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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 8 2861-2867
Copyright © 1998 by The Endocrine Society

Original Studies

Developmental Maturation of Baboon Placental Trophoblast: Expression of Messenger Ribonucleic Acid and Protein Levels of Cytosolic and Secretory Phospholipases A21

Miriam D. Rosenthal, Eugene D. Albrecht and Gerald J. Pepe

Departments of Biochemistry (M.D.R.) and Physiology (G.J.P.), Eastern Virginia Medical School, Norfolk, Virginia 23501; and the Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction (E.D.A.), University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Gerald J. Pepe, Ph.D., Department of Physiology, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. E-mail: GJP{at}borg.evms.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the human placenta at term exhibits high levels of phospholipase A2 (PLA2) enzyme activity, our understanding of the ontogenesis, regulation, and specific roles of placental phospholipases A2 remains relatively incomplete. Using the baboon as the experimental model, the present study determined whether the levels of the messenger ribonucleic acid (mRNA) for the cytosolic (cPLA2) and/or type IIa, nonpancreatic secretory (sPLA2) enzymes are developmentally regulated and modulated by glucocorticoid treatment. Total RNA was extracted from whole villous placenta obtained on days 60 (early; n = 3), 100 (mid; n = 3), and 165 (late; n = 4) of gestation (term = day 184) from untreated baboons and on day 100 (n = 4) after maternal administration on days 60–99 of betamethasone (3 mg/day). The complementary DNA to cPLA2 recognized a single 2.9-kb mRNA transcript in both baboon and human placenta. Mean (±SE) levels of cPLA2 mRNA, expressed, in arbitrary units as a ratio to that of ß-actin, were similar at early (0.19 ± 0.05) and midgestation (0.34 ± 0.17) and increased (P < 0.005) 10-fold (2.53 ± 0.53) by late gestation. Levels of cPLA2 protein were also greater (P < 0.05) on day 165 (2.6 ± 0.3 arbitrary units) than on day 60 (0.6 ± 0.2). Like that in the human, the baboon placenta contained very high levels of a single 0.9-kb mRNA transcript for sPLA2. In contrast to that of cPLA2, normalized levels of sPLA2 mRNA were similar at all three time points and were associated with high levels of sPLA2 protein throughout gestation. Treatment with betamethasone increased (P < 0.02) cPLA2 mRNA levels on day 100 by over 4-fold, but had no effect on sPLA2 mRNA levels. Additional studies indicated that the mRNAs for sPLA2 and cPLA2 were detected in an enriched fraction of nontrophoblast cells isolated by collagenase dispersion and Percoll density centrifugation. The mRNA for cPLA2 was also expressed in cytotrophoblast and syncytiotrophoblast cell fractions. Collectively, these findings indicate that the baboon placenta expresses mRNA and protein for both the cytosolic and secretory PLA2 enzymes, and that expression of cPLA2 is developmentally regulated and modulated by glucocorticoids. We previously demonstrated an estrogen-dependent developmental increase in placental expression of specific components of the progesterone steroidogenic pathway during the second half of baboon pregnancy. The current findings indicate that the developmental increase in placental function also includes expression of at least one specific PLA2 enzyme controlling arachidonic acid mobilization and eicosanoid synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN HUMAN and nonhuman primates, the placenta plays an active role in producing hormones essential to pregnancy maintenance and fetal development. This is reflected by a progressive developmental increase in placental expression of key components of the progesterone steroidogenic pathway, e.g. the P-450 cholesterol side-chain cleavage system (1), enzymes of cortisol/cortisone metabolism (2), and key protein hormones such as chorionic somatomammotropin (3). Enhanced gene expression is observed both during the transformation of cytotrophoblasts into syncytiotrophoblasts and during the subsequent functional maturation of the syncytiotrophoblasts (4, 5). The developing placenta is, in turn, a target tissue for regulation by hormones, including cortisol and estrogen (6).

In addition to steroid and protein hormones, the placenta actively synthesizes several cyclooxygenase metabolites of arachidonic acid, including thromboxane A2, prostacyclin, and PGE2 (7, 8). PGs have been shown to play an autocrine/paracrine role in pregnancy maintenance in part by modulating uteroplacental immune and inflammatory responses (9). Placental thromboxane has also been postulated to modulate maternal and fetal hemodynamics (10). Recent studies indicate that the human placenta synthesizes a wide variety of noncyclooxygenase eicosanoids, including the lipoxygenase metabolite 12-HETE (hydroxyeicosa tetraenoic acid) and cytochrome P450 metabolites at levels comparable to those of the PGs (11). Although the contributions of these latter arachidonate metabolites to placental development are still unclear, 12-HETE has been shown to modulate vasoconstriction in the human placenta (12).

The synthesis of all eicosanoids requires the activation of one or more phospholipase A2 (PLA2) enzymes that catalyze the release of free arachidonic acid from the sn-2 position of membrane phospholipids. At least two distinctly different calcium-dependent PLA2 enzymes have been shown to contribute to the synthesis of these autocoid modulators (13, 14). One is a 14-kDa nonpancreatic secretory protein (group IIa or sPLA2) that has been localized to a number of cell types, including platelets (15) and intestinal Paneth cells (16), and is also present in plasma and synovial fluid (17). The other is an 85-kDa cytosolic PLA2 (cPLA2), which is expressed in many cell types (18). cPLA2 is specific for arachidonyl-containing phospholipids and has been shown to be a key intracellular mediator of hormone-stimulated eicosanoid synthesis (19).

The human placenta at term exhibits high levels of PLA2 enzymatic activity (13) and expresses type IIa sPLA2 messenger ribonucleic acid (mRNA) (20). Moreover, explants of term human placentas release sPLA2 enzymatic activity in vitro (21). Expression of mRNA for cPLA2 has also recently been demonstrated in human term placenta and membranes (22). Despite their importance, however, our understanding of the ontogenesis, regulation, and specific roles of placental PLA2 in pregnancy remains incomplete. Our laboratories have shown that the baboon is a valuable nonhuman primate model for the study of the endocrinology of human pregnancy, including the developmental maturation of placental trophoblasts. Therefore, the present study was designed to determine which phospholipase A2 genes are expressed in the baboon placenta and to ascertain whether levels of mRNA for cPLA2 and/or type IIa sPLA2 are developmentally regulated. Because expression of both sPLA2 and cPLA2 has been shown to be modulated by antiinflammatory agents such as glucocorticoids (23, 24, 25), and placentas were available from betamethasone-treated baboons at midgestation (26), we also investigated whether placental PLA2 gene expression is modulated by betamethasone.


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

Female baboons (Papio anubis) obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX) and weighing 10–15 kg were housed individually in air-conditioned quarters as described previously (27). Females were paired with males for 5 days at the anticipated time of ovulation, as determined by menstrual cycle history and turgescence of the external sex skin. Baboons were cared for and used strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals (Publication 85–23, 1985). The experimental protocol employed was approved by the institutional animal care and use committee of Eastern Virginia Medical School.

On days 60, 100, and 165 of gestation (term = day 184) baboons were anesthetized with halothane-nitrous oxide, and laparotomy and hysterotomy were performed. After delivery of the fetus, the placenta was removed and stripped of associated membranes, and tissue pieces were frozen in liquid nitrogen for subsequent Northern/Western analysis. Midgestation (day 100) placentas were also obtained from baboons that were treated with 3.0 mg betamethasone (Celestone-Soluspan, Schering Corp., Orange, NJ) administered sc to the mother after ketamine (10 mg/kg BW; Ketalar, Parke-Davis, Detroit, MI) sedation daily on days 60–99 gestation as described previously (26).

Tissue samples were also obtained from an adult, nonpregnant baboon that had been killed. A term human placenta was obtained after uncomplicated vaginal delivery and was similarly frozen in liquid nitrogen for subsequent analysis. Prior approval was obtained from the institutional review board, Eastern Virginia Medical School. U937 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured as described previously (28).

Northern analysis of sPLA2 and cPLA2 mRNA

Total RNA was extracted from placental samples and from heart, liver, spleen, and kidney of a nonpregnant adult female baboon essentially as described previously (1, 4). Tissues were homogenized in 4 mol/L guanidine isothiocyanate, 25 mm sodium acetate (pH 6.0), and 0.83% ß-mercaptoethanol at 4 C, and RNA was extracted with chloroform-isoamyl alcohol (24:1, vol/vol), essentially as described by Chirgwin et al. (29). The aqueous layer was forced through a 23-gauge needle, layered onto a 5.7 mol/L cesium chloride gradient, and centrifuged at 179,000 x g for 21 h at 23 C.

Approximately 30 µg total RNA were denatured in 50% formamide, 2.2 mol/L formaldehyde, and 20 mmol/L 3-[N-morpholino]propane sulfonic acid, pH 7.0, and size-fractionated by electrophoresis in a 1.0% agarose gel containing 2.2 mol/L formaldehyde. RNA was transferred overnight by capillary action onto a nylon membrane (Magnagraph, MSS, Westboro, MA) using 20 x SSC (1 x SSC contains 0.15 mol/L sodium chloride and 15 mmol/L sodium citrate, pH 7.0) and immobilized by baking for 1 h at 80 C.

Plasmids containing complementary DNAs (cDNAs) for human sPLA2 (group IIa) and cPLA2, generously provided by Dr. Lisa Marshall (Smith Kline Beecham, King of Prussia, PA), were purified and treated with appropriate restriction endonucleases to yield a full-length cDNA for sPLA2 (800 bp) and 780 bp of the 5'-terminus of the 2.9-kb cPLA2 cDNA. The cDNA for human ß-actin (no. 65128) was obtained from American Type Culture Collection. Membranes were prehybridized for 4 h at 42 C in a solution containing 5 x SSC, 50% formamide, 0.1% polyvinylpyrrolidone, 0.1% BSA, 0.1% Ficoll, 0.1% SDS, denatured salmon sperm DNA (0.25 mg/mL), yeast transfer RNA (50 µg/mL), and 0.050 mol/L sodium phosphate, pH 6.5. Hybridization was performed overnight in fresh buffer containing 32P-labeled cDNA probe, prepared by incubation with 120 µCi [{alpha}-32P]deoxy-CTP (6000 mCi/mmol; New England Nuclear, Boston, MA) and a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). After hybridization, the membranes were washed four times (5 min each) at room temperature in 2 x SSC containing 0.1% SDS, then twice (15 min each) at 50 C in 0.1% SSC and 0.1% SDS. The membranes were exposed (-80 C) for 2–96 h (depending on level of signal) to medical x-ray film. Before successive probing with a second cDNA probe, membranes were washed for 1 h at 65 C in 50% (vol/vol) formamide and 10 mmol/L sodium phosphate buffer, pH 6.5, and then washed for 15 min at 23 C in 0.1% SDS in 2 x SSC. After film development, samples were quantified by one-dimensional densitometry in an LKB Ultroscan XL Enhanced Laser densitometer (LKB, Bromma, Sweden) equipped with computer Gel Scan XL Software.

Western analysis

For detection of cPLA2 protein, samples of whole villous placenta were homogenized (three times, 15 s each time, at 10-s intervals) on ice with a Brinkmann Polytron (Brinkmann Instruments, Westbury, NY) in sample buffer (Ca2+- and Mg2+-free phosphate-buffered saline with 1% cholic acid, 0.1% SDS, and 1 mmol/L ethylenediamine tetraacetate) to which was added a protease inhibitor cocktail consisting of phenylmethylsulfonylfluoride (0.1 mg/mL), aprotinin (10 µg/mL), and soybean trypsin inhibitor (0.1 mg/mL). The protein concentration was determined by the bicinchoninic acid procedure (Sigma Chemical Co., St. Louis, MO).

For analysis of sPLA2 protein, acid extracts were prepared by homogenizing tissue samples (as described above) in equal volumes of 0.36 N sulfuric acid containing 2 mol/L NaCl, and then stirring for 4 h at 4 C (30). After centrifugation for 1 h at 28,000 x g, the supernatants were dialyzed to pH 4.5 with 10 mmol/L sodium acetate and concentrated under vacuum.

Samples for electrophoresis were prepared by addition of 5 x Laemmli buffer (31) to a final concentration of 1 x, heating at 100 C for 2 min, and centrifugation (10,000 x g, 2 min). The samples were then loaded (30 µg protein/lane) onto preformed SDS-polyacrylamide gels (8% for detection of cPLA2; 15% for sPLA2) in a Bio-Rad mini-protein II electrophoresis chamber (Bio-Rad Laboratories, Richmond, CA) containing chilled 0.025 mol/L Tris, 0.192 mol/L glycine, and 0.1% SDS buffer (pH 8.3). Samples were electrophoresed at 110 V and wet transferred with a Mini Trans Blot transfer unit (110 V, 75 min) in 0.192 mol/L glycine-0.025 mol/L Tris buffer (pH 8.3) containing 20% (vol/vol) methanol onto Immobilon-P membranes (Micron Separations, Westborough, MA).

For detection of cPLA2, membranes were blocked for 1 h with constant agitation at 22 C in buffer I [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, and 0.1% Tween-20] containing 5% nonfat dried milk (Bio-Rad). The membranes were then incubated overnight at 4 C with monoclonal mouse IgG2b antibody to the amino-terminal domain of human cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:1000 in buffer I containing 0.1% IGEPAL (Sigma) and 5% dried milk. Membranes were washed and incubated for 1 h at 22 C with rabbit anti-mouse IgG horseradish peroxidase-conjugated second antibody. After washing, enhanced chemiluminescence reagent (ECL, Amersham Life Technologies, Aylesbury, UK) was applied to the membranes for 1 min, and the wrapped membranes were visualized by 5- to 30-s exposures to medical x-ray film. After film development, the blots were quantified using a Bioimage analyzer (Bioimage, Ann Arbor, MI).

For detection of sPLA2, membranes were blocked for 1 h at 37 C in buffer I containing 3% BSA (Sigma). The membranes were then incubated for 1 h at room temperature with polyclonal (rabbit) antibody to human vertebral disc sPLA2, provided by Dr. Richard Franson (Virginia Commonwealth University, Richmond, VA). The antibody was purified using ImmunoPure Plus Immobilized protein A (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s directions and diluted to 5 µg/mL in buffer I containing 0.05% IGEPAL and 1.5% BSA. The membranes were then washed and incubated for 1 h at room temperature with donkey antirabbit IgG horseradish peroxidase-conjugated second antibody in buffer I containing 1.0% BSA. After washing, the enhanced chemiluminescence procedure was performed as described above. Recombinant human sPLA2 used as a standard (50 ng/lane) was provided by Dr. Lisa Marshall (Smith Kline Beecham).

Placental cell dispersion

Enriched fractions of cytotrophoblast, syncytiotrophoblast, and nontrophoblast cells from late gestation baboon placenta were prepared using modifications of the method of Kliman et al. (32) as previously adapted for use with baboon tissues (33). Briefly, sections of whole villous placenta were rinsed in cold sterile calcium- and magnesium-free Hanks’ salts (HBSS) and dispersed by incubation for 35 min at 37 C in bicarbonate-buffered HBSS containing collagenase (0.1%), hyaluronidase (0.1%), deoxyribonuclease (0.01%), trypsin inhibitor (0.023%), and FBS (0.1%). The digest was then iced, strained through Nitrex cloth, and centrifuged at 500 x g for 15 min at 4 C. The resulting pellets were resuspended in HBSS, and aliquots were layered over 5–70% Percoll gradients, which were centrifuged at 1125 x g for 20 min at 23 C. The layers containing primarily syncytiotrophoblasts (density, 1.014–1.021), cytotrophoblasts (density, 1.048–1.066), and nontrophoblast cells (two bands at intermediate densities) were transferred to separate tubes of HBSS and centrifuged at 500 x g for 15 min. Total RNA was prepared from the resultant pellets as described above.

Statistical analysis

Data were analyzed by unpaired t tests or ANOVA, with post-hoc comparison of the means using adjusted t tests with P values corrected by the Bonferroni method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Baboon placental cPLA2 and sPLA2 mRNA levels

Figure 1Go shows Northern blot analysis of total RNA from various baboon tissues as well as term human placenta and human promyelocytic U937 cells probed with cDNAs to the 85-kDa cPLA2 and group IIa sPLA2. The cDNA probe for cPLA2 recognized a single 2.9-kb transcript in baboon tissues similar to that in human U937 cells. The level of cPLA2 mRNA in baboon placenta exceeded those in kidney and other organs examined. The cDNA probe for type IIa sPLA2 recognized a single 0.9-kb transcript in both human and baboon placenta. As seen in the blot, the message for sPLA2 was present at high levels in late gestation placenta. Although some sPLA2 also appeared to be expressed in adult heart, the baboon liver, kidney, and spleen samples were essentially negative. However, ß-actin mRNA was expressed in all samples (data not shown). Based upon the film exposure times required for detection of comparably labeled PLA2 probes, the levels of cPLA2 mRNA in baboon placenta were substantially less than those of type IIa sPLA2.



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  		Figure 1. Expression of cytosolic cPLA2 and type IIa sPLA2 mRNA in baboon placenta and adult baboon tissues. Northern blot of total RNA (~30 µg/lane) from adult baboon heart, liver, kidney, and spleen, baboon and human whole villous placenta, and U937 cells was initially probed with human cDNA for sPLA2. The membrane was then stripped and reprobed for cPLA2. The molecular size of transcripts was determined from the migration pattern of a 0.28- to 6.6-kb RNA ladder. x-Ray film exposure was 4 h for sPLA2 and 72 h for cPLA2.

 
Developmental changes in steady state levels of PLA2 mRNA

As shown in Fig. 2AGo, expression of cPLA2 mRNA in placentas from early (day 60) and midgestation (day 100) was minimal relative to that in late gestation (day 165). These differences were also observed when the cPLA2 mRNA levels were expressed as a ratio of ß-actin mRNA, the levels of which declined in late gestation as we have previously shown in whole villous baboon placenta (4). Aggregate analysis (Fig. 2BGo) indicated that mean (±SE) cPLA2/ß-actin levels increased (P < 0.005, by ANOVA) over 10-fold from 0.19 ± 0.05 arbitrary units early in gestation to 2.53 ± 0.53 arbitrary units at late gestation.



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  		Figure 2. Developmental expression of cPLA2 and type IIa sPLA2 mRNAs in baboon placental whole villous tissue. A, Northern blot of total RNA (30 µg/lane) in placentas obtained from baboons at early (day 60), mid- (day 100), and late (day 165) gestation (term = 184 days), which was initially probed with cDNA for sPLA2, then stripped and reprobed with cDNAs to cPLA2 and ß-actin. B, Means (±SE) of the ratios of cPLA2/ß-actin mRNA levels (arbitrary units) in early (n = 3), mid- (n = 3), and late (n = 4) gestation whole villous baboon placentas analyzed by autoradiodensitometry. Groups with different letter superscripts differ from each other (P < 0.05) as determined by ANOVA and post-hoc corrected Bonferroni P values.

 
In contrast to the results for cPLA2, however, mRNA for sPLA2 appeared to be expressed at relatively high levels early in gestation (Fig. 2Go). Moreover, aggregate analysis (data not shown) indicated that mean sPLA2 mRNA levels and ratio to ß-actin did not change with advancing gestation.

Developmental changes in cPLA2 protein levels

Western analysis of proteins from baboon placenta confirmed the presence of cPLA2 protein (Fig. 3AGo), which was detected as a major band with an expected apparent molecular size of 110 kDa (18, 19). Moreover, levels of cPLA2 in whole villous placenta (Fig. 3BGo) increased with advancing gestation and were approximately 4-fold greater (P < 0.05) on day 165 (2.5 ± 0.3 arbitrary units) than on day 60 (0.6 ± 0.2).



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  		Figure 3. Developmental expression of cPLA2 protein in baboon placental whole villous tissue. A, Representative Western blot of protein homogenates (30 µg/lane) from whole villous placentas obtained at early, mid-, and late gestation, incubated with monoclonal antibody to human cPLA2, and detected by the enhanced chemiluminescence procedure. B, Mean (±SE) of cPLA2 expression levels (arbitrary units) at early (n = 4), mid- (n = 4), and late (n = 4) gestation. Values with different superscripts differ from each other (P < 0.05) were determined by ANOVA and post-hoc corrected Bonferonni P values.

 
Western immunoblot of sPLA2 protein

A polyclonal antibody prepared against human vertebral disc sPLA2 and previously shown not to cross-react with pancreatic PLA2 (34) was used to detect sPLA2 protein in baboon tissue. Figure 4AGo shows that the antibody recognized a 14-kDa sPLA2 band in an acid extract of term human placenta and a similarly sized protein in baboon placenta obtained in late gestation. The expression of sPLA2 was tissue specific, as sPLA2 protein was not detected in baboon heart, kidney, or spleen. As shown in Fig. 4BGo, levels of sPLA2 protein were similar in acid extracts from two early, two mid-, and two late gestation placentas.



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  		Figure 4. A, Western blot analysis of secretory PLA2 protein in baboon placenta and adult tissues. Acid extracts (40 µg/lane) of adult baboon heart, kidney, and spleen, and baboon and human whole villous placenta were probed with polyclonal antibody to human vertebral disc sPLA2 and detected by the enhanced chemiluminescence procedure. Recombinant human sPLA2 (50 ng/lane) was used as a standard. B, Western blot of sPLA2 protein in acid extracts of placentas obtained from baboons at early (n = 2), mid- (n = 2), and late (n = 2) gestation.

 
Effects of betamethasone on sPLA2 and cPLA2 mRNA levels

Figure 5Go shows the Northern blot analysis of placental whole villous tissue obtained at midgestation from untreated baboons and animals in which the mother was administered betamethasone on days 60–99 of gestation. Glucocorticoid treatment markedly enhanced the expression of mRNA levels of cPLA2. Aggregate analysis (Fig. 5BGo) indicated that mean (±SE) cPLA2/ß-actin mRNA levels were increased approximately 4-fold (P < 0.016, by unpaired t test) by betamethasone. By contrast, steady state type IIa sPLA2 mRNA levels (Fig. 5AGo) were not significantly altered by betamethasone treatment (ratio to ß-actin, 0.91 ± 0.20 vs. 1.45 ± 0.59 for controls).



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  		Figure 5. Effects of maternal betamethasone administration at midgestation on expression of cPLA2 and type IIa sPLA2 in baboon placenta. A, Northern blot of total RNA (30 µg/lane) in placentas obtained on day 100 from baboons untreated or treated with betamethasone (3.0 mg/day) administered to the mother on days 60–99 of gestation. The membrane was probed successively for sPLA2, cPLA2, and ß-actin as described in Fig. 2Go. B, Means (±SE) of the ratios of cytosolic phospholipase A2/ß-actin mRNA levels in placentas obtained from midgestation control animals (n = 4) and after maternal betamethasone administration (n = 4) analyzed by autoradiodensitometry. *, Value differs from the control at P < 0.016 (by unpaired t test).

 
Distribution of PLA2 mRNAs in placental cells

To investigate which cells expressed sPLA2 and cPLA2 mRNA, late gestation baboon placentas were digested with collagenase and fractionated by Percoll gradient centrifugation. This procedure yields three major fractions, a substantial, relatively buoyant syncytiotrophoblast band, a smaller band of relatively more dense cytotrophoblasts, and an intermediate fraction that contains a not fully characterized mixture of nontrophoblast cells, including endothelial cells, fibroblasts, and macrophages (32). Figure 6Go shows a representative Northern blot from three of four independent placental preparations. As shown in the figure, the mRNA for sPLA2 was expressed primarily in the nontrophoblast cell population. However, the mRNA for cPLA2 was expressed in trophoblast as well as nontrophoblast cells.



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  		Figure 6. Northern blot analysis of the mRNAs for cytosolic and type IIa secretory phospholipases A2 in different cell populations within baboon placenta. Enriched fractions of syncytiotrophoblast (S), nontrophoblast (N), and cytotrophoblast (C) cells were prepared by collagenase dispersion and Percoll density centrifugation. The Northern blot of total RNA (~20 µg/lane) from cells prepared from three late gestation placentas was probed for type IIa sPLA2 and cPLA2 as described in Fig. 1Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that late gestation baboon placenta contains substantial levels of mRNA for both the 85-kDa cytosolic or cPLA2 and the type IIa 14-kDa secretory or sPLA2. Our results also indicate that cPLA2 gene expression is developmentally regulated, with levels of cPLA2 message and protein increasing significantly with advancing gestation. Taken together, the observations that cPLA2 steady state mRNA and protein levels increased late in gestation, and that the levels of expression in the placenta were markedly higher than those in other organs are consistent with a role for cPLA2 in synthesis of placental eicosanoids, and thus in the autocrine and/or paracrine modulation of placental function.

Our results also indicate that placental steady state cPLA2 mRNA levels were increased in animals treated with betamethasone at midgestation. Down-regulation of cPLA2 and/or sPLA2 mRNA expression in many cell types by glucocorticoids is a well documented antiinflammatory mechanism (18, 24, 25). In amnion cells, however, dexamethasone has been shown to stimulate synthesis of cyclooxygenase-2, indicating that glucocorticoids may also exhibit positive regulatory effects on enzymes of eicosanoid synthesis, particularly in intrauterine tissues (35). Furthermore, glucocorticoid-induced premature labor has been shown to up-regulate cPLA2 mRNA levels in ovine endometrium (36). Whether the observed effect of betamethasone on placental cPLA2 mRNA was direct or elicited via modulation of other hormones or paracrine/autocrine factors remains to be elucidated.

The placenta is a complex organ containing a variety of specialized cell types that may possess differing capabilities for PLA2-mediated arachidonate mobilization and subsequent eicosanoid synthesis. Many of these, such as endothelial cells, smooth muscle, and macrophages are not unique to the placenta. Importantly, however, preparations of isolated trophoblasts and cultured trophoblast cells have both been shown to actively synthesize arachidonate metabolites such as PGE2, thromboxane A2, and 12-HETE (7). Although the physiological role(s) of these active lipids in placental function remains to be elucidated, increased trophoblast synthesis of thromboxane A2 and lipid peroxides may contribute to placental vasoconstriction in preeclampsia (10, 37). Our observations that both cytotrophoblast and syncytiotrophoblast cell fractions express the mRNA for cPLA2, but little or no type IIa sPLA2, would suggest that cytosolic or cPLA2 may play a role in mediating eicosanoid synthesis in placental trophoblasts.

The findings of the present study also indicate that type IIa sPLA2 mRNA is expressed in very high levels in placenta as early as day 60 of gestation, and that gene expression is accompanied by substantial synthesis of immunoreactive protein. Although the role(s) of sPLA2 in primate placenta during gestation remains to be elucidated, the multiple contributions of sPLA2 in other organ sites suggest several possibilities. In other tissues, secretory PLA2, acting as ectoenzymes, have been shown to initiate and/or enhance arachidonate mobilization and thus contribute to cellular eicosanoid synthesis (16). sPLA2 also appears to contribute to antimicrobial activity and the removal of injured cells during inflammation (38). Alternatively, sPLA2 could contribute to the extensive membrane remodeling and membrane fusion that occur during placental growth and differentiation. Indeed, recent studies on ovarian phospholipases A2 led to the suggestion that type IIa sPLA2 may participate in the rapid cellular and tissue remodeling that occur during the periovulatory period (39, 40).

In conclusion, the present study shows relatively high levels of mRNA for both the 85-kDa cytosolic or cPLA2 and the 14-kDa type IIa secretory or sPLA2 in baboon placenta. Steady state levels of cPLA2 mRNA and protein exhibit a developmental increase during gestation similar to that for enzymes that contribute to placental synthesis of steroid and protein hormones. Moreover, cPLA2 expression appears to be modulated by glucocorticoids. Taken together, these observations support the hypothesis that hormonal regulation of cPLA2 gene expression enhances eicosanoid synthesis in the developing placenta. By contrast, the maintenance of relatively high levels of type IIa sPLA2 mRNA throughout gestation and its expression primarily in nontrophoblast cells of the placenta support a distinctly different role for this PLA2 enzyme in primate pregnancy.


   Acknowledgments
 
The authors greatly appreciate the assistance of Ms. Kathleen Dunstan in performing the cPLA2 Western blot analyses, Mr. Nicholas Zachos in preparation of the figures, and Ms. Sandra Huband in preparation of the final manuscript. The authors thank Dr. Lisa Marshall, Smith Kline Beecham (King of Prussia, PA) for the cDNAs for human cPLA2 and type IIa sPLA2, Dr. Richard Franson of Virginia Commonwealth University for the antibody to human sPLA2, and Ms. Marcia Burch and Dr. Laura Moen for their excellent technical advice.


   Footnotes
 
1 This work was supported by NIH Research Grant R01 HD-13294. Back

Received January 15, 1998.

Revised April 7, 1998.

Accepted May 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Babischkin JS, Pepe GJ, Albrecht ED. 1996 Developmental expression of placental trophoblast P-450 cholesterol side chain cleavage, adrenodoxin, and {Delta}5-3ß-hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy. Placenta. 17:595–602.[CrossRef][Medline]
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  3. Golos TG, Durning M, Fisher JM, Fowler PD. 1993 Cloning of four growth hormone/chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta. Endocrinology. 133:1744–1752.[Abstract]
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