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Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology (J.S.B., E.D.A.), Center for Studies in Reproduction, University of Maryland School of Medicine, Baltimore, Maryland 21201; and Department of Physiology (G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501
Address all correspondence and requests for reprints to: Eugene D. Albrecht, Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11–017, 655 West Baltimore Street, Baltimore, Maryland 21201.
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Abstract |
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Placentas were maintained in situ after fetectomy, and placental villi were comprised of syncytiotrophoblasts that seemed morphologically normal, based on their histology and immunocytochemical expression of pregnancy-specific-ß1-glycoprotein. In untreated baboons, peripheral serum estradiol increased with advancing gestation, and mean (±SE) concentrations were 1.22 ± 0.05 ng/ml on days 101–160 of gestation. After fetectomy serum estradiol concentrations decreased to 24% (P < 0.01) of normal. Androstenedione or estradiol administration after fetectomy increased serum estradiol levels to values that were 57% (P < 0.01) of, or 90% (P < 0.001) greater than intact controls, respectively, .
Placental P-450scc specific activity, determined on a mitochondrial-enriched fraction of villous tissue, was 281.1 ± 15.0 pmol pregnenolone plus progesterone formed per mg mitochondrial protein in untreated control baboons. Fetectomy resulted in a 52% decrease (P < 0.001) in placental P-450scc activity. Administration of androstenedione or estradiol after fetectomy increased P-450scc activity to values that were not significantly different from control. P-450scc mRNA levels were quantified by competitive RT-PCR. P-450scc mRNA levels in placental villous tissue of fetectomized baboons was 38% lower (P < 0.01) than that in the intact controls (110.9 ± 5.9 attomoles/µg RNA). The administration of androstenedione after fetectomy restored P-450scc mRNA to a level that was not different from the untreated controls. The results of this study show that there was close association between the levels of estrogen and the specific activity of and the mRNA levels for placental P-450scc in the second half of baboon pregnancy. Therefore, we propose that the P-450scc enzyme that catalyzes the conversion of substrate cholesterol to pregnenolone is regulated, for the most part, by estrogen in the primate placenta.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our present studies are focused on elucidating the subcellular mechanisms by which estrogen regulates P-450scc activity and whether the developmental increase in placental P-450scc mRNA is dependent upon estrogen. We have shown previously that removal of the fetus, but not the placenta, i.e. fetectomy, at midgestation in baboons (11) eliminates fetal adrenal C19-steroid estrogen precursors and results in a decline in placental estrogen formation. However, after fetectomy the placenta remains in situ, viable, and functional with respect to the potential for formation of estrogen from aromatizable C19-steroids (12) and syncytiotrophoblast-specific proteins (13). Thus, in the present study, we determined the mRNA levels for and activity of the P-450scc within the placenta after fetectomy and androstenedione or estradiol administration to ascertain the role of estrogen on placental P-450scc expression in the second half of baboon pregnancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Baboons either served as untreated intact controls or underwent fetectomy using halothane (1.5%): nitrous oxide (0.5 1/min): oxygen (2.0 1/min) anesthesia on day 100 of gestation as described previously (11). Fetectomized animals either were not further treated, received 50 mg pellets of the estrogen precursor androstenedione (Innovative Research, Toledo, OH) implanted sc in the mother in increasing numbers between days 100 and 155 of gestation (one on day 100, two on day 110, and three each on days 120, 130, 140, 150, and 155), or were injected sc with estradiol benzoate in 0.5 cc sesame oil in increasing amounts of 0.5–2.5 mg per day between days 101–160 of gestation. Intact animals also received androstenedione implants, as described above, on days 100–155 to serve as steroid-injected controls. On day 160 of gestation, baboons were anesthetized with halothane, a laparotomy and hysterotomy performed, and placentas were removed. Placentas were rinsed in ice-cold 0.9% saline and cleared of membranes and decidua, and villous tissue was immediately processed for the determination of P-450scc activity or frozen in liquid nitrogen for the analysis of P-450scc mRNA levels. Because the baboons used for the determination of placental P-450scc activity were studied before the P-450scc mRNA assay or RNA collections were implemented in our laboratory, an additional set of intact and fetectomized baboons was used for P-450scc mRNA analysis. Moreover, because similar P-450scc activity results were obtained with androstenedione and estradiol treatment, only one of these regimens, i.e. androstenedione administration, was used for the study of P-450scc mRNA levels. Animals were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (Publication No 85–23, 1985). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Immunocytochemistry of
pregnancy-specific-ß1-glycoprotein (SP1)
Placental villous sections were stained with hematoxylin and
eosin or affixed to coverslips for immunocytochemical analysis of
SP1, as detailed previously (14). The primary antiserum
against human SP1 (1:1200, Dako, Santa Barbara, CA) was
visualized using goat antirabbit antisera, rabbit peroxidase,
antiperoxidase, and hydrogen peroxidase:
3,3'-diaminobenzidine-tetrahydrochloride:Tris solution.
Competitive RT-PCR
A sensitive competitive RT-PCR assay was established to quantify
mRNA in whole villous tissue of the present study and for purposes of
future studies of low RNA levels in purified trophoblast cell
fractions.
Preparation of RNA. Sections of whole villous tissue were homogenized in 4 M guanidine isothiocyanate extracted with chloroform: isoamylalcohol, and total RNA obtained via cesium chloride gradient centrifugation.
P-450scc primers sequence and synthesis. Oligonucleotide primers synthesized by Life Technologies, Inc. (Grand Island, NY) were selected from the cDNA sequence of human P-450scc (15) and flanked a portion of the sequence that spans exons 3 and 4 and overlapped intron 3 (16). Primer 1: downstream, 5'-GTCCCATGCAGCCACATGGTCCTTCGGCATCA-ATGAATCGCTGGG-3' (position: 857–833 linked to 752–733). Primer 2: upstream, 5'-AATTTAATACGACTCACTATAGGGATGGAAGAAAGACCGGGTGGC-3' (position: T7 polymerase sequence [underlined] linked to 483–502). Primer 3: downstream, 5'-ATGCAGCCACATGGTCCTTC-3' (position: 852–833). Primer 4: upstream, 5'-TGGAAGAAAGACCGGGTGGC-3' (position 483–502).
Construction of internal standard RNA. P-450scc-competitive reference standard (CRS) was prepared adapting the method of Riedy et al. (17), using RT-PCR to generate the cDNA template, followed by transcription with T7 polymerase. Total RNA (5 µg) from baboon placenta was reversed transcribed at 37 C for 60 min in a reaction mixture (20 µl) containing 1 mM each of dATP, dCTP, dGTP, dTTP (Promega Corp., Madison, WI), 1 mM dithiothreitol, 200 U SUPERSCRIPT RNase H- RT (Life Technologies), 50 U RNasin (Promega), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 pmol primer 1. After 60 min, the RT mixture was cooled to 4 C and then added to a PCR reaction mixture (80 µl) containing 8 mM Tris-HCl (pH 8.3), 0.9 mM MgCl2, 40 mM KCl, 2.5 U cloned Thermus aquaticus DNA polymerase (Amplitaq, Perkin-Elmer/Cetus, Norwalk, CT), and 10 pmol primer 2. PCR was performed in a programmable thermal cycler (MJ Research, Cambridge, MA) and the sample amplified in 25 sequential cycles at 94 C for 1 min, 60 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. A portion of the PCR reaction mixture was fractionated by electrophoresis in a 2% agarose gel and visualized in ethidium bromide. The amplified product (320 bp) contains sequences for the T7 polymerase and has an 80-bp deletion as compared with the target (wild type) mRNA strand. The PCR product was gel purified using the QIAEX DNA gel extraction kit (Qiagen Inc., Chatsworth, CA). P-450scc-CRS was synthesized from 200 ng cDNA template using the MEGAscript T7 in vitro transcription kit (Ambion Inc., Austin, TX).
Competitive RT-PCR. A constant amount of total RNA (2.5 µg) from villous tissue was added to the RT mixture containing 2-fold serial dilutions of the P-450scc-CRS (500 -125 attomoles) in the presence of 10 pmol of primer 3. Upon completion of the RT, 10 pmol primer 4 was added for the PCR reaction. Two negative controls, in which either RNA or RT were omitted from the reaction, were also performed. The PCR products were fractionated by electrophoresis in a 2% agarose gel, stained in ethidium bromide, visualized with a UV transilluminator, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA).
Quantification of P-450scc. Photographs (negative image), representing amplified products, were analyzed by autoradiographic scanning using a model 620 Video Densitometer (Bio-Rad, Richmond, CA). The intensity of the amplified products was represented as the relative area under each sample band. A correction factor (18) was used to correct for the size difference between P-450scc-CRS cDNA and the P-450scc target cDNA: corrected relative area = relative area x P-450scc target bp length/P-450scc-CRS bp length. The logarithm (log) of the ratio of P-450scc-CRS area to P-450scc target area was plotted as a function of the concentration of P-450scc-CRS added to each PCR reaction. The concentration of P-450scc mRNA was determined where the ratio of P-450scc-CRS and P-450scc target areas was equal to 1 (i.e. equivalence point).
Validation of assay. To ensure that the P-450scc-CRS and
P-450scc target template amplified with similar efficiencies, their
amplification kinetics were compared as described (19). Total RNA (2.5
µg) from whole villous tissue and 275 attomoles of P-450scc-CRS were
reversed transcribed and amplified by PCR. The log of the P-450scc-CRS
and P-450scc target areas was plotted as a function of the cycle number
(Fig. 1
). Amplification proceeded with the same
efficiency for both templates because the resultant PCR products
accumulated in a parallel manner throughout both the exponential and
nonexponential phases.
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Cyanoketone, an inhibitor of 
5-3ß-hydroxysteroid
dehydrogenase, was not used in the assay to prevent the conversion of
pregnenolone to progesterone because this synthetic steroid binds to
oxidized placental cytochrome P-450 and inhibits activity. Rather,
P-450scc activity was expressed as the net amount of pregnenolone plus
progesterone formed, as determined by subtracting the zero-time steroid
values from those obtained after the incubation period.
RIA of steroids
One milliliter of the mitochondrial assay incubate was extracted
two times with 5 ml of diethyl ether, and pregnenolone and progesterone
content were determined by 3H RIA, basically as previously
described in our laboratory (22). Prior comparison of results for
pregnenolone and progesterone assay with and without Sephadex LH-20
affinity chromatography of mitochondrial incubates indicated that
purification of aliquots before RIA was unnecessary.
Serum estradiol was determined by RIA using solid-phase 125I-RIA (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA).
Statistical analysis
The data were analyzed by ANOVA with post hoc
comparison of the means by Newman-Keuls multiple comparison test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The overall mean (±SE) serum
estradiol level in untreated control baboons on days 101–160 of
gestation was 1.22 ± 0.05 ng/ml (Fig. 3).
Fetectomy was associated with a marked decrease in serum estradiol
concentration (Fig. 2) to a mean between days 101–160 of gestation of
0.30 ± 0.03 ng/ml, or 24% of that (P < 0.01) in
untreated controls at the same interval (Fig. 3). Administration of
androstenedione after fetectomy partially restored serum estradiol
concentrations to a mean on days 101–160 of 0.69 ± 0.03 ng/ml,
or 57% of normal (P < 0.01, Figs. 2 and 3). The
administration of estradiol after fetectomy resulted in a mean
(±SE) serum estradiol concentration of 2.32 ± 0.11
ng/ml, a value 90% greater (P < 0.001) than in
untreated, intact animals. Treatment of intact baboons with
androstenedione resulted in a mean serum estradiol concentration of
2.82 ± 0.20 ng/ml on days 101–160, a value 2-fold greater
(P < 0.01) than in untreated controls (data not shown
in figure).
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). Placental weights after fetectomy and administration
of androstenedione or estradiol were 54% and 55% (P
< 0.01), respectively, also lower than in controls at term, and only
approximately 18% larger than that previously determined in untreated
baboons at midgestation (85 ± 7).
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), seemed to remain intact and very
similar in appearance to that in the untreated controls (Fig. 4A).
Moreover, syncytiotrophoblasts of intact baboons (Fig. 4C) and animals
that had undergone fetectomy (Fig. 4D) exhibited an equally extensive
level of reactivity with antisera to syncytiotrophoblast-specific
SP1. Consequently, on the basis of their capacity to
aromatize androstenedione to estradiol and express SP1,
syncytiotrophoblasts of fetectomized baboons were functional.
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Mean (±SE) specific activity of placental P-450scc in
mitochondria of untreated baboons was 281.1 ± 15.0 pmol
pregnenolone plus progesterone formed per mg protein. Fetectomy
resulted in a 52% decrease (P < 0.001) in placental
P-450scc activity (133.7 ± 22.1, Fig. 5). The
administration of androstenedione after fetectomy increased P-450scc to
a level (226.0 ± 35.5) that was greater (P <
0.05) than with fetectomy alone and 80% of, and not significantly
different from, that observed in the intact controls. Mean P-450scc
activity in baboons treated with estradiol benzoate after fetectomy was
337.0 ± 39.9, a value greater (P < 0.001) than
with fetectomy alone and similar to that of the untreated intact
controls. Androstenedione treatment of intact baboons resulted in
P-450scc levels (212.8 ± 58.9) that were not significantly
different from normal.
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. The expected 370-bp P-450scc PCR target product
generated from villous total RNA and 290 bp-PCR product generated from
P-450scc-CRS were expressed (Fig. 6A). In contrast, there was no PCR
product formed when RNA or RT were omitted from the reaction (not
shown). The slopes of the log of the CRS to target areas plotted as a
function of CRS added were similar for trophoblast RNA obtained from
intact (slope = 1.22) and fetectomized (slope = 1.08) baboons
(Fig. 6B), indicating no difference in amplification efficiency.
However, analysis of the equivalence points from each plot indicated
that placental P-450scc mRNA levels were different in intact and
fetectomized baboons.
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). Fetectomy resulted in a P-450scc mRNA level
(69.2 ± 7.0 attomoles/µg RNA) that was 38% lower
(P < 0.01) than in the intact controls. Administration
of androstenedione after fetectomy restored P-450scc mRNA to a level
(96.4 ± 9.1) that was greater (P < 0.05) than
with fetectomy alone and similar to that observed in the untreated
controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the P-450scc complex consists of the cytochrome P-450scc terminal oxidase, adrenodoxin reductase flavoprotein, and adrenodoxin iron-sulfur protein (1), the changes in P-450scc activity elicited by estrogen in baboons of the present study potentially could have reflected regulation of one or more of these P-450scc enzyme components. However, we previously showed that the developmental increase in the mRNA levels for P-450scc in baboon syncytiotrophoblasts was not associated with a change in the mRNA levels for adrenodoxin (9). In addition, the content of mitochondrial cytochrome P-450, 80–100% of which is involved in cholesterol side-chain cleavage in the placenta (24), was significantly reduced as determined by spectral analysis in placental tissue of antiestrogen-treated baboons (10). Therefore, on the basis of these observations plus the comparable change in mRNA levels and activity of P-450scc in animals of the present study, we suggest that the terminal oxidase is the primary component of the P-450scc system that is regulated by estrogen within syncytiotrophoblasts in the second half of baboon pregnancy.
In addition to the regulatory action on the P-450scc enzyme, we recently have shown that estrogen also increased low-density lipoprotein (LDL) receptor mRNA levels and LDL uptake within syncytiotrophoblasts during baboon and human pregnancy. Thus, there was an ontogenetic increase in LDL receptor mRNA levels in (25) and LDL uptake by (14) syncytiotrophoblasts in pregnant baboons, which was blocked by inhibiting the formation (23) or action (26) of estrogen. In human syncytiotrophoblasts in culture, estradiol increased the uptake and use of LDL cholesterol for progesterone production (27). Because the progressive increase in LDL receptor and P-450scc mRNA expression with advancing baboon pregnancy occurred specifically within the syncytiotrophoblasts (9, 25), we have proposed that there is an estrogen-dependent developmental increase in functional differentiation of syncytiotrophoblasts in the second half of primate pregnancy, which is manifested as an increase in key components of the progesterone biosynthetic pathway. Apparently, this provides the necessary substrate for the increase in placental progesterone production that is typical of primate pregnancy (6).
Estrogen also has been shown to regulate P-450scc activity and/or expression in porcine granulosa cells (28) and the rabbit corpus luteum (29) and, along with FSH, induce the mRNA for P-450scc in vitro and in vivo in rat granulosa cells (30). Moreover, in the laboratory rat and rabbit, species in which the corpus luteum is the principal source of progesterone during pregnancy, estrogen also regulates additional components of the progesterone biosynthetic pathway (reviewed in Refs. 31–33). For example, high-density lipoprotein uptake (34), de novo cholesterol biosynthesis (35), and mitochondrial sterol carrier protein 2 content (32) were stimulated by estrogen in the corpus luteum during rat pregnancy. Cholesterol transfer within the mitochondria and/or between storage sites also was regulated by estrogen in the rabbit corpus luteum (36). Consequently, cellular uptake and intracellular trafficking of cholesterol substrate, as well as P-450scc-catalyzed cholesterol metabolism, seem to be regulated by estrogen in both the laboratory rodent and primate. Apparently, although two completely different reproductive organs are involved in progesterone production during pregnancy in these species, estrogen has a similar key regulatory role.
Although placental P-450scc activity in fetectomized baboons was restored by androstenedione to a level similar to that in intact animals late in gestation, increasing serum estrogen levels 2-fold above normal by administration of estradiol to fetectomized or androstenedione to intact animals, did not further elevate placental P-450scc. Similar observations were observed for placental LDL uptake in baboons (23). Therefore, we suggest that placental LDL uptake and P-450scc activity are maintained at the normal levels observed in untreated controls near term by the amounts of estrogen typically present in vivo but that these aspects of the progesterone pathway cannot be enhanced above normal by further increases in the concentrations of estrogen. The latter may result from down regulation of the estrogen receptor, which is expressed in syncytiotrophoblasts of the human placenta (37, 38), by the high levels of estrogen that are present in late gestation.
The immunocytochemical expression of placental SP1, the capacity to aromatize androstenedione to estradiol, the secretion of pregnancy-associated placental peptide-A (13), and the increase in P-450scc in response to estrogen shown in the present study are consistent with continued functional capacity of the placental syncytiotrophoblast after fetectomy in baboons. The syncytial trophoblast also seemed normal in the human placenta despite fetal death (39). However, the arrest of placental growth after fetectomy, as shown in baboons of the present study, may have reflected devascularization and, consequently, impaired development of the fetal mesenchyme (39) resulting from the loss of fetal villous blood flow (40). This suggests that factors of fetal origin other than, or in addition to, estrogen may be involved in growth and development of the fetal mesenchymal-derived vascular system; however, additional study is needed to examine this possibility.
In summary, the results of the present study show that there was a close association between the levels of estrogen and the mRNA levels for, and specific activity of, the P-450scc enzyme in the placenta during the second half of baboon pregnancy. On the basis of the present and recently published studies, we propose that estrogen regulates the biosynthesis of progesterone within the placenta by actions on the uptake of cholesterol substrate in the form of LDL and catalytic conversion of cholesterol to pregnenolone, thereafter, via the P-450scc enzyme.
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Acknowledgments |
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Footnotes |
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Received June 10, 1996.
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References |
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5-3ß-hydroxysteroid
dehydrogenase/isomerase messenger ribonucleic acids during baboon
pregnancy. Placenta, in press
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