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Articles |
a Departments of Obstetrics/Gynecology,
b Physiology,
c Anatomy, and
d the Interdisciplinary Program in Molecularand Cellular Biology, Tulane University School of Medicine, New Orleans, Louisiana 70112
e Tulane Regional Primate Research Center,Covington, Louisiana 70433
f Department of Medicine, University of California-San Francisco, and Veterans Affairs Medical Center, San Francisco, California 94121
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interrelationships among pathways determining free cholesterol availability appear to be important for the regulation of optimal placental progesterone production. Thus, pregnant women deprived of optimal LDL-cholesterol availability [4], or lacking LDL receptors due to genetic defects [5], exhibit concentrations of progesterone adequate for pregnancy maintenance. Therefore, other pathways in the placenta must be sufficient to support a significant, albeit reduced, level of progesterone production [4]. In this respect, both human choriocarcinoma cells [6, 7] and normal trophoblast cells [8] in culture secreted significant amounts of progesterone in the absence of LDL. Under these conditions, cholesterol for progesterone synthesis must have been derived from de novo synthesis and/or from the hydrolysis of cholesterol ester stores. In the baboon, an excellent model for human pregnancy [9], we have reported that although maternal progesterone levels decline with LDL-cholesterol withdrawal, cholesterol availability in the syncytiotrophoblast is sufficient to maintain a level of progesterone production adequate for pregnancy maintenance [10]. Although LDL receptor and HMG-CoA reductase mRNAs increased and ACAT-1 mRNA declined in a syncytiotrophoblast-enriched fraction as a result of decreased LDL-cholesterol availability, no quantitative changes were apparent in whole villous tissue [11], suggesting that the regulation of cholesterol metabolism in the steroidogenically active syncytiotrophoblast may differ significantly from that in proliferative nonendocrine placental cell types. Collectively, these studies suggest that in vivo, the primate syncytiotrophoblast may derive significant amounts of the cholesterol necessary for optimal progesterone production from sources other than the preferred LDL receptor pathway. The aim of this study, therefore, was to further investigate the potential contributions of the LDL receptor pathway, de novo cholesterol synthesis (HMG-CoA reductase), and the ACAT/CEH axis to the availability of cholesterol for placental progesterone synthesis in the baboon, with advancing gestational age.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals were maintained and utilized strictly in accordance with USDA 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. Eleven 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 [9–13]. A 12L:12D 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. Placentae were collected upon cesarean delivery, as previously described [11], early in gestation on Days 60–61 (n = 3), at midgestation on Days 98–102 (n = 4), or late in gestation on Days 160–167 (n = 4). Normal term in the baboon is ~184 days [9]. Samples designated as "early" were collected approximately 30–35 days after the luteal-placental shift [14].
Preparation of a Syncytiotrophoblast-Enriched Fraction
Samples of placental villous tissue were flash-frozen in liquid nitrogen for storage at -80°C. Remaining villous tissue was chilled on ice, and cells were dispersed as we have described previously [9–11, 13, 14]. Briefly, villous tissue was minced in calcium- and magnesium-free Hanks' Balanced Salt Solution (HBSS; Gibco, Grand Island, NY), and cells were dispersed in HBSS containing 0.1% collagenase (type 1A; 420 U/mg; Sigma Chemical Co., St. Louis, MO), 0.1% hyaluronidase (type I-S; 300 U/mg; Sigma), 0.01% DNase I (2000 Kunitz U/mg; Sigma), 1.0% lipoprotein-free fetal bovine serum (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 was sequestered via centrifugation. We have reported that, as determined via immunohistochemistry, baboon placental cells isolated by this method are primarily syncytiotrophoblasts [12].
RNA Extraction and Initial Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated according to Chomczynski and Sacchi [15] and Chirgwin et al. [16], as adapted for use in our laboratory [11]. Briefly, 50–100 mg of placental villous tissue or syncytiotrophoblast cells was homogenized (Ultra-Turrax T 25; Janke & Kunkel, Staufen, Germany) in 1 ml Trizol Reagent (Life Technologies, Gaithersburg, MD). After incubation at room temperature, chloroform was added and the tubes were agitated. After centrifugation (12 000 x g, 4°C), RNA was precipitated with isopropyl alcohol, washed with 75% ethanol, dried, and resuspended in diethylpyrocarbonate-treated distilled water. To preclude possible DNA contamination, RNA was treated with DNase (0.25 U/µl; Promega, Madison, WI). After incubation (37°C, 30 min), the reaction was terminated (75°C, 5 min). The RNA was then precipitated with isopropyl alcohol at 4°C, washed with 75% ethanol, dried, and resuspended in diethylpyrocarbonate-treated distilled water. RNA concentrations were determined at 260 nm absorbance (DU640 Spectrophotometer; Beckman Instruments, Fullerton, CA) and stored at -70°C.
Oligonucleotide primers were synthesized (Tulane Molecular Biology Consortium, Department of Biochemistry, Tulane Medical School) according to the published sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; [17, 18]), HMG-CoA reductase [17, 19], and LDL receptor [17, 20]. PCR primer sequences and product sizes for LDL receptor and HMG-CoA reductase were described in our previous report [11]. Primer sequences for ACAT PCR amplification were 5'-TATGAAGGAAGTTGGCAGTC-3' and 5'-GAGAATGAGGAGGGCAATA-3' for 5' primer and 3' primer, respectively. They were designed (Oligo Primer Analysis Software 5.0; National Biosciences, Plymouth, MN) according to the human cDNA sequence ([21]; GenBank accession #L21934). Total RNAs extracted from both human and baboon placentae were amplified by RT-PCR, and products generated were 259 base pairs. The RT-PCR products of ACAT primers were sequenced (Biotech Core, Palo Alto, CA). In the PCR portion, the human ACAT PCR product was 100% homologous to the human cDNA sequence, and the baboon's was 97% homologous to the human's.
Complementary DNAs were synthesized using the SuperScript Kit (Life Technologies), as we have previously reported [11, 22]. The PCR proceeded according to the GeneAmp PCR Reagent kit protocol (Perkin-Elmer/Cetus, Norwalk, CT) and was performed in a Temp-Tronic thermocycler (Barnstead/Thermolyne, Dubuque, IA). Conditions for PCR were as follows: LDL receptor (30 cycles for amplification, denaturation at 94°C for 90 sec, hybridization at 58°C for 60 sec, extension at 72°C for 60 sec); HMG-CoA reductase (35 cycles for amplification, denaturation at 94°C for 90 sec, hybridization at 58°C for 60 sec, extension at 72°C for 60 sec); ACAT (40 cycles for amplification, denaturation at 95°C for 30 sec, hybridization at 55°C for 30 sec, extension at 72°C for 60 sec); GAPDH (24 cycles for amplification, denaturation at 94°C for 30 sec, hybridization at 58°C for 60 sec, extension at 72°C for 60 sec). 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.
Quantitative Competitive RT-PCR
Quantitation of RT-PCR results was achieved using the PCR Mimic Construction Kit (Clontech, Palo Alto, CA), as we have described previously [22]. Thus, one set of primers was used to amplify both the target gene cDNA and Mimic, a nonhomologous DNA fragment engineered to contain specific target gene sequences necessary for recognition by the gene-specific primers for the genes of interest, i.e., "mimicking" the target cDNA [23, 24]. The Mimic DNA effectively competes with the target cDNA for the same primers, thereby serving as an internal standard. Preliminary competitive PCR was performed using 10-fold serial dilutions of 1 attomol Mimic against a constant amount of cDNA for the gene of interest. Results from these initial dilutions identified the range for subsequent 2-fold dilutions necessary for optimal quantitation. Products were resolved by 2% agarose gel electrophoresis, and band intensities were analyzed using the Alpha Imager 2000 digital analysis system with Alpha Imager 2000 software (Alpha Innotech, San Leandro, CA). Final concentrations were expressed as attomol/µg total RNA (1 attomole = ~6 x 105 molecules).
Radioenzymatic Analyses
Cell-free homogenates were prepared on the basis of the method previously described for rat liver [25, 26]. To prepare the placental homogenate, villous tissue was homogenized in 2.5 volumes of microsome buffer (0.10 M sucrose, 0.05 M KCl, 0.04 M KH2PO4, and 0.03 M EDTA, pH 7.4) using a motor-driven glass-polytetrafluoroethylene Potter-Elvehjem homogenizer. Homogenates were centrifuged (1000 x g, 10 min) to remove whole cells and debris, and the supernatant was saved for analyses. The method for preparing syncytiotrophoblast cell extracts was modified from Brown et al. [27]. Briefly, syncytiotrophoblasts were dissolved in buffer (100 µl buffer/30 mg cell pellet) containing 50 mM KH2PO4 (pH 7.4), 5 mM dithiothreitol (DTT), 5 mM EDTA, 0.2 M KCl, and 0.5% Nonidet P40. After incubation for 30 min at 4°C and 10 min at 37°C, the suspension was centrifuged (10 000 x g, 1 min). Protein levels were determined according to the method of Bradford [28]. Enzyme activities were assessed according to Erickson et al. [25] and Smith et al. [26]. All assay conditions (incubation time, protein, etc.) were such that measurements were made in the linear ranges. Briefly, HMG-CoA reductase was assayed in 200 µl microsome buffer, including 40 mM glucose-6-phosphate, 3 mM nicotinamide adenine dinucleotide phosphate, 10 mM DTT, 250 mM NaCl, 30 mM EDTA, 1 unit glucose-6-phosphate dehydrogenase, samples containing 0.5 to 1 mg protein, and 30 nM (150 000 dpm) DL-[3,14C]3-hydroxy-3-methylglutaryl-CoA (NEN Life Science Products, Boston, MA) substrate. The limit of detection for the assay was estimated to be approximately 10 pmol mevalonate formed. The assay time was 20 min.
ACAT activity was quantitated in an assay volume of 0.2 ml, which contained 0.5–1.0 mg of placental homogenate protein, 95 µl ACAT buffer (0.25 M sucrose, 1 mM EDTA, 0.1 M Tris, pH 7.5), 335 µg fatty acid-free BSA, and 5 nmol [14C]oleyl-CoA (38 000 dpm/nmol; Amersham Life Science, now Amersham Pharmacia Biotech, Piscataway, NJ). Assay of the activity at apparent Vmax was accomplished by addition of exogenous cholesterol in the form of cholesterol:egg phosphatidyl choline (1:8 by weight) liposomes prepared by bath sonication (Laboratory Supplies Company, Hicksville, NY). Routinely, 8 µg cholesterol per assay was used. The limit of detection for the assay was estimated to be approximately 1 pmol cholesteryl oleate formed. The assay time was 2 min.
Neutral CEH activity was determined using cholesterol [14C]-oleate:egg phosphatidyl choline liposomes (1:50 by weight) prepared by sonication. Placental homogenate were assayed in a total assay volume of 0.3 ml buffer containing 60 mM Tris-HCl, pH 8.0, and 6 mM mercaptoethanol, liposomes (cholesterol-[14C]-oleate; 4 µg, 30 537 dpm/nmol), and homogenate protein (0.5–1.0 mg). The limit of detection for the assay was estimated to be approximately 0.1 pmol oleate formed. The assay time was 30 min.
Western Immunoblot Analysis
Syncytiotrophoblast cell lysates were prepared essentially as described above, except that buffers contained additional protease inhibitors. The cell lysis buffer contained 50 mM KH2PO4, pH 7.4, 5 mM DTT, 200 mM KCl, 5 mM EDTA, 2 mM EGTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 1 mM PMSF, and 1% Nonidet P40. Cell lysates were subjected to Western immunoblot analysis as described previously [26, 29]. Briefly, samples were denatured in a mixture containing 10% glycerol, 4.5% SDS, 5% ß-mercaptoethanol, 62.5 mM Tris-HCl (pH 6.8), 1.5 mM EDTA, and 0.01% bromophenol blue; they were then electrophoretically separated on a 7.5% SDS-polyacrylamide gel (12–15 mA/gel at 4°C, 1.5–2 h). Proteins were transferred electrophoretically from SDS slab gels onto nitrocellulose paper in a Trans-Blot cell apparatus (Bio-Rad Laboratories, Richmond, CA) at 100 voltage (4°C, 1 h) with an electrode buffer consisting of 20 mM Tris-HCl, 150 mM glycine, and 20% methanol.
The Western-Light Plus Chemiluminescent Detection System (Tropix, Bedford, MA) was used for detection of proteins on nitrocellulose membranes. After protein transfer, the nitrocellulose membranes were incubated in 10 ml blocking buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 137 mM NaCl, 0.2% I-Block, and 0.1% Tween 20) for 60 min at room temperature, then overnight at 4°C. Nitrocellulose blots were washed 3 times for 15 min each in 25 ml of wash buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 137 mM NaCl, and 0.1% Tween 20) between successive steps. HMG-CoA reductase antiserum [29] was kindly provided by Dr. Gene C. Ness, University of South Florida, Tampa. Membranes were incubated with HMG-CoA reductase antiserum (1:1000) for 1 h, with secondary antibody (biotinylated goat anti-rabbit IgG, 1:30 000 dilution) for 30 min, and then with streptavidin-alkaline phosphatase conjugate (1:20 000 dilution) for 20 min at room temperature. Nitrocellulose membranes were washed 3 times for 15 min in washing buffer and 2 times for 2 min with single-strength assay buffer (20 mM Tris-HCl, pH 9.8, 1 mM MgCl2). Membranes were incubated with the chemiluminescent substrate (supplied in the kit) for 5 min and exposed to radiograph film (HyperFilm; Amersham). After processing, images of films were captured digitally, and the band densities were quantitated by computer imaging (Alpha Imager 2000; Alpha Innotech).
Statistical Analysis
Standard least-squares ANOVA with multiple comparison of means by the least significant difference method, using the General Linear Models procedure of the Statistical Analysis System [30], was employed to assess the effects of advancing gestation on measured parameters. A significant difference was understood to exist when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After semiquantitative assessment of band densities for mRNA transcripts for genes of interest relative to GAPDH, a constitutively expressed "housekeeping" gene, quantitative assessment of mRNA levels in syncytiotrophoblast cells was made via PCR Mimic methodologies. As shown in Table 1, semiquantitative comparison of transcripts for LDL receptor and HMG-CoA reductase in villous tissue revealed no statistically significant differences as a result of gestational age. However, differences were observed in relative amounts of mRNA for leptin receptor in enriched fractions of syncytiotrophoblast cells collected in early pregnancy when compared to either mid or late pregnancy (P < 0.05). By quantitative competitive RT-PCR (Fig. 1), LDL receptor mRNA (mean ± SEM) in syncytiotrophoblast cells increased (P < 0.05), from 1.79 ± 0.28 x 10-3 attomol/µg total RNA early in gestation, to 25.8 ± 11.4 in mid and 24.1 ± 7.48 in late gestation. In contrast, mRNA levels for HMG-CoA reductase in villous tissue did not change as a result of gestational age, while mRNA levels in syncytiotrophoblast cells were similar in early, mid, and late pregnancy with a nonstatistically significant trend upward with advancing gestation. No differences in ACAT-1 mRNA levels were found, or trends detected, in either whole villous tissue or syncytiotrophoblast cells as a consequence of gestational age. As quantitated by competitive RT-PCR, transcript abundances in syncytiotrophoblast cells collected throughout pregnancy were 6.52 ± 2.49 x 10-3 attomol/µg total mRNA for HMG-CoA reductase and 2.33 ± 0.05 for ACAT-1.
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Enzyme Activities in Placental Villous Tissue
HMG-CoA reductase activities in baboon placental homogenates from early, mid, and late pregnancy were 155 ± 27.4, 131 ± 16.3, and 64.7 ± 18.5 pmol mevalonate formed/min per milligram protein, respectively (Fig. 2A), with those from early and mid pregnancy higher than those from late pregnancy (P < 0.05). ACAT activities in baboon placental homogenates (Fig. 2B) were 3.55 ± 0.35, 2.81 ± 0.50, and 1.34 ± 0.09 pmol cholesterol oleate formed/min per milligram protein, respectively, with those from early and mid pregnancy higher (P < 0.05) than those from late pregnancy. CEH activities in placental homogenates from early, mid, and late pregnancy were 0.182 ± 0.015, 0.170 ± 0.08, and 0.196 ± 0.019 pmol oleate formed/min per milligram protein, respectively. No statistically significant differences were detected in CEH activities as a result of gestational age.
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Enzyme Activities and Protein Levels in Syncytiotrophoblasts
ACAT and CEH activities in syncytiotrophoblast cell lysates were below the minimal detectable range for our assay system. However, HMG-CoA reductase activities in cell lysates taken at early, mid, and late pregnancy (Fig. 3) were 35.2 ± 4.13, 21.8 ± 4.01, and 15.5 ± 3.74 pmol mevalonate formed/min per milligram protein, respectively. Thus, activity was higher in early pregnancy than in either mid (P < 0.05) or late (P < 0.01) pregnancy.
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Syncytiotrophoblast cells were collected upon cesarean delivery, frozen in liquid nitrogen, and stored at -70°C prior to cell lysate extraction and immunoblot analysis. As in rat liver microsomes (positive control; [29]), two HMG-CoA reductase bands (100 kDa and 70 kDa) were detected in syncytiotrophoblast cell lysates. Densitometric analysis of immunoblots (average pixel values after background correction) for HMG-CoA reductase (100-kDa band) showed that the protein level for HMG-CoA reductase in syncytiotrophoblast cells from early pregnancy was higher (P < 0.05) than that from either mid or late pregnancy (Fig. 4, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placental HMG-CoA reductase and ACAT activities declined with advancing baboon gestation. A commensurate decrease in HMG-CoA reductase protein, as determined by immunoblot analysis, also was observed with advancing gestation and was similar to the decline in enzyme activity measured radioenzymatically. Therefore, our results in the baboon agree with those in the human [32], where the specific activity of HMG-CoA reductase in a microsomal fraction prepared from early placenta was dramatically greater than that at term. Maternal progesterone levels remain relatively constant throughout most of baboon pregnancy, while lipoprotein-cholesterol concentrations are high in the maternal circulation throughout pregnancy and increase only modestly with increasing gestational age [9]. Therefore, our current results indicate a propensity for enhanced de novo cholesterol biosynthesis in the syncytiotrophoblast during early pregnancy, perhaps necessary for optimal progesterone synthesis at this time. This relationship, when considered along with our previous report of enhanced LDL receptor and HMG-CoA reductase mRNAs and decreased ACAT mRNA, resulting from maternal lipoprotein-cholesterol withdrawal [11], suggests a control mechanism that is sensitive to relative changes in available free cholesterol.
The present in vivo results support the concept that the baboon placenta can utilize cholesterol produced de novo for progesterone production, especially in early pregnancy. Similarly, Simpson et al. [33] demonstrated that in the absence of LDL, human choriocarcinoma cells secreted approximately 50% of that progesterone normally produced after LDL-cholesterol addition, thus indicating the availability of alternative sources. In agreement with this interpretation, progesterone production by human syncytiotrophoblasts cultured in the absence of LDL was approximately 40% of that measured when LDL was present in culture medium [34]. Estradiol further stimulated basal progesterone formation in the absence of LDL. Under these conditions, cholesterol for progesterone synthesis must have been derived from de novo synthesis and/or from cholesterol ester stores. In a comparable in vivo study, it was demonstrated that only two thirds of the placental progesterone produced in women infused with radiolabeled cholesterol could be attributed to lipoprotein-cholesterol derived from the maternal circulation [1, 35]. The remaining one third of the cholesterol utilized by the syncytiotrophoblast for progesterone biosynthesis likely originated from de novo synthesis and/or from esterified cholesterol stores. Therefore, taken together, the evidence strongly suggests that non-lipoprotein cholesterol-yielding pathways have the potential to provide significant amounts of cholesterol substrate for placental progesterone production during primate pregnancy.
In the current study, CEH activity in placental homogenates did not differ between early, mid, and late pregnancy. ACAT activity in placental homogenates, however, declined with advancing gestation. Simpson and Burkhart [36] reported that ACAT activity in human placental microsomes in vitro was inhibited by some steroid hormones, including progesterone and estradiol. While no significant increase or decline in maternal serum progesterone levels occurs over the final two thirds of baboon pregnancy, estradiol concentrations increase progressively from midgestation until term [12]. Thus, the decrease in ACAT activity in baboon placenta with advancing gestation may reflect an inhibition exerted by estradiol. If so, the effect of estradiol may be biphasic, acting to supply cholesterol substrate to the baboon syncytiotrophoblast via up-regulation of LDL receptor transcription [9, 31], then inhibiting ACAT activity, in order to ensure a continuing supply of free cholesterol precursor by reducing the sequestration of cholesterol into the pool of stored ester. The suggestion of a biphasic effect of estrogen must await further validation, however, because although estrogen clearly up-regulates LDL receptor action in baboon placental trophoblast [9, 10, 13] in vivo, and in human placental cells in vitro [34], it was reported to have no effect on ACAT activity in human syncytiotrophoblasts in culture [8].
It has been reported that ACAT, as well as its product, cholesteryl esters, is present in low levels in human placenta [36]. CEH activity in human placenta is also extremely low [37, 38]. In the present study, low overall ACAT and CEH activities in the baboon placental homogenate mimicked the human situation. Further, both ACAT and CEH activities in syncytiotrophoblast cell lysates were below the minimum detectable range of our assay systems, while HMG-CoA reductase activities in syncytiotrophoblast cells were detectable at the picomolar level. Therefore, the ACAT/CEH axis may not play a major role in regulating the availability of free cholesterol necessary for progesterone production in the primate syncytiotrophoblast. ACAT activities in whole villous tissue in the present study were poorly correlated with mRNA concentrations, suggesting that regulation was posttranscriptional. A similar lack of association between mRNA concentrations and protein levels has been reported by others in other tissues and cell types. Rea et al. [39] found that hepatic ACAT mRNA expression was not correlated with cellular cholesterol esterification, and Matsuda et al. [40] reported that when the cholesterol level within HepG2 cells (human hepatoblastoma cell line) was high, both whole-cell ACAT activity and microsomal ACAT activity increased. In contrast, cholesterol depletion of HepG2 cells resulted in a decrease in ACAT activity, although RT-PCR and Northern blotting revealed that neither cholesterol loading nor cholesterol depletion altered the amount of ACAT mRNA.
In summary, the expression of LDL receptor mRNA in syncytiotrophoblast cells, collected and fractionated at three periods during pregnancy, increased with advancing gestational age and may reflect a similar change in vivo. Although HMG-CoA reductase mRNA concentrations remained unchanged in syncytiotrophoblast cells, both HMG-CoA reductase activity and protein declined with advancing gestation. Further, although CEH activity in placental homogenates was unchanged throughout pregnancy, ACAT activities in placental homogenates from early and mid gestation were significantly higher than those from late gestation. The relative contributions of cholesterol substrate from the LDL receptor and the synthetic pathways were not assessed directly in the present study. However, increased HMG-CoA reductase activity in early pregnancy, together with relatively low LDL receptor levels, suggests that de novo synthesis is a potentially important source of free cholesterol for progesterone synthesis in early gestation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michael C. Henson, Department of Obstetrics/Gynecology, Tulane University School of Medicine, 1430 Tulane Avenue, Room 4500, New Orleans, LA 70112-2699. FAX: 504 584 1846; michael.henson{at}tulane.edu
Accepted: August 2, 1999.
Received: May 4, 1999.
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