1 Department of Biomedical Sciences, Laboratory for Pregnancy and Newborn Research, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401; 2 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and 3 Department of Obstetrics and Gynecology, University Hospital Utrecht, 3584 CX Utrecht, The Netherlands
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ABSTRACT |
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In this study, we characterized the changes in the extracellular matrix proteoglycan decorin in pregnant intrauterine tissues in late gestation and in association with labor and delivery in sheep. In addition, we examined the effects of estradiol and progesterone on regulation of decorin mRNA expression in myometrium from the nonpregnant ovariectomized sheep. Using suppression subtractive hybridization in combination with Northern blot analysis, we identified a significant increase in decorin mRNA in the pregnant sheep myometrium during labor. The abundance of decorin mRNA paralleled myometrial contractility. The increase in decorin mRNA during labor was only demonstrated in the myometrium; no increase was observed in the endometrium or fetal membranes. Estradiol upregulated decorin mRNA and may act as a potential stimulator responsible for the increased decorin in the myometrium during parturition. The ovine decorin cDNA spans 1288 nt, includes 1083 nt of coding sequence predicted to encode a protein of 360 amino acids, 119 nt of 5'-untranslated region (UTR) and 86 nt of 3'-UTR. Over the coding region, the protein shares 79-96% nt sequence identity and 73-94% identity in the deduced amino acid sequence with homologous mammalian sequences. Using cloned decorin cDNA, we observed that the fibroblasts are the predominant cell type in the pregnant sheep myometrium containing decorin mRNA. These data suggest that increased decorin synthesis participates in the matrix changes that may play a role in myometrial activation.
myometrium; endometrium; fetal membranes; parturition; estradiol; progesterone; sheep
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INTRODUCTION |
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THE ONSET OF LABOR IS ASSOCIATED with both myometrial activation and stimulation involving upregulation or downregulation of a cassette of genes. Each gene in this cassette contributes to some extent in the switch in myometrial contractility patterns from the contracture pattern that occurs throughout pregnancy to the well-established contraction pattern seen during the progression of established labor (12). Altered abundance of members of this myometrial cassette of genes is a prerequisite for labor and delivery. Myometrial activation before and during labor appears to play a critical role in connecting fetal signals that indicate fetal readiness for birth to maternal factors that carry labor forward to expel the fetus.
The myometrium is composed of bundles of smooth muscle cells embedded in an extracellular matrix together with blood vessels and nerves supplying or passing through the muscle (6). Studies on myometrial activation associated with parturition have focused primarily on myometrial smooth muscle cell function (17, 33, 36), whereas investigation of concurrent changes in myometrial extracellular matrix proteins is limited. Using suppression subtractive hybridization (SSH), we identified a marked parturition-related increase at both the mRNA and protein levels in one extracellular matrix protein, thrombospondin-1 (TSP1), in the pregnant ovine myometrium (37), which is consistent with one recent report of increased TSP1 during labor in human myometrium (19). These data suggested that changes in uterine extracellular matrix proteins may also play an important role associated with myometrial activation during labor.
The elucidation of the labor-related changes in TSP1 demonstrated that SSH is a powerful and sensitive technique for rapidly identifying changes in abundance of mRNAs for individual genes associated with important physiological events. SSH is a powerful method for studying the changing pattern of gene expression that allows identification of changes in both known and previously undescribed mRNAs in relation to significant in vivo events. In the current study, we used SSH to characterize another important myometrial extracellular matrix protein, decorin, in relation to parturition. We also determined decorin mRNA expression in the pregnant sheep endometrium and fetal membranes. Furthermore, we cloned and sequenced decorin cDNA from a pregnant ovine myometrial cDNA library. The cDNA sequence of decorin has not been characterized in sheep, and decorin expression in the myometrium and intrauterine tissues in relation to labor has not been examined in any species.
Decorin, a ubiquitous interstitial proteoglycan, belongs to an expanding family of gene products involved in the control of cell proliferation and regulation of extracellular matrix assembly (15, 25). Decorin is a small proteoglycan with a protein core of 40-45 kDa. The central region of decorin is made up of 10 tandem repeats, with most of the leucine residue-rich motifs in the conserved positions, which have been implicated in modulating protein-protein interactions (22). Decorin's ability to interact with collagen (31) and growth factors (25) has suggested that decorin may play a role in relation to the processes involved in the ripening of the pregnant cervix (21, 24, 27). Very limited information is available regarding the regulation of decorin expression in different physiological situations. Cytokines, such as interleukin-1, have been demonstrated to upregulate decorin mRNA expression in arterial smooth muscle cells (9). However, the mechanism regulating decorin expression in pregnant or nonpregnant uterine tissues is not clear.
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MATERIALS AND METHODS |
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Animals and tissue collection. Twenty-eight pregnant Rambouillet-Dorset ewes bred on a single occasion and carrying fetuses of known gestational age (GA) were studied. Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee. The Cornell facilities are approved by the American Association for the Accreditation of Laboratory Animal Care. At 120 days GA, ewes from which tissues were obtained were instrumented with electromyogram (EMG) leads sewn into the myometrium and fetal and maternal carotid arterial and jugular venous catheters to monitor patterns of myometrial contractility and fetal well-being (20). Labor was defined as having occurred when the myometrial EMG record showed a clear switch from contractures to contractions followed by contraction activity for at least 5 h.
Myometrium was obtained from ewes during labor in a comparative study to determine whether there were any differences in labor induced by the infusion of betamethasone (Celestone phosphate, Schering; n = 5). Betamethasone (10 µg/h) was administered intravenously into the fetal jugular vein continuously over a period of 48 h in a total dosage of 0.48 mg to precipitate betamethasone-induced premature labor (BIL). Ewes underwent necropsy at 130 days GA when they went into labor as a result of the betamethasone administration. Myometrium was also obtained from contemporary control ewes (n = 5) at the same stage of gestation (130 days GA). These ewes were designated as early controls not in labor (ECNL). Fetuses of ECNL ewes were infused with physiological saline. These ewes were not in labor since the myometrial EMG showed only contractures and no contractions. Tissues were also collected from ewes in spontaneous term labor (STL) at 145-147 days GA (n = 6) and term control ewes not in labor (TCNL, n = 6) at a similar GA (143-147 days).Nimesulide infusion. Nimesulide (D-5648; Sigma Chemical, St. Louis, MO) was prepared in DMEM (D-5648; Sigma Chemical) at a concentration of 1.25 mg/ml. Nimesulide infusion to the ewe (30-mg bolus iv, followed by 6-h infusion, 30 mg/h) commenced 9 h after onset of STL at 147-148 days GA (n = 6).
Steroid effects on regulation of decorin mRNA expression in the nonpregnant ovariectomized ewe myometrium. Twenty nonpregnant ewes were ovariectomized on the day of ovulation following removal of progesterone sponges used to synchronize estrus. Forty days later, ewes received one of the following treatments: saline controls (n = 5), estradiol (Sigma Chemical) infused intravenously for 2 days (50 µg/day, n = 5), an intravaginal progesterone sponge for 10 days (containing 0.3g progesterone, n = 5), an estradiol plus progesterone group in which the intravaginal progesterone sponge was in place for 10 days and estradiol (50 µg/day) was infused intravenously on days 9 and 10 with the progesterone sponge still in place. Progesterone sponges were purchased from Carter Holt Harvey Plastic Products (Hamilton, New Zealand) and produced plasma concentrations in the range 1.5-2 ng/ml (provided by manufacturer). All animals were infused with the same rate of physiological saline as the control group.
Myometrium, endometrium, amnion, and chorion were always removed from the ventral aspect of the midportion of the body of the uterus at necropsy under halothane general anesthesia. Uterine wall strips were rapidly dissected into their component layers. Myometrium, endometrium, amnion, and chorion were frozen in liquid nitrogen for later RNA extraction. Another similar portion of the myometrium was frozen in liquid nitrogen-cooled isopentane for later in situ hybridization analysis. Frozen tissues were stored atConstruction of subtracted cDNA library: total RNA and poly(A)+ RNA isolation. Total RNA was isolated from myometrium, endometrium, amnion, and chorion in STL and TCNL as described previously (34). Poly(A)+ RNA from myometrium was extracted from total RNA using the Micro-FastTrack kit as suggested by the manufacturer (Invitrogen, San Diego, CA). The poly(A)+ RNA from the two groups (TCNL and STL) was purified in parallel using the same reagents and protocol.
cDNA subtraction library. SSH (8) was used to construct a subtraction library to identify upexpressed genes in the STL myometrium as described previously (37). The library was made using a PCR-Select cDNA Subtraction Kit according to the protocol provided by the manufacturer (CLONTECH Laboratories, Palo Alto, CA). Briefly, cDNA was synthesized from 2 µg of poly(A)+ RNA from STL and TCNL myometrium. For construction of a library enriched for transcripts upregulated in STL myometrium, poly(A)+ RNA from STL and TCNL were designated as "tester" and "driver," respectively. The present study reports on the analysis of decorin, one of the transcripts expressed at a higher level in the STL myometrium identified by SSH.
The subtracted and amplified cDNA were ligated into a pCR2.1 vector using a T/A cloning kit (Invitrogen). Positive clones were identified by differential screening of the subtracted library using both forward-subtracted and reverse-subtracted probes (derived by switching between tester and driver populations for another subtractive hybridization). The two probes labeled with [Cloning and sequencing of decorin cDNA from ovine myometrial cDNA
library.
An ovine myometrial cDNA library was custom-made (CLONTECH
Laboratories) from poly(A)+ RNA isolated from the pregnant
sheep myometrium during labor. Because the clone (sequence 1-999,
GenBank accession no. AF125041) from the subtracted cDNA library lacked
the 3'-end sequence of the cDNA, PCR cloning was used to retrieve
the sequence from the myometrial cDNA library. A gene-specific forward
primer (DC-F1) synthesized from the sequence of the subtractive clone
was used in combination with a vector reverse primer (SA-R1) to amplify the remaining downstream sequences from the myometrial cDNA library. PCR was done using 0.4 µM each of primer pair (Table
1) in a volume of 50 µl containing 50 mM
KCl, 10 mM Tris · HCl (pH 8.3), 2 mM
MgCl2, and 0.2 mM each for dATP, dCTP, dGTP, and dTTP. The reaction was carried out for 30 cycles at an annealing temperature of
60°C for 30 s, a polymerization temperature of 72°C for 2 min, and a heat-denaturation temperature of 94°C for 30 s in a thermal cycler (Gene Amp PCR system 2400; Perkin Elmer-Cetus, Norwalk, CT). The
PCR-amplified DNA fragments were cloned in a pCR 2.1 vector using the
T/A cloning kit (Invitrogen) under the conditions recommended by the
manufacturer.
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Northern blot analysis. Samples of total RNA (40 µg/lane) from myometrium, endometrium, amnion, and chorion were denatured in 17.4% (vol/vol) formaldehyde, 50% (vol/vol) formamide, 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA (pH 7.0) for 5 min at 65°C and separated on a 1% (wt/vol) agarose/0.66 M formaldehyde gel. Ethidium bromide-stained rRNA bands were visualized (ultraviolet) to insure that RNA degradation had not occurred and that an equal amount of RNA had been loaded into each lane. After electrophoresis, RNA was transferred to a nylon membrane (Gene Screen Plus) by capillary blotting for 24 h in 10× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Completion and uniformity of transfer were assessed by determining transfer of 28S and 18S rRNA from the gel. Membranes were prehybridized at 42°C for 5 h in hybridization solution [50% (vol/vol) deionized formamide, 50 mM sodium phosphate, 0.8 M NaCl, 2% (wt/vol) SDS, 100 µg salmon sperm DNA/ml, 20 µg tRNA/ml, and 1× Denhardt's solution (50× = 1% solution of BSA, Ficoll, and polyvinylpyrrolidone)].
The cloned ovine decorin cDNA (GenBank accession no. AF12504) was labeled with [In situ hybridization. In situ hybridization was performed as described previously (32) with the following modification: frozen sections of pregnant sheep myometrium were cut and mounted on poly-L-lysine-coated slides. Frozen tissue sections were then fixed in 4% paraformaldehyde for 10 min at room temperature followed by two washes in 1× PBS at room temperature. Prehybridization was performed at 42°C for 1 h in prehybridization buffer containing 50% formamide, 5× SSPE (1× SSPE = 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA), 0.1% SDS, 0.1% (vol/vol) Denhardt's solution, 200 µg denatured salmon testis DNA/ml, and 200 µg tRNA/ml. Hybridization was carried out for 18 h at 42°C in hybridization buffer [prehybridization buffer + 4% (wt/vol) dextran sulfate] containing 1.5× 106 cpm 35S-labeled ovine decorin cDNA probe/section. After hybridization, the sections were rinsed at room temperature for 2 h in 2× SSC, 2 h in 1× SSC, 1 h in 0.5× SSC, and finally for 1 h in 0.5× SSC at 37°C. The sections were then dehydrated by passing through an alcohol series containing 300 mM ammonium acetate and coated with liquid photographic emulsion (NTB2, Kodak). After 10 days of exposure, the sections were developed and stained with hematoxylin and eosin.
In controls, serial sections were treated with pancreatic RNase A (20 µg/ml) for 30 min at room temperature before hybridization. After enzyme treatment, the sections were rinsed in three changes of 2× SSC (5 min each) and hybridized with the [Statistical analysis. Decorin mRNA concentration in each Northern blot (following normalization of the content of decorin mRNA to 18S rRNA in individual samples) was expressed as a ratio of decorin mRNA to 18S rRNA. Differences between different groups for Northern blot analysis were analyzed by ANOVA. Data throughout are presented as means ± SE.
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RESULTS |
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Cloning and characterization of ovine decorin cDNA.
Ovine decorin cDNA was assembled from overlapping fragments obtained
from both subtracted and nonsubtracted ovine myometrial cDNA libraries.
The characterized region of decorin cDNA spans 1288 nt (Genbank
accession no. AF125041), includes 1083 nt of coding sequence predicted
to encode a protein of 360 amino acids (relative molecular weight
39,977), 119 nt of 5'-untranslated region (UTR) and 86 nt of
3'-UTR including the poly(A)+ tail. Over the coding
region, ovine decorin shares 79-96% nt sequence
identity, and 73-94% identity in the deduced amino acid sequence
with homologous mammalian sequences, with the highest identities (96%
and 94%) with bovine decorin at the nucleotide and amino acid levels,
respectively (Fig. 1). The level of
conservation of amino acids among mammalian species is very high,
except for a hypervariable region near the NH2-terminal
region (Fig. 1, 39-51 in sequence). A potential glycosaminoglycan
attachment site was found at Ser34-Gly35,
followed by two cysteine residue clusters
(Cys55, Cys59, Cys61,
Cys68, and Cys314, Cys347) flanking
a series of leucine-rich repeats with consensus sequence L-X-X-L-X-L-X-X-N-X-L/I
(residues 108-119, 132-142, 148-158, and 172-182).
Two putative cAMP- and cGMP-dependent protein kinase phosphorylation
sites were located in the cysteine cluster flanking region and in the
COOH terminal. Three potential N-linked oligosaccharide attachment sites were also placed in the COOH-terminal region of the
protein (Fig. 1).
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Identification of decorin as one of the genes that is upregulated in
the myometrium during labor.
Decorin was identified as one of the upregulated genes from the
subtraction cDNA library representing transcripts expressed at higher
levels in the myometrium during STL compared with gestation matched
controls. Northern blot analysis was performed to further confirm the
upregulation of decorin mRNA in the pregnant sheep myometrium during
labor. Decorin mRNA was significantly increased in ovine myometrial
tissue in STL as well as in BIL (Fig. 2), indicating strong induction of decorin mRNA in this tissue associated with labor (P < 0.01). After nimesulide infusion, decorin
mRNA was significantly decreased below levels seen in STL in the
myometrium (Fig. 2; P < 0.05) but remained significantly
higher than ewes TCNL (P < 0.01). In contrast with the
myometrium, there were no significant changes of decorin mRNA during
STL compared with TCNL in the pregnant sheep endometrium and fetal
membranes (Fig. 3).
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Effect of estradiol and progesterone on myometrial decorin mRNA
expression.
After estradiol treatment of nonpregnant ovariectomized ewes, decorin
mRNA concentration analyzed by Northern blot analysis increased
significantly (P < 0.05) in the myometrium (Fig.
4), whereas progesterone treatment alone
had no significant effect on decorin mRNA abundance. Progesterone did
not antagonize estradiol's stimulatory effect on decorin mRNA
expression when used in combination with estradiol.
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In situ hybridization.
In situ hybridization demonstrated that decorin mRNA was mainly
associated with fibroblast-type cells in the myometrium (Fig. 5A). However, some of the
myometrial cells also contained decorin mRNA (data not shown).
Control sections treated with RNase A showed no specific signals (Fig.
5B).
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DISCUSSION |
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In the present study, we characterized decorin cDNA and quantified decorin mRNA expression in pregnant sheep myometrium, endometrium, and fetal membranes as well as nonpregnant sheep myometrium. Two major findings are apparent. First, there was a significant increase of decorin mRNA in pregnant sheep myometrium during STL as well as glucocorticoid-induced premature labor, indicating that the increase in decorin is a common feature associated with the switch of myometrial activity from contractures to contractions. Second, our data show that, although endometrium and fetal membranes are exposed to a similar hormonal milieu associated with labor, no similar increase in decorin mRNA occurred in these tissues. The demonstration of tissue-specific changes in decorin mRNA suggests differential regulation of decorin mRNA expression in these intrauterine tissues and further indicates that the change in decorin mRNA may play a role in the altered physiology of the myometrium associated with the myometrial contraction. However, the mechanism(s) that underlies the differential regulation of decorin mRNA expression in the different intrauterine tissues is not clear. These results indicate that cell-type-specific subcellular signals are important to direct responses to changes in physiological state and hormonal balance in some cell types while leaving the decorin synthetic activity of other cells unaltered. This form of differential regulation is of fundamental importance for a ubiquitous cell function such as decorin mRNA synthesis.
It is interesting to note that inhibition of myometrial contraction by
nimesulide resulted in the suppression of myometrial decorin mRNA
expression during labor. The nimesulide infusion regimen we used
resulted in inhibition of myometrial contractility (29) and a
significant inhibition of PGE2 and cortisol production in
the fetal circulation (30). Both the decreased fetal plasma cortisol
and PGE2 production that accompany
administration of nimesulide decrease placental 17-hydroxylase mRNA
expression observed in our previous study (18), which may result in a
decreased placental conversion of progesterone to estrogen. The further downstream changes induced by the altered estrogen-to-progesterone ratio result in suppressed expression of uterine labor-related genes,
such as oxytocin receptor, estrogen receptor, and associated proteins
(35) and decorin as observed in the current study. These changes lead
to reduced myometrial contraction and arrest the progression of labor.
During pregnancy and parturition, profound changes occur in the uterus including extensive growth, remodeling, and activation. In this regard, most studies of labor have focused on myometrial smooth muscle cell function, e.g., smooth muscle cell communication-gap junction (11, 17), ion channels (2, 14), oxytocin receptor (7, 10, 36, 38), and prostaglandin receptor changes (3, 4, 26). Very few studies have been conducted to examine extracellular matrix proteins in association with myometrial activation during pregnancy and labor (19). Our current data together with our previous finding of labor-related changes in myometrial TSP1 (37) indicate that uterine activation is not restricted to myometrial smooth muscle cells but also involves extracellular matrix proteins.
Decorin is a small dermatan sulfate proteoglycan. In the cervix, decorin coats collagen fibrils and is involved in collagen formation (13, 28, 31). An increased ratio of decorin to collagen has been observed in active labor in the human cervix, indicating that more decorin is available per collagen molecule during labor (24). In the cervix, this increased decorin during labor may compete with the decorin bound to collagen, effectively disrupting the organization between the collagen fibrils, with resultant disorganization of collagen (23). The potential function of increased decorin in relation to myometrial activation, however, is not clear.
Parturition in sheep is accompanied by both a rise in estradiol and a
fall in progesterone in maternal plasma, together with changes in a
large number of other potential regulators (5, 16). Nimesulide infusion
resulted in decreased placental 17-hydroxylase expression
accompanied by decreased expression of decorin mRNA in the pregnant
sheep myometrium during labor, an observation consistent with
involvement of estrogen and progesterone in the regulation of
myometrial decorin mRNA expression. We further tested the hypothesis
that estradiol and/or progesterone are involved in the regulation of
decorin mRNA expression by conducting studies in ovariectomized
nonpregnant sheep treated with estradiol and/or progesterone.
Estradiol-dependent induction of decorin mRNA was observed in the
myometrium. Progesterone alone had no significant effect on myometrial
decorin mRNA expression. In addition, progesterone did not antagonize
estradiol's positive effect on decorin expression when used in
combination with estradiol. These results further support the view that
the changes in decorin mRNA in uterine tissues of pregnant sheep in
labor are regulated by the changes in plasma estradiol and progesterone
concentrations that precede labor in sheep.
The present study is the first to characterize the complete decorin cDNA sequence in this important experimental species. The level of conservation of amino acids among mammalian species is very high, except for a hypervariable region near the NH2-terminal region (39-51 in sequence). A potential glycosaminoglycan attachment site was found at Ser34-Gly35, followed by two cysteine residue clusters (Cys55, Cys59,Cys61, Cys68, and Cys314, Cys347) flanking a series of leucine-rich repeats with consensus sequence L-X-X-L-X-L-X-X-N-X-L/I (residues 108-119, 132-142, 148-158, and 172-182). Two putative cAMP- and cGMP-dependent protein kinase phosphorylation sites were located in the cysteine cluster flanking region and in the COOH terminal. Three potential N-linked oligosaccharide attachment sites were also placed in the COOH-terminal region of the protein.
Characterization of decorin cDNA provides a powerful tool to further analyze the expression of decorin mRNA by Northern blot analysis and in situ hybridization. Using our cloned decorin cDNA probe, we found the localization of decorin mRNA is mainly associated with fibroblast cells in the pregnant sheep myometrium. This observation further supports our hypothesis that remodeling of extracellular matrix of the pregnant myometrium constitutes a part of the mechanism for uterine activation during labor.
These studies provide further evidence that SSH is a powerful technique that enables the study of differentially regulated genes during labor. In addition, we demonstrated that 1) ovine decorin cDNA shares a high identity among mammalian species; 2) increases in decorin mRNA are myometrium specific and associated with myometrial activation during both STL and BIL in sheep, and the abundance of myometrial decorin mRNA parallels the labor-related changes in myometrial contractility patterns; and 3) myometrial fibroblast cells are responsible for producing decorin.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Child Health and Human Development Grant HD-21350.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. W. Nathanielsz, Dept. of Biomedical Sciences, Laboratory for Pregnancy and Newborn Research, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853-6401 (E-mail: pwn1{at}cornell.edu).
Received 5 April 1999; accepted in final form 5 October 1999.
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