less, ovarian cholesterol esters 12-fold less, and plasma progesterone concentrations 4-fold less than in nontreated controls. Ovarian HMG-CoA reductase activity increased 20-fold during 4-APP administration. When 4-APP treatment was discontinued, all concentrations returned to those of nontreated controls within 5 days (37). Similarly, later studies determined that luteal cells from superovulated, 4-APP-treated rats released 2-fold less progesterone into medium than did cells from nontreated rats, an effect that was partially overcome by coculture with exogenous lipoproteins (38). Correspondingly, luteal HMG-CoA reductase mRNA levels were significantly enhanced by 4-APP administration (39). It was concluded that 4-APP effectively deprived steroidogenic tissues of cholesterol precursor, and that the availability of blood-borne cholesterol regulates the rate of de novo cholesterol and progesterone biosynthesis in the rodent corpus luteum. Subsequent experiments determined that ACAT activity declined in 4-APP-treated superovulated rats to approximately 15% of that measured in nontreated controls, whereas iv infusion of human lipoproteins reversed the effects of 4-APP on HMG-CoA reductase and ACAT levels, and raised plasma cholesterol 6-fold (40). Thus, use of 4-APP for investigation of the interrelated steroidogenic pathways vital to reproduction has exclusively used rodents, species that are totally dependent upon luteal progesterone for pregnancy maintenance throughout gestation. Experiments using primates, which depend upon the placenta for the production of progesterone throughout most of gestation, have not been performed until now.

In the present study, maternal LDL-cholesterol was approximately 9-fold lower in pregnant baboons that received 4-APP, than in untreated controls, whereas concomitantly measured progesterone concentrations were 3.5 times lower in 4-APP-treated animals. Further, progesterone levels in 4-APP-treated animals were slightly higher than those that we have previously reported for baboons deprived of cholesterol substrate via inhibition of placental LDL receptor-mediated uptake, baboons that nonetheless successfully maintained their conceptuses (8, 9, 10). The current experiment, therefore, marks the first demonstrated 4-APP-induced decline in progesterone production in a primate, and establishes the drug’s efficacy as a tool for future investigations of mechanisms regulating progesterone biosynthesis during baboon pregnancy. Further, in comparison to the constitutive expression of GAPDH, the relative abundances of LDL receptor and HMG-CoA reductase mRNAs in syncytiotrophoblast were enhanced as a result of 4-APP administration, whereas corresponding ACAT mRNA appeared diminished as a result of treatment. Although these results must be interpreted with caution due to the semiquantitative nature of the RT-PCR methodology employed, these effects represent predictable outcomes for tissues expressing coordinated regulation of cholesterol-sensitive pathways and constitute the first in vivo demonstration of such mechanisms regulating progesterone biosynthesis and cholesterol homeostasis in the nonhuman primate maternal-fetoplacental unit. Future studies will determine the potential for secondary cholesterol-yielding mechanisms to return maternal progesterone concentrations in 4-APP-treated animals to concentrations approaching those in normal pregnancy, or to maintain them at depressed levels capable of maintaining pregnancy.

Estrogen mediates placental progesterone biosynthesis in the baboon placental syncytiotrophoblast (1), and we have demonstrated that this regulation does not occur with the terminal conversion of pregnenolone to progesterone, via enhancement of {Delta}5-3ß-hydroxysteroid dehydrogenase activity (41), but rather at the LDL receptor level (8, 9, 10). Comparable studies, utilizing human placental cell cultures in which the stimulatory effect of endogenous estrogen on radiolabeled LDL uptake was attenuated by the potent antiestrogen, 4-hydroxytamoxifen (42), or in which LDL uptake and attendant progesterone release were augmented by coculture with exogenous estrogen (43), strongly suggest the existence of a similar regulatory mechanism in human syncytiotrophoblast and reaffirms the baboon as an excellent model for human pregnancy. Further, we have reported that LDL uptake by the baboon syncytiotrophoblast increases over the course of gestation in response to the increase in maternal estrogen levels typical of advancing primate pregnancy (14). This finding is especially relevant in respect to Albrecht et al. (15), who reported an increase in LDL receptor mRNA levels in baboon syncytiotrophoblast, commensurate with the suggested estrogen-responsive increase in LDL uptake by syncytiotrophoblast cells with advancing gestation. At first analysis, these results appear at odds with reports that [125I]LDL binding to placental microvilli did not differ in the first through third trimesters of human pregnancy (44), or that LDL receptor mRNA concentrations in whole human placental villous tissue were significantly greater early in gestation than at term (45). Although the potential for species differences exist, it must be noted that although LDL receptor mRNA concentrations in a purified syncytiotrophoblast cell fraction increased several-fold with progressive baboon pregnancy, LDL receptor mRNA levels measured in whole villous tissue remained relatively unchanged (15). Thus, in the baboon, the apparently constitutive expression of the LDL receptor in whole villous tissue may have concealed the developmental nature of LDL receptor expression evident in syncytiotrophoblast-enriched fractions collected with advancing gestation. In respect to the steroidogenically active syncytiotrophoblast, therefore, this finding agrees with the increase in [125I]LDL uptake reported earlier to occur with progressive baboon pregnancy (14), and suggests that the accurate quantitation of mRNAs vital to progesterone biosynthesis in unprocessed whole human or baboon villous tissues may be similarly confounded by divergent rates of expression among proliferative nonendocrine components of the placenta. This rationale is further supported by findings of the present study, which suggest substantial changes in LDL receptor, HMG-CoA reductase, and ACAT mRNAs in syncytiotrophoblast as a result of lipoprotein-cholesterol withdrawal, but not in whole villous tissue. These results imply a divergence in the regulation of both primary and secondary cholesterol-yielding mechanisms that may be inherent between steroidogenic, e.g. syncytiotrophoblasts, and nonsteroidogenic, e.g. connective tissue, placental cell subpopulations.

In summary, 4-APP administration to pregnant baboons dramatically decreased lipoprotein-cholesterol levels in the present study. Further, this decrease in peripheral LDL-cholesterol substrate resulted in a significant decline in circulating maternal progesterone during a period in gestation when the placental syncytiotrophoblast is normally responsible for production of the progesterone necessary to maintain pregnancy. Additionally, the potential enhancement of LDL receptor and HMG-CoA reductase mRNAs and the decline in ACAT mRNA in the baboon syncytiotrophoblast, suggested to result from lipoprotein inhibition, constitutes the first demonstration of this compensatory mechanism in the primate placenta. The physiological relevance of the relationship between the preferred LDL-cholesterol pathway and these secondary cholesterol-yielding mechanisms remains to be elucidated, as does a complete understanding of the disparities in expression of mRNAs in syncytiotrophoblast vs. whole villous tissue. Future studies will investigate these issues and better define the capabilities of both de novo cholesterol and deesterification pathways to contribute to the pool of cholesterol substrate available for progesterone biosynthesis during primate pregnancy.


   Acknowledgments
 
We express our sincere appreciation to Dr. Steven M. Hill of the Department of Anatomy, Tulane University School of Medicine, for his advice concerning RT-PCR methodologies; to Drs. Rudolph P. Bohm, Jr. and Marion S. Ratterree, Tulane Regional Primate Research Center, for their surgical expertise; and to Mrs. Nathlynn Dellande for her assistance with the graphic design of figures for this manuscript.


   Footnotes
 
1 This work was supported by NIH Research Grant R29 HD32502 to M.C.H. and by P51 RR00164–32 to the Tulane Regional Primate Research Center. It was presented in part at the 77th Annual Meeting of The Endocrine Society. Back

Received September 19, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pepe GJ, Albrecht ED 1994 Actions of placental and fetal adrenal steroid hormones in primate pregnancy. Endocr Rev 16:608–648[Abstract/Free Full Text]
  2. Carr BR, MacDonald PC, Simpson ER 1982 The role of lipoproteins in the regulation of progesterone secretion by the human corpus luteum. Fertil Steril 38:303–311[Medline]
  3. Hellig H, Gattereau D, Lefebvre Y, Bolte E 1970 Steroid production from plasma cholesterol. I. Conversion of plasma cholesterol to placental progesterone in humans. J Clin Endocrinol Metab 30:624–631[Abstract/Free Full Text]
  4. Winkel CA, Snyder JM, MacDonald PC, Simpson ER 1980 Regulation of cholesterol and progesterone synthesis in human placental cells in culture by serum lipoproteins. Endocrinology 106:1054–1060[Abstract/Free Full Text]
  5. Brown MS, Goldstein JL 1986 A receptor-mediated pathway for cholesterol homeostasis. Science 232:34–47[Free Full Text]
  6. Parker, Jr, CR, Illingworth DR, Bissonette J, Carr BR 1986 Endocrine changes during pregnancy in a patient with homozygous familial hypobetalipoproteinemia. N Engl J Med 314:557–560[Medline]
  7. Boguslawski W, Sokowlowski W 1984 HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy. Int J Biochem 16:1023–1026[CrossRef][Medline]
  8. Henson MC, Babischkin JS, Pepe GJ, Albrecht ED 1988 Effect of the antiestrogen ethamoxytriphetol (MER-25) on placental low density lipoprotein uptake and degradation in baboons. Endocrinology 122:2019–2026[Abstract/Free Full Text]
  9. Henson MC, Pepe GJ, Albrecht ED 1991 Regulation of placental low density lipoprotein uptake in baboons by estrogen: dose-dependent effects of the anti-estrogen ethamoxytriphetol (MER-25). Biol Reprod 45:43–48[Abstract]
  10. Albrecht ED, Henson MC, Pepe GJ 1991 Regulation of placental low density lipoprotein (LDL) uptake in baboons by estrogen. Endocrinology 128:450–458[Abstract/Free Full Text]
  11. Gibori G, Chen Y-DI, Khan I, Azhar S, Reaven GM 1984 Regulation of luteal cell lipoprotein receptors, sterol contents and steroidogenesis by estradiol in the pregnant rat. Endocrinology 114:609–617[Abstract/Free Full Text]
  12. Kovanen PT, Brown MS, Goldstein JL 1970 Increased binding of low density lipoprotein to liver membranes from rats treated with 17 {alpha}-ethinyl estradiol. J Biol Chem 254:11367–11373[Free Full Text]
  13. Veldhuis JD, Gwynne JT, Azimi P, Garney J, Juchter D 1985 Estrogen regulates low density lipoprotein metabolism by swine granulosa cells. Endocrinology 117:1321–1327[Abstract/Free Full Text]
  14. Henson MC, Pepe GJ, Albrecht ED 1992 Developmental increase in placental low density lipoprotein uptake during baboon pregnancy. Endocrinology 130:1698–1706[Abstract/Free Full Text]
  15. Albrecht ED, Babischkin JS, Koos RD, Pepe GJ 1995 Developmental increase in low density lipoprotein messenger ribonucleic acid levels in placental syncytiotrophoblasts during baboon pregnancy. Endocrinology 136:5540–5546[Abstract]
  16. Gwynne JT, Strauss III JF 1982 The role of lipoproteins in steroidogenesis and cholesterol metabolism in steroidogenic glands. Endocr Rev 3:299–329[Abstract/Free Full Text]
  17. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources rich in ribonuclease. Biochemistry 18:5294–5299[CrossRef][Medline]
  18. Matsuda H, Haramata H, Miyazaki A, Sakai M, Chang CCY, Chang T-Y, Kobori S, Shichiri M, Horiuchi S 1996 Activation of acyl-coenzyme A:cholesterol acyltransferase activity by cholesterol is not due to altered mRNA levels in HepG2 cells. Biochim Biophys Acta 1301:76–84[Medline]
  19. Chang CCY, Huh H-Y, Cadigan KM, Chang T-Y 1993 Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol Chem 268:20747–20755[Abstract/Free Full Text]
  20. Wang AM, Doyle MV, Mark DF 1989 Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 86:9717–9721[Abstract/Free Full Text]
  21. Tso JY, Sun X-H, Kao T, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2458–2502
  22. Luskey KL, Stevens B 1985 Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. J Biol Chem 260:10271–10277[Abstract/Free Full Text]
  23. Yamamoto T, Davis CG, Brown MS, Schneider WJ, Casey ML, Goldstein JL, Russell DW 1984 The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell 39:27–38[CrossRef][Medline]
  24. Noma A, Okabe H, Netsu-Nakayama K, Ueno Y, Shinohara H 1979 Improved method for simultaneous determination of cholesterol in high- and low-density lipoproteins. Clin Chem 25:1480–1481[Abstract/Free Full Text]
  25. Henson MC, Shi W, Greene SJ, Reggio BC 1996 Effects of pregnant human, nonpregnant human, and fetal bovine sera on human chorionic gonadotropin, estradiol, and progesterone release by cultured human trophoblast cells. Endocrinology 137:2067–2074[Abstract]
  26. SAS Institutes Inc 1989 SAS/STAT User’s Guide: Statistics, Version 6, ed 4. SAS Institutes, Cary, NC, p 846
  27. Rudling M 1992 Hepatic mRNA levels for the LDL receptor and HMG-CoA reductase show coordinate regulation in vivo. J Lipid Res 33:493–501[Abstract]
  28. Torbert JA, Bell GD, Birtwell J, James I, Kukovita WR, Pryor JS, Buntinx A, Holmes IB, Chao YS, Bolognese JA 1982 Cholesterol lowering effects of mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in healthy volunteers. J Clin Invest 69:913–919
  29. Grundy SM, Bilheimer DW 1984 Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by mevinolin in familial hypercholesterolemia heterozygotes: effects on cholesterol balance. Proc Natl Acad Sci USA 81:2538–2542[Abstract/Free Full Text]
  30. Simpson ER, Porter JC, Milewich L, Bilheimer DW, MacDonald PC 1978 Regulation by plasma lipoproteins of progesterone biosynthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured human choriocarcinoma cells. J Clin Endocrinol Metab 47:1099–1105[Abstract/Free Full Text]
  31. Moise J, Ilekis J, Scommegna A, Benveniste R 1986 The effect of dehydroepiandrosterone sulfate on de novo and low-density lipoprotein-stimulated progesterone secretion by human choriocarcinoma JEG-3 cells. Am J Obstet Gynecol 154:1080–1085[Medline]
  32. Simpson ER, Bilheimer DW, MacDonald PC, Porter JC 1979 Uptake and degradation of plasma lipoproteins by human choriocarcinoma cells in culture. Endocrinology 104:8–16[Abstract/Free Full Text]
  33. Davis EM, Plotz EJ, Leroy GV, Gould RG, Werbin H 1956 Hormones in human reproduction. I. Metabolism of progesterone. Am J Obstet Gynecol 72:740–755
  34. Grimes RW, Pepe GJ, Albrecht ED 1990 Progesterone (P4) formation and activities of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, acylcoenzyme-A:cholesterol acyl transferase (ACAT), and cholesterol ester hydrolase (CEH) in human trophoblasts cultured with estradiol (E2). Biol Reprod [Suppl 1]42:145 (Abstract)
  35. Boguslawski W, Sokowlowski W 1984 HMG-CoA reductase activity in the microsomal fraction from human placenta in early and term pregnancy. Int J Biochem 16:1023–1026
  36. Simpson ER, Burkhart MF 1980 Acyl coA:cholesterol acyl transferase activity in human placental microsomes: inhibition by progesterone. Arch Biochem Biophys 200:79–85[CrossRef][Medline]
  37. Christie MH, Strauss III JF, Flickinger GL 1979 Effect of reduced blood cholesterol on sterol and steroid metabolism by rat luteal tissue. Endocrinology 105:92–98[Abstract/Free Full Text]
  38. Bruot BC, Wiest WG, Collins DC 1982 Effects of low density and high density lipoproteins on progesterone secretion by dispersed corpora luteal cells from rats treated with aminopyrazolo-(3, 4-d) pyrimidine. Endocrinology 110:1572–1578[Abstract/Free Full Text]
  39. Puryear TK, McLean MP, Khan I, Gibori G 1990 Mechanism for control of hydroxymethylglutaryl-coenzyme A reductase and cytochrome P-450 side chain cleavage message and enzyme in the corpus luteum. Endocrinology 126:2910–2918[Abstract/Free Full Text]
  40. Schuler LA, Toaff ME, Strauss III JF 1981 Regulation of ovarian cholesterol metabolism: control of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase. Endocrinology 108:1476–1486[Abstract/Free Full Text]
  41. Henson MC, Pepe GJ, Albrecht ED 1987 Transuterofetoplacental conversion of pregnenolone to progesterone in antiestrogen-treated baboons. Endocrinology 121:1265–1271[Abstract/Free Full Text]
  42. Marshburn PW, Shi W, Greene SJ, Reggio BC, Henson MC Low density lipoprotein (LDL) uptake by human trophoblast cells cultured in human or fetal bovine sera: regulation by estrogen. Program of the District VII American College of Obstetricians and Gynecologists Annual Meeting, Nashville TN, 1995, p 134 (Abstract)
  43. Grimes RW, Pepe GJ, Albrecht ED 1996 Regulation of human placental trophoblast low-density lipoprotein uptake in vitro by estrogen. J Clin Endocrinol Metab 81:2675–2679[Abstract]
  44. Alsat E, Mondon F, Malassine A, Rebourcet R, Goldstein S, Cedard L 1987 Identification of distinct receptors for native and acetylated-low-density lipoprotein in human placental microvilli. Trophoblast Res 2:17–27
  45. Furuhashi M, Seo H, Mizutani S, Narita O, Tomoda Y, Matsui N 1989 Expression of low density lipoprotein receptor gene in human placenta during pregnancy. Mol Endocrinol 3:1252–1256[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
S. Tartakover-Matalon, N. Cherepnin, M. Kuchuk, L. Drucker, I. Kenis, A. Fishman, M. Pomeranz, and M. Lishner
Impaired migration of trophoblast cells caused by simvastatin is associated with decreased membrane IGF-I receptor, MMP2 activity and HSP27 expression
Hum. Reprod., April 1, 2007; 22(4): 1161 - 1167.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
I. Kenis, S. Tartakover-Matalon, N. Cherepnin, L. Drucker, A. Fishman, M. Pomeranz, and M. Lishner
Simvastatin has deleterious effects on human first trimester placental explants
Hum. Reprod., October 1, 2005; 20(10): 2866 - 2872.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Kawai, K. F. Swan, A. E. Green, D. E. Edwards, M. B. Anderson, and M. C. Henson
Placental Endocrine Disruption Induced by Cadmium: Effects on P450 Cholesterol Side-Chain Cleavage and 3{beta}-Hydroxysteroid Dehydrogenase Enzymes in Cultured Human Trophoblasts
Biol Reprod, July 1, 2002; 67(1): 178 - 183.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. C. Henson and V. D. Castracane
Leptin in Pregnancy
Biol Reprod, November 1, 2000; 63(5): 1219 - 1228.
[Abstract] [Full Text]

Home page
 Nucleic Acids ResHome page
M. Brigotti, D. Carnicelli, P. Accorsi, S. Rizzi, L. Montanaro, and S. Sperti
4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) as a novel inhibitor of the RNA and DNA depurination induced by Shiga toxin 1
Nucleic Acids Res., June 15, 2000; 28(12): 2383 - 2388.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
A. E. Green, J. S. O'neil, K. F. Swan, R. P. Bohm Jr., M. S. Ratterree, and M. C. Henson
Leptin Receptor Transcripts Are Constitutively Expressed in Placenta and Adipose Tissue with Advancing Baboon Pregnancy
Experimental Biology and Medicine, April 1, 2000; 223(4): 362 - 366.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
W. Shi, K. F. Swan, S. R. Lear, J. S. O'Neil, S. K. Erickson, and M. C. Henson
Regulation of Pathways Determining Cholesterol Availability in the Baboon Placenta with Advancing Gestation
Biol Reprod, December 1, 1999; 61(6): 1499 - 1505.
[Abstract] [Full Text]

Home page
 Exp. Biol. Med.Home page
R. M. Harrison, P. P. Phillippi, K. F. Swan, and M. C. Henson
Effect of Genistein on Steroid Hormone Production in the Pregnant Rhesus Monkey
Experimental Biology and Medicine, October 2, 1999; 222(1): 78 - 84.
[Abstract] [Full Text]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. C. Henson, V. D. Castracane, J. S. O’Neil, T. Gimpel, K. F. Swan, A. E. Green, and W. Shi
Serum Leptin Concentrations and Expression of Leptin Transcripts in Placental Trophoblast with Advancing Baboon Pregnancy
J. Clin. Endocrinol. Metab., July 1, 1999; 84(7): 2543 - 2549.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Henson, M. C.
Right arrow Articles by Swan, K. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Henson, M. C.
Right arrow Articles by Swan, K. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals