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Prostaglandins and Leukotriene B₄ Are Potent Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 2 Activity in Human Choriocarcinoma JEG-3 Cells¹

^a The Lawson Research Institute, St. Joseph's Health Centre, Departments of Obstetrics and Gynaecology and Physiology, University of Western Ontario, London, Ontario, Canada N6A 4V2

	ABSTRACT

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The 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is responsible for the inactivation of glucocorticoids. This is the predominant isozyme in the human placenta, where it is proposed to protect the fetus from high levels of maternal cortisol. In the present study, we examined the effects of eicosanoids on the activity of 11ß-HSD2 in human choriocarcinoma JEG-3 cells, a well-established model for placental trophoblasts. Treatment of JEG-3 cells for 24 h with either prostaglandin (PG) E₂ or F₂ attenuated 11ß-HSD2 activity (~40%). Paradoxically, indomethacin, an inhibitor of cyclooxygenases, inhibited (~40%) rather than stimulated the activity of this enzyme. This indicated that the arachidonic acid metabolism may be diverted to other pathway(s), the products of which may inhibit 11ß-HSD2 activity. To determine whether the lipoxygenase pathways were involved, the cells were treated with nordihydroguaretic acid (NDGA), a blocker of all three (5-, 12-, and 15-) lipoxygenases. NDGA caused a 3-fold increase in 11ß-HSD2 activity. To further delineate which specific lipoxygenase pathway was involved, the cells were incubated with zileuton, a selective inhibitor of 5-lipoxygenase. This resulted in a similar increase in 11ß-HSD2 activity, suggesting that the products of this pathway (e.g., leukotrienes) may be involved. Given that leukotriene B₄ (LTB₄) is the most biologically active product of the 5-lipoxygenase pathway, we treated the cells with LTB₄, which inhibited 11ß-HSD2 activity in a time- and dose-dependent manner with a maximal effect (60% reduction) at 10 nM for 9 h. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that 11ß-HSD2 mRNA levels were not altered by the addition of LTB₄, PGE₂, or PGF₂, indicating an effect at the posttranscriptional level. In conclusion, these results demonstrate that prostaglandins and LTB₄ are potent inhibitors of 11ß-HSD2 activity in JEG-3 cells, suggesting that placental 11ß-HSD2 activity is modulated by these locally produced eicosanoids. This is the first time that the products of arachidonic acid metabolism have been found to regulate the activity of 11ß-HSD2.

	INTRODUCTION

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Glucocorticoids are essential for normal fetal organ growth and maturation, but excessive exposure to glucocorticoids in utero leads to intrauterine growth restriction (IUGR) and possibly affects postnatal life [1, 2]. Fetal exposure to glucocorticoids is controlled by many factors, including fetal adrenal production, circulating corticosteroid-binding globulin concentrations, intracellular metabolism, and the transplacental transfer of maternal glucocorticoids [3]. The placental 11ß-hydroxysteroid dehydrogenase type 2 enzyme (11ß-HSD2), which converts maternal cortisol to its inactive metabolite, cortisone, plays a pivotal role in restricting the passage of maternal cortisol to the fetus [4]. Therefore, it is proposed that placental 11ß-HSD2 serves as a barrier to protect the fetus from high levels of maternal glucocorticoids [5]. Recent studies have provided circumstantial evidence in support of this hypothesis.

Immunohistochemistry studies have localized 11ß-HSD2 exclusively in the syncytiotrophoblast, the site of maternal-fetal exchange [6]. In both humans and rats, there is a positive correlation between placental 11ß-HSD2 activity and birth weight [7, 8]. Moreover, placental 11ß-HSD2 activity is attenuated in pregnancies complicated with IUGR [9, 10], and IUGR is a characteristic feature of the apparent mineralocorticoid excess [11], a syndrome resulting from 11ß-HSD2 deficiency [12].

Previous studies have shown that progesterone, estradiol, and nitric oxide attenuate, while agonists of the protein kinase A pathway and retinoic acid up-regulate 11ß-HSD2 activity and mRNA in cultured primary human placental trophoblast and/or human choriocarcinoma JEG-3 cells [13–17]. Several elegant studies in the baboon have also provided convincing evidence that estrogen increases the placental 11ß-HSD dehydrogenase activity (cortisol to cortisone) both in vivo and in vitro [18–20].

Given that eicosanoids, such as prostaglandins and leukotrienes, are produced in the placenta and are intimately involved in pregnancy and parturition [21, 22], we hypothesized that these locally produced factors regulate the activity of placental 11ß-HSD2. In the present study, we examined the effects of the products of cyclooxygenase and lipoxygenase pathways in arachidonic acid metabolism on 11ß-HSD2 activity in JEG-3 cells. This syncytiotrophoblast-like cell line has retained the ability to convert cortisol to cortisone and therefore has been used widely for investigation of the regulation of 11ß-HSD2 [13, 14, 17, 23]. Moreover, JEG-3 cells produce a range of placental hormones/factors, including prostaglandins and leukotrienes [24–26]. Here we present the first evidence that prostaglandin (PG) E₂ and F₂ and leukotriene B₄ (LTB₄) inhibit 11ß-HSD2 activity without affecting its steady-state mRNA level in JEG-3 cells.

	MATERIALS AND METHODS

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Reagents and Supplies

[1,2,6,7-³H(N)]Cortisol (80 Ci/mmol) was purchased from Du Pont Canada Inc. (Markham, ON, Canada). Nonradioactive steroids were obtained from Steraloids Inc. (Wilton, NH). LTB₄ was purchased from Sigma Chemical Company (St. Louis, MO). Zileuton was obtained as a kind gift from Abbott Laboratories (Abbott Park, IL). Nordihydroguaretic acid (NDGA) was purchased from Cayman Chemicals (Ann Arbor, MI). Polyester-backed thin-layer chromatography (TLC) plates were obtained from Fisher Scientific Ltd. (Unionville, ON, Canada). All solvents used were OmniSolve grade from BDH Inc. (Toronto, ON, Canada). General molecular biology reagents were from Gibco BRL (Burlington, ON, Canada) or Pharmacia Canada Inc. (Baie D'Urte, PQ, Canada). The JEG-3 cell line was purchased from American Type Culture Collection (Rockville, MD). Cell culture supplies were obtained from Gibco BRL or Fisher Scientific. PGE₂ and PGF₂ were generously donated by Dr. T.G. Kennedy (University of Western Ontario, London, ON, Canada).

Cell Culture and Treatments

JEG-3 cells were cultured in minimum essential medium Eagle (MEM) supplemented with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, and penicillin/streptomycin. Cells were maintained in T-25 Corning (Corning, NY) flasks at 37°C in a 95% air:5% CO₂ humidified incubator. The medium was changed every other day. The cells were passaged as required. To study the effects of eicosanoids on 11ß-HSD2 activity, cells were plated onto 12-well Corning plates and cultured to confluence. The cells were then cultured in serum-free medium for 24 h before treatment, and all the treatments were carried out under serum-free conditions. Cells in triplicate wells were exposed to given doses of eicosanoids or inhibitors for 24 h or as stated otherwise. Controls (also in triplicate) were treated similarly but without the addition of eicosanoids or inhibitors. For 11ß-HSD2 mRNA analysis, cells were subjected to identical culture and treatment conditions as described above except that they were maintained in T-25 flasks.

Assay of 11ß-HSD2 Activity—Radiometric Conversion Assay

At the end of treatment, the cells were washed three times in serum-free medium to remove the treatment compound, in an attempt to exclude its having a possible competitive inhibition. The level of 11ß-HSD2 activity in intact cells was determined by measuring the rate of cortisol to cortisone conversion, as described previously [17]. Briefly, the cells were incubated for 4 h at 37°C in serum-free medium containing ~100 000 cpm [³H]cortisol and 10 nM unlabeled cortisol. At the end of incubation, the medium was collected, and steroids were extracted. The extracts were dried, and the residues were resuspended. A fraction of the resuspension was spotted on a TLC plate, which was developed in chloroform/methanol (9:1, v:v). The bands containing the labeled cortisol and cortisone were identified by UV light of the cold carriers, cut out into scintillation vials, and counted in Scintisafe Econol 1 (Fisher Scientific, Toronto, ON, Canada). The rate of cortisol to cortisone conversion was calculated, and the blank values (defined as the amount of conversion in the absence of cells) were subtracted and expressed as a percentage of control. Results are shown as mean ± SEM. Under conditions of the present study, the basal level of 11ß-HSD2 activity in JEG-3 cells was 8–12%. One-way ANOVA followed by Dunnett's test was used to determine statistical differences. Significance was set at p < 0.05.

Analysis of 11ß-HSD2 mRNA Semiquantitative Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)

To determine whether changes in 11ß-HSD2 activity following the different treatment regimes were associated with alterations in 11ß-HSD2 mRNA, the relative abundance of 11ß-HSD2 mRNA in JEG-3 cells was assessed by an established semiquantitative RT-PCR protocol, as described [17]. Briefly, total RNA was extracted from cultured cells using an RNeasy kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's instructions. Before use, the RNA samples (1–2 µg) were checked by agarose gel electrophoresis in the presence of formaldehyde, and the integrity of the RNA was assessed by the presence of two sharp bands representing 28S and 18S rRNA after staining with ethidium bromide. One microgram of total RNA was reverse-transcribed using a standard oligo-dT primer in a total volume of 20 µl. An aliquot (2 µl) of the RT reaction products was then subjected to a standard PCR (95°C, 55 sec; 55°C, 55 sec; 72°C, 1 min; 32 cycles) using sequence-specific primers (forward primer, 5'-AGTAGTTGCTGATGCGGA; reverse primer, 5'-CATGCAAGTGCTCGATGT) that correspond to nucleotides 624–641 and 1004–1021 in the published human 11ß-HSD2 cDNA [27], respectively. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a control. The same PCR conditions were used for GAPDH except that a cycle number of 25 instead of 32 was used. The primers for GAPDH (forward primer, 5'-ACCACAGTCCATGCCATCAC; reverse primer, 5'-TCCACCACCCTGTTGCTGTA) were obtained from Clontech Laboratories Inc. (Palo Alto, CA), and they correspond to nucleotides 586–605 and 1018–1037 in the published human GAPDH cDNA [28]. The PCR conditions for 11ß-HSD2 were chosen after performing preliminary studies demonstrating that the amount of RT products (2 µl) and the cycle number (32) were within the linear range (Fig. 1). Similar experiments were performed to determine the PCR conditions for GAPDH (data not shown).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Establishment of conditions for semiquantitative RT-PCR. Total cellular RNA was extracted from JEG-3 cells, 1 µg of which was used as a template to produce cDNA as described in Materials and Methods. Different amounts (0.7, 2, and 6 µl) of cDNA was subjected to 32 cycles of PCR, and 2 µl of cDNA was subjected to different cycles (28, 32, and 36) of PCR, using specific primers to amplify a 397-base pair product encoding the human 11ß-HSD2.

To verify the RT-PCR products and to assess the relative abundance of 11ß-HSD2 mRNA, a fraction of the RT-PCR products was then subjected to a standard Southern blot analysis, using ³²P-labeled human 11ß-HSD2 cDNA and ³²P-labeled human GAPDH cDNA as probes. A total of 3 independent experiments were conducted, and data from a representative experiment are shown.

	RESULTS

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Effects of PGE₂, PGF₂, and Indomethacin on 11ß-HSD2 Activity

To study the effects of prostaglandins, JEG-3 cells were treated with 10 ng/ml of either PGE₂ or PGF₂ for 24 h. Significant inhibition (~40%) of 11ß-HSD2 activity was observed for both prostaglandins. When the cells were treated for 24 h with 1 µg/ml of PGE₂ or PGF₂, a similar inhibition was observed (Fig. 2). To investigate the effects of blocking prostaglandin synthesis, JEG-3 cells were treated for 24 h with 10 µM indomethacin, an inhibitor of the cyclooxygenase enzymes. Paradoxically, indomethacin inhibited (~40%) rather than stimulated the activity of this enzyme, indicating that another pathway(s) of arachidonic acid metabolism may exert more potent inhibitory effects on 11ß-HSD2 activity, or that other prostanoids (PGI₂, PGD₂, or thromboxanes) may exert a stimulating effect that may override prostaglandin-induced inhibition (Figs. 2 and 3).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Effects of PGE2, PGF2, and indomethacin on 11ß-HSD2 activity. JEG-3 cells were incubated with 10 ng/ml (open bars) or 1 µg/ml (solid bars) of either PGE2 or PGF2, or 10 µM indomethacin for 24 h in the absence of serum. At the end of treatment, the level of 11ß-HSD2 activity in intact cells was determined by measuring the rate of cortisol (10 nM) to cortisone conversion. Bars represent the mean ± SEM of 4 independent experiments, each performed in triplicate. Results are expressed as the percentage of the 11ß-HSD2 activity in JEG-3 cells without any treatment. * p < 0.05 and ** p < 0.01 when compared with controls.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Overview of the major pathways of arachidonic acid metabolism. Indomethacin blocks the cyclooxygenase pathway, NDGA blocks all three lipoxygenase pathways, and zileuton blocks specifically the 5-lipoxygenase pathway. PLA2, phospholipase A2; COX, cyclooxygenase; 15-LO, 15-lipoxygenase; 12-LO, 12-lipoxygenase; 5-LO, 5-lipoxgenase; PGs, prostaglandins; TXs, thromboxanes; PGI2, prostacyclin; LTs, leukotrienes; EETs, epoxyeicosatrienoic acids; DHTs, dihydroxyeicosatrienoic acids; CYTO P-450, cytochrome P450; HETE, hydroxyeicosatetraenoate.

Effects of NDGA and Zileuton on 11ß-HSD2 Activity

To determine whether the lipoxygenase pathways were involved in attenuating 11ß-HSD2 activity, the cells were treated for 24 h with 1 µM NDGA, an inhibitor of all three (5-, 12-, and 15-) lipoxygenase pathways. NDGA caused a 3-fold increase in 11ß-HSD2 activity (Fig. 4). To examine possible interactions between the products of cyclooxygenases and lipoxygenases, NDGA was used in combination with indomethacin. Although not statistically significant, there was an apparent potentiation of NDGA-induced stimulation of 11ß-HSD2 by the addition of indomethacin (data not shown). To further delineate which specific lipoxygenase pathway was involved in attenuating 11ß-HSD2 activity, JEG-3 cells were treated for 24 h with 10 µM zileuton, an inhibitor of the 5-lipoxygenase pathway (Fig. 3). This resulted in a 3-fold increase in 11ß-HSD2 activity, similar to that observed when the cells were treated with NDGA (Fig. 4).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Effects of NDGA and zileuton on 11ß-HSD2 activity. JEG-3 cells were incubated with 1 µM NDGA or 10 µM zileuton for 24 h in the absence of serum. At the end of treatment, the level of 11ß-HSD2 activity in intact cells was determined by measuring the rate of cortisol (10 nM) to cortisone conversion. Bars represent the mean ± SEM of 3–6 independent experiments, each performed in triplicate. Results are expressed as the percentage of the 11ß-HSD2 activity in JEG-3 cells without any treatment. * p < 0.05 and ** p < 0.01 when compared with controls.

Effects of LTB₄ on 11ß-HSD2 Activity

Given that LTB₄ is the most biologically active product of the 5-lipoxygenase pathway, JEG-3 cells were treated with 10 nM LTB₄ for different lengths of time (3, 6, 9, 12, and 24 h). Significant inhibition of 11ß-HSD2 activity was observed by 3 h, with a maximal inhibition (~60%) by 9 h in culture (Fig. 5). When the cells were exposed to different concentrations of LTB₄, ranging from 0.1 to 100 nM for 9 h, there was a dose-dependent decrease in the level of 11ß-HSD2 activity with a maximal response at 10 nM (Fig. 6).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Inhibition of 11ß-HSD2 activity by LTB4—time course. JEG-3 cells were incubated with 10 nM LTB4 in the absence of serum for the indicated times. At the end of treatment, the level of 11ß-HSD2 activity in intact cells was determined by measuring the rate of cortisol (10 nM) to cortisone conversion. Bars represent the mean ± SEM of 3–4 independent experiments, each performed in triplicate. Results are expressed as the percentage of the 11ß-HSD2 activity in JEG-3 cells without any treatment. * p < 0.05 and ** p < 0.01 when compared with controls.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Inhibition of 11ß-HSD2 activity by LTB4—dose-response curve. JEG-3 cells were incubated for 9 h with the indicated concentrations of LTB4 in the absence of serum. At the end of treatment, the level of 11ß-HSD2 activity in intact cells was determined by measuring the rate of cortisol (10 nM) to cortisone conversion. Bars represent the mean ± SEM of 3–4 independent experiments, each performed in triplicate. Results are expressed as the percentage of the 11ß-HSD2 activity in JEG-3 cells without any treatment. * p < 0.05 and ** p < 0.01 when compared with controls.

Effects of PGE₂, PGF₂, and LTB₄ on 11ß-HSD2 mRNA

In order to determine whether decreases in 11ß-HSD2 activity following PGE₂, PGF₂ and LTB₄ treatment are due to changes in 11ß-HSD2 mRNA, the relative level of 11ß-HSD2 mRNA was assessed by semiquantitative RT-PCR analysis. Treatment of JEG-3 cells with 1 µg/ml and 5 µg/ml of either PGE₂ or PGF₂ for 24 h, or with 10 nM LTB₄ from 1.5 to 6 h, did not alter the levels of 11ß-HSD2 mRNA (Fig. 7).

View larger version (44K): [in this window] [in a new window]   FIG. 7. Effects of PGE2, PGF2, and LTB4 on 11ß-HSD2 mRNA. Autoradiographs of the semiquantitative RT-PCR analysis of 11ß-HSD2 mRNA in JEG-3 cells after treatment with 1 µg/ml and 5 µg/ml of PGE2 or PGF2 for 24 h, or with 10 nM LTB4 for different lengths of time (1.5, 3, and 6 h) in serum free medium. At the end of treatment, total cellular RNA was extracted and subjected to a semiquantitative RT-PCR, followed by Southern blotting, as described in Materials and Methods. A total of 3 independent experiments were conducted, and the results from one representative experiment are shown.

	DISCUSSION

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In the present study, we have demonstrated for the first time that prostaglandins and LTB₄ are potent inhibitors of 11ß-HSD2 bioactivity in JEG-3 cells, a model for the human placental trophoblast. If these eicosanoids inhibit 11ß-HSD2 activity in the human placenta in vivo, the present findings suggest that these locally produced eicosanoids could potentially increase the transplacental transfer of maternal cortisol to the fetus and thus influence fetal development.

Given that both human placental trophoblast and JEG-3 cells endogenously produce PGE₂, PGF₂, and LTB₄, in addition to a range of other hormones/factors and enzymes [21, 24–26, 29], the use of JEG-3 cells in the present study is appropriate and relevant to human placental 11ß-HSD2 physiology. In the present study, we demonstrated that LTB₄ at nanomolar concentrations, which are well within the physiological levels found in human umbilical plasma [30], was effective in attenuating 11ß-HSD2 activity in JEG-3 cells. Similarly, we observed a significant decrease in 11ß-HSD2 activity in these cells when treated with 10 ng/ml of PGE₂ or PGF₂. This concentration is comparable with that found in human placental tissue homogenates at term [31]. Therefore, the present findings suggest that these locally produced eicosanoids may inhibit the activity of placental 11ß-HSD2 under physiological conditions.

Prostaglandins have previously been shown to play a key role in the initiation of labor by enhancing uterine contractility [32, 33]. They are produced locally during pregnancy by maternal myometrium and decidua, and by fetal membranes and placenta [31]. Similarly, placental 11ß-HSD2 is present from early gestation and remains active throughout pregnancy and during labor [34–36]. If the inhibitory effects of prostaglandins on 11ß-HSD2 occur in the human placenta in vivo, this would lead to greater amounts of maternal cortisol crossing the placenta into fetal circulation. The increased fetal exposure to maternal cortisol by prostaglandins could adversely affect fetal organ growth and maturation [1, 2]. Likewise, fetal protection from maternal glucocorticoids by the placental 11ß-HSD2 could also be jeopardized by elevated intrauterine LTB₄ concentrations, since, as our present study has demonstrated, at physiological concentrations, LTB₄ exerts potent inhibitory effects on 11ß-HSD2 activity in JEG-3 cells. Within the intrauterine environment, LTB₄ is produced by the placenta and fetal membranes [22]. However, another important source of leukotrienes is the production by leukocytes and macrophages, which are often recruited in response to an infection, usually causing inflammation [37]. It is noteworthy that recent clinical reports have associated fetal growth restriction with inflammation. In one study, it was shown that 41% of normotensive mothers who had lesions of decidual vasculopathy and/or chronic inflammation had very low birth weight infants [38]. Other studies have correlated IUGR with massive infiltration of mononuclear cells in the intervillous space (massive chronic intervillositis) [39]. During an infection and inflammation, this additional source of LTB₄ could potentially cause greater transfer of maternal glucocorticoids across the placenta to the fetus, exerting deleterious effects on fetal development. It will be important to determine if indeed placental 11ß-HSD2 activity is attenuated under these pathological conditions.

In the present study, we have also demonstrated that there are no corresponding changes in the steady-state level of 11ß-HSD2 mRNA in JEG-3 cells after treatment with PGE₂, PGF₂, or LTB₄, indicating an effect at the posttranscriptional level. Although it remains possible that these eicosanoids may interact directly with the 11ß-HSD2 enzyme as inhibitors of enzyme activity, this is unlikely given that the cells were washed three times to remove these compounds before the addition of the substrate for 11ß-HSD2, in an attempt to exclude their having a possible competitive inhibition. There are two potential mechanisms by which these eicosanoids inhibit 11ß-HSD2 activity in JEG-3 cells. It is conceivable that these eicosanoids may inhibit the translation of 11ß-HSD2 protein. Alternatively, they may be involved in the posttranslational modification of 11ß-HSD2 protein. Obviously, further studies are required to determine the precise mechanisms by which this inhibition is achieved.

Indomethacin, a blocker of the cyclooxygenase pathway and thus prostanoid synthesis, resulted in an unexpected inhibition of 11ß-HSD2 activity. There are two potential explanations for this apparent paradox. In a recent study, it has been shown that there is more metabolism of arachidonic acid via the lipoxygenase pathway than the cyclooxygenase pathways in JEG-3 cells [26]. Therefore, when the cyclooxygenase pathway is blocked, it is possible that more arachidonic acid would be available for metabolism to other lipoxygenase and cytochrome P450 pathway products, which would attenuate 11ß-HSD2 activity in JEG-3 cells. Alternatively, other prostanoids, such as prostacyclin, prostaglandin D₂, and/or thromboxanes may stimulate 11ß-HSD2 to a greater extent than the inhibitory effect observed by prostaglandins E₂ and F₂. Therefore, when indomethacin is used to block all prostanoid synthesis, this stimulation would be removed, resulting in a decrease in 11ß-HSD2 activity. Although our present results provide support for the former explanation, the possibility of the two operating concomitantly cannot be ruled out.

In summary, the present study has demonstrated that prostaglandins and LTB₄ are potent inhibitors of 11ß-HSD2 activity in JEG-3 cells, suggesting that placental 11ß-HSD2 is under the control of locally produced eicosanoids. Whatever their mechanisms of action, this is the first report providing direct evidence that the products of arachidonic acid metabolism regulate the activity of 11ß-HSD2.

	FOOTNOTES

 
¹ This work was supported by the Canadian MRC (Grant MT-12100 to K.Y.). D.B.H. was supported partially by an AEF Grant from Department of Obstetrics and Gynaecology, the University of Western Ontario. K.Y. is an Ontario Ministry of Health Career Scientist.

² Correspondence: K. Yang, Lawson Research Institute, 268 Grosvenor Street, London, ON, Canada N6A 4V2. FAX: 519 646 6110; kyang{at}julian.uwo.ca

Accepted: February 15, 1999.

Received: October 2, 1998.

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