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Laboratory for Pregnancy and Newborn Research (W.X.W., Q.Z., X.M., P.W.N.), Department Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401; and Department of Obstetrics and Gynecology (N.U.), Faculty of Medicine, University of Tokyo, 113-0033 Tokyo, Japan
Address all correspondence and requests for reprints to: Dr. Peter W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Department Physiology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401. E-mail: pwn1{at}cornell.edu
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Abstract |
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Introduction |
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The switch in myometrial contractility patterns, from contractures to contractions (2, 3), is accompanied by myometrial activation (1) involving changes in expression of a collection of contraction-associated proteins. Contraction-associated proteins that have been investigated during late gestation and labor in sheep and other species include: the estrogen receptor (4, 5); the steroid receptor-associated proteins-heat shock proteins 70 and 90 (Hsp 70 and 90) (6); oxytocin (OT) (7, 8, 9); agonist receptors—OT receptors (10, 11, 12, 13), PG receptors (14, 15, 16), gap junction proteins (17, 18), and ion channels (19, 20). Once activated, the myometrium can respond to paracrine and endocrine stimulation by uterotonic agonists (e.g. OT and PGs) that bring labor to a successful conclusion.
Based on these observations, others and we (1, 21) have proposed that the onset of labor is associated with preparation of the myometrium, with resultant changes in a cassette of genes (up- or down-regulation). Each member of the cassette of genes contributes, to some extent, in the switch in myometrial contractility patterns from contractures to contractions, as well as the progression of established labor. Altered abundance of members of this cassette of genes in the myometrium is a prerequisite for labor and delivery. Myometrial activation before and during labor seems 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.
Understanding of the molecular basis of myometrial activation is incomplete. More information is needed on the genes involved during labor and the factors regulating their expression. To understand the molecular basis of myometrial activation, the relevant subsets of differentially expressed genes associated with the various stages of parturition must be identified, cloned, and studied in detail. Suppression subtractive hybridization (SSH) is a powerful approach to identify and isolate complementary DNAs (cDNAs) of differentially expressed genes. In the present study, we employed this sensitive and powerful technique to identify unknown factor(s), which may play important roles in the process of parturition. Improved understanding of the cellular processes that occur at labor will advance our knowledge of the key regulatory mechanisms involved in preparation for and development of parturition.
Using SSH, we identified a marked increase at the messenger RNA (mRNA) level associated with parturition in one extracellular matrix protein, thrombospondin-1 (TSP1, a glycoprotein) in the pregnant ovine myometrium. The increase in TSP1 mRNA level was accompanied by increased TSP1 protein level, measured by Western blot analysis. We also examined the cellular distribution of TSP1 mRNA, in relation to its protein expression in the pregnant sheep myometrium, using in situ hybridization and immunocytochemistry.
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Materials and Methods |
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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-Plough Corp. (Kenilworth, NJ), n = 5]. Betamethasone (10 µg/h) was administered iv 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 (BPL). Ewes underwent necropsy at 130 dGA, 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 dGA). 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, because the myometrial EMG showed only contractures and no contractions. Tissues were also collected from ewes in spontaneous term labor (STL) at 145–147 dGA (n = 6), and term control ewes not in labor (TCNL; n = 6) at the same gestational age (143–147 dGA).
Nimesulide infusion. Nimesulide (D-5648; Sigma Chemical Co., St. Louis, MO) was prepared in DMEM at a concentration of 1.25 mg/ml. Nimesulide infusion to the ewe (n = 6) iv (30 mg bolus, followed by 6 h infusion, 30 mg/h) commenced 9 h after onset of labor, at 147–148 dGA.
Myometrium was always removed from the ventral aspect of the midportion of the body of the uterus and frozen in liquid nitrogen for later RNA extraction. Another similar portion of the myometrium was embedded in Tissue Freezing Medium (Triangle Biomedical Science, Durham, NC) and frozen in liquid-nitrogen-cooled isopantane for later study by in situ hybridization analysis and immunocytochemistry. Frozen tissues were stored at -80 C.
Construction of subtracted cDNA library
Total RNA and poly-A+ RNA isolation. Total RNA
was isolated from myometrium of the sheep in STL and TCNL, as described
previously (4). Briefly, total RNA was isolated from frozen tissues by
homogenization in 4.2 M guanidinium thiocyanate solution.
RNA was pelted through a 5.7 M cesium chloride cushion. The
RNA purity and recovery of each tissue was determined by UV
spectrophotometry (260 and 280 nM). Poly-A+ RNA
was extracted from total RNA using the MicroFastTrack 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 (23) was used to construct subtraction library to identify up-expressed genes in the STL myometrium. The library was made using PCR-Select cDNA Subtraction Kit, according to the protocol provided by the manufacturer (CLONTECH Laboratories, Inc., Palo Alto, CA). Briefly, the SSH protocol is as follows: poly-A+ RNA from STL and TCNL were designated as tester and driver, respectively. The double-strand cDNAs were synthesized from 2 µg myometrial poly-A+ RNA obtained from both STL and TCNL ewes. The synthesized double-strand cDNAs were then digested with restriction enzyme RsaI. The digested blunt ends of the tester cDNA (from STL) were divided into two parts and ligated with two different cDNA adaptors. Two cycles of hybridization were followed after the ligation. In the first cycle, excess driver cDNAs (from TCNL) were added to the two populations of tester cDNAs after denaturation, to achieve equalization and enrichment of differentially expressed sequences in the hybridization solution. During the second hybridization process, denatured fresh driver cDNAs were added into the two tester cDNA populations again and allowed to anneal, to further enrich the differentially expressed sequences. The final hybridization solution (also called the subtracted library) was employed as a template to amplify the differentially expressed sequences in the tester population by using a set of PCR primers and was followed by nested PCR primers. Both sets of PCR primers were annealed to the different sites of ligated adaptors. The secondary PCR amplification, by using nested primers, considerably enriched the differentially expressed sequences and reduced background of PCR product (23).
The amplified cDNA fragments from the secondary PCR were ligated into a
pCR2.1 vector using a T/A cloning kit (Invitrogen).
Positive clones were identified by differentially screening the
subtracted library using both forward-subtracted and reverse-subtracted
probes (derived by switching between tester and driver populations for
another subtractive hybridization). The forward-subtracted and
reverse-subtracted probes, labeled with 
-32P
deoxycycidine triphosphate (dCTP), were hybridized with the replicate
cDNA library dot blots (Gene Screen Plus membrane, NEN
Research Products, DuPont NEN, Boston, MA). Positive
clones, identified by differential screening, were cultured in LB
medium to obtain plasmid DNA for sequencing. The plasmid DNA was
purified using the Biorobot system (Qiagen, Chatsworth,
CA), and sequencing was done by Taq cycle sequencing using
DyeDeoxy terminators in an automated sequencer (ABI 377 DNA sequencer;
PE Applied Biosystems, Foster City, CA) at the core
sequencing facility of Cornell University. The cloned fragments were
sequenced from at least two different clones and from both the coding
and noncoding strands. The acquired sequence data were aligned against
the GenBank nucleotide database at the National Center for
Biotechnology Information (National Institutes of Health, Bethesda, MD)
using Blastn to search for sequence matches.
Identification of TSP1 as an up-regulated gene in parturition. The Blastn search revealed that three of the up-regulated gene clones were homologous to ovine TSP1. Because it was isolated from the tester cDNA population (STL myometrium), it represented the up-regulated gene from the myometrium of the pregnant sheep during labor. The acquired sequence data were aligned against the GenBank nucleotide database and revealed that the clones contain partial coding and most downstream sequences of TSP1 mRNA. The cloned ovine TSP1 fragment was then used as a cDNA probe for Northern blot analysis and in situ hybridization, as described below.
Northern blot analysis
Total RNA was prepared from individual tissues, as described
above. Samples of total RNA (30 µg/lane) from each tissue were
denatured in 17.4% (vol/vol) formaldehyde, 50% (vol/vol) formamide,
20 mM 3-(N-morpholino) propanesulfonic acid), 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 ribosomal RNA (rRNA) bands
were visualized (UV) to ensure 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; DuPont NEN, Wilmington, DE) by
capillary blotting for 24 h in 10 x SSC (1 x SSC is
0.15 M NaCl and 0.015 M Na 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 transfer RNA/ml, and 1 x Denhard’s (50x = 1% solution of
BSA, Ficoll, and polyvinylpyrrolidone).
The cloned ovine TSP1 cDNA fragment was labeled with
-32P dCTP (for Northern blot analysis) and labeled with
-35S dCTP (for in situ hybridization) using
the random priming method (DuPont NEN) to specific
activities of approximately 1 x 109 cpm/µg and used
at a final concentration of 1 x 106 cpm specific
probe/ml of hybridization solution.
Hybridization was carried out at 42 C for 20 h in hybridization solution containing the specific TSP1 cDNA probe. Membranes were washed sequentially in 2 x SSC at room temperature for 10 min, and 0.5 x SSC with 0.1% SDS at 65 C for 30 min. Kodak X-OMAT AR film (obtained from Sigma Chemical Co.) was exposed to the membrane with intensifying screens at -80 C. Exposure durations were varied to achieve hybridization signals within the limited linear range for densitometry.
Membranes were stripped of TSP1 probe by boiling in 0.1 x SSC
with 0.1% (wt/vol) SDS for 30 min and rehybridized with
-32P dCTP-labeled 18S cDNA probe to normalize TSP1 mRNA
levels. Autoradiographed signals were quantified by Scan
densitometry.
In situ hybridization
In situ hybridization was performed as described
previously (13), with some modification. Frozen sections of pregnant
sheep myometrium were cut and mounted on
poly-L-lysine-coated slides. Tissue sections were fixed in
freshly prepared 4% paraformaldehyde in 10 mM PBS (pH 7.2)
for 10 min, followed by 2 x PBS wash, 5 min each, at room
temperature. Prehybridization was performed at 42 C for 1 h in
prehybridization buffer containing 50% formamide, 5 x SSPE
(1 x SSPE being 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 transfer RNA/ml. Hybridization was carried out for
18 h at 42 C in hybridization buffer [prehybridization buffer
plus 4% (wt/vol) dextran sulfate] containing 1.0 x
106 cpm 35S-labeled ovine TSP1 cDNA
probe/section. After hybridization, the sections were rinsed at room
temperature for 2 h in 2 x SSC, 2 h in 1 x SSC,
1 h in 0.5 x SSC, and finally for 1 h in 0.5 x
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
exposure, the sections were developed and stained with hematoxylin and
eosin.
Controls
Serial sections were treated with pancreatic ribonuclease
(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 x SSC (5 min each) and hybridized with the
-35S dCTP-labeled ovine TSP1 cDNA probe, as described
above.
Western blot analysis
Cytosolic extracts. Myometrium was ground into small pieces
and then homogenized in Tris buffer [50 mM Tris (pH 7.4),
10 mM EDTA, and 1 mM diethyldithi-ocarbamic
acid], containing 2 mM octyl glucoside, and was
centrifuged at 30,000 x g for 20 min at 4 C. The
30,000 x g supernatant (cytosol) was retained and
stored at -80 C until used for electrophoretic analyses. The protein
concentration was determined by the method of Bradford (Bio-Rad Laboratories, Inc., Hercules, CA).
The cytosolic proteins (50 µg/lane) were then separated by 10% SDS-PAGE and were electrophoretically transferred to a nylon membrane (Imobilon, Millipore Corp., Bedford, MA) using a transfer blot cell (Bio-Rad Laboratories, Inc.). The filters for immunostaining were blocked with 2% BSA in 10 mM Tris-Cl buffer containing 0.1% Tween-20. After blocking, the blots were washed three times with wash buffer (5 min each), containing 10 mM Tris-Cl and 0.1% Tween-20, and were incubated with a mouse monoclonal anti-TSP1 antibody (NeoMarkers, Fremont, CA; Cat No: MS-421-P1, clone A6.1, 1:400 dilution) at 4 C overnight. The blots were then washed, incubated with peroxidase-conjugated sheep antimouse IgG (Amersham Life Sciences, Arlington Heights, IL) at room temperature for 1 h. After each antibody incubation, the blots were washed three times (15 min each) in wash buffer. The protein bands were visualized using an enhanced chemiluminescence Western blotting detection kit (Amersham). The molecular size of the proteins was determined by running standard molecular weight marker proteins (Bio-Rad Laboratories, Inc.) in an adjacent lane.
Immunocytochemistry
Frozen sections (4 µm) of ovine myometrial tissue were
immunostained for TSP1 using the avidin-biotin immunoperoxidase method.
Briefly, sections were fixed in acetone for 10 min at room temperature
and then rinsed twice in 0.05 M Tris buffered saline, 5 min
each. Unless otherwise specified, all slides were sequentially
incubated, for various times, at room temperature, with each of the
following reagents: 1) 3% (vol/vol) H2O2 in
methanol for 30 min to block endogenous peroxidase activity; 2) 25%
(vol/vol) normal horse serum and 5% (wt/vol) BSA in 0.05 M
Tris-Cl with 0.15 M NaCl, pH 7.6, for 1 h; 3) mouse
monoclonal anti-TSP1 antibody (1:400 dilution) described above as used
for Western blotting, for 20 h at 4 C; 4) biotinylated horse
antimouse IgG (Vector Laboratories, Inc., Burlingame, CA)
for 0.5 h; 5) avidin-biotin complex (Vector Laboratories, Inc.): 40 µl of each in 5 ml 0.05 M Tris-Cl, pH
7.6, for 0.5 h; and 6) 3,3-diaminobenzidine tetrahydrochloride
(Sigma Chemical Co.) for 10 min: 4 µg/10 ml 0.05
M Tris buffer (pH 7.6), to which was added 0.2 ml 3%
H2O2. After each incubation, the slides were
washed with Tris buffered saline for 15 min, except for step 2). The
slides were then mounted in Permount with or without hematoxylin
counterstaining. Specificity of immunostaining for the TSP1 was
confirmed by three approaches: 1) omission of the primary antibody; 2)
incubation of the slides with the normal mouse serum instead of the
primary antibody; and 3) Western blot analysis to determine the
specificity of the antibody.
Statistical analysis
After normalization of the content of TSP1 mRNA to 18S rRNA in
individual samples, TSP1 mRNA concentration in each Northern blot was
expressed as a ratio of TSP1 mRNA to 18S rRNA. Differences between
different groups for Northern blot or Western blot analysis were
analyzed by ANOVA followed by multiple comparison using a Tukey-Kramer
procedure. Data throughout are presented as the mean ±
SEM.
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Results |
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demonstrates the
electrophoretic analysis of the secondary PCR product of SSH. There was
a different banding pattern between the STL myometrial subtracted
sample (lane 1), compared with the STL myometrial unsubtracted sample
(lane 2). A 1.2-kb band, indicated by the white
arrow, along with other bands of different sizes, can be
identified clearly in the STL myometrial subtracted sample, indicating
a great enrichment of differentially expressed genes in the subtracted
sample. A dot blot of differential screening is shown in Fig. 2. Duplicate blots were hybridized with
forward-subtracted probes and reverse-subtracted probes, respectively.
Dots hybridized with reverse-subtracted probes corresponded to
background (Fig. 2). Clones hybridized only with forward-subtracted
probes represent mRNAs that were differentially expressed in the tester
(STL) population of myometrium (Fig. 2). Three of the clones
representing the TSP1 gene were identified by sequence analysis and
Blastn search.
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), indicating strong
induction of an approximately 6.5-kb TSP1 mRNA in the myometrium in
premature, as well as term, labor (P < 0.01). After
nimesulide infusion, TSP1 mRNA was significantly decreased below levels
seen in STL in the myometrium (Fig. 4)
(P < 0.05) but remained significantly higher than TCNL
ewes not in labor (P < 0.01).
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). An additional band at 140-kDa was
observed in one myometrial sample. The immunoreactive band of TSP1
increased significantly (P < 0.001) in BPL or STL,
which corresponds with the changes in TSP1 gene transcription observed
by Northern blot analysis.
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). However, some of myometrial cells
also contained TSP1 mRNA (Fig. 6B). Control sections treated with RNase
A showed no specific signals (Fig. 6C). Dense immunostaining for TSP1
was detected in the fibroblast-type cells, as well as in the
extracellular matrix of the pregnant sheep myometrium (Fig. 6, D1 and
D2). Some of the myometrial cells were also stained for TSP1 (Fig. 6E).
When myometrial sections were reacted with the normal mouse serum as a
replacement for the primary TSP1 antibody, the positive staining for
TSP1 was abolished (Fig. 6F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the cell biology of TSP1 has been extensively studied at the molecular and cellular levels, changes in TSP1 in critical uterine tissues have not been studied in relation to pregnancy and parturition in any species. Throughout pregnancy, the myometrium is undergoing extensive biochemical and physiological changes. Myometrial activity initially remains relatively quiescent throughout pregnancy, demonstrating only irregular, long-lasting epochs of activity (contractures). At STL, the myometrium becomes active, developing regular contractions to expel the fetus (2). The genes controlling these changes are poorly understood. SSH is a powerful and sensitive technique for rapidly identifying changes in abundance of mRNAs for individual genes during physiological events. It is a powerful method for studying the changing pattern of gene expression that allows identification of both known and previously undescribed mRNAs.
Numerous cDNA subtraction methods have been reported (30, 31, 32). They are usually inefficient for obtaining low-abundance transcripts, and they require considerable amounts of poly-(A)+ RNA, involve multiple or repeated subtraction steps, and are labor intensive (23). The mRNA differential display (33) and RNA fingerprinting by arbitrary primed PCR (34) are potentially faster methods for identifying differentially expressed genes. However, both of these methods have a high level of false positives (35, 36) and are biased for high copy number mRNA (37).
Our study represents the first use of the SSH technique to examine transcript changes at the time of parturition. We demonstrated a marked increase of TSP1 associated with STL in myometrium collected from the central body of the pregnant uterus. Up-regulation of TSP1 mRNA in the pregnant sheep myometrium at STL, identified by SSH, was confirmed by Northern blot analysis. The demonstration that similar changes in TSP1 gene expression are associated with both STL and BPL supports the view that increased uterine TSP1 production plays an important role in the uterine changes that accompany labor, regardless of the manner in which labor is initiated.
It is interesting to note that inhibition of myometrial contraction by nimesulide resulted in the suppression of myometrial TSP1 expression during labor. The nimesulide infusion regimen that we used resulted in inhibition of myometrial contractility (38) and significantly inhibited PG production in the fetal circulation (39). Thus, our findings provide further evidence of coupling of PG concentration and the progress of labor with the active synthesis of uterine labor-related genes, including TSP1. These findings indicate that uterine activation and stimulation are both required for the normal progression of labor.
We also report here that the increased TSP1 mRNA is carried to the protein level. TSP1 mRNA rose concurrently with TSP1 protein. The molecular weights we observed are consistent with the report that TSP1 is readily proteolyzed in tissue lysates, resulting in bands on Western blot of lower molecular weight (120 or 140K) than the intact 180K subunit (from the data sheet provided by the TSP1 antibody vendor). The mechanisms that up-regulate TSP1 production during labor are not defined. Parturition in sheep is accompanied by both a rise in estradiol and fall in progesterone in maternal plasma, together with changes in a large number of other potential regulators. The involvement of estradiol or/and progesterone in the regulation of increased TSP1 during labor awaits further study.
Increased TSP1 mRNA in the uterine lower segment, identified by Northern blot analysis, has been found in association with human labor (40). In our study, we were able to evaluate changes in the body of the uterus, which plays a more active role in labor than the lower segment. Differential molecular mechanisms are associated with the regional control of contractility of the pregnant uterus; for example, inhibitory and contractile PG receptors are differentially distributed in the uterine corpus and fundus, compared with lower segment (41). Therefore, the information obtained from uterine lower segment cannot be simply applied to the uterine corpus and fundus. In the current study, the increase in TSP1 mRNA was confirmed by three different molecular approaches: SSH, dot blot analysis, and Northern blot analysis. In addition, we quantitatively demonstrated, by Western blot analysis, that the increased TSP1 mRNA was accompanied by increased TSP1 protein. The function of up-regulated myometrial TSP1 during parturition is not clear. Because TSP1 increases with labor in human myometrium (40), we hypothesize that TSP1 may act as an indirect regulator of myometrial contractility. Further studies are needed to clarify the potential function of myometrial TSP1 in myometrial activation.
In the present study, we immunolocalized the TSP1 in the pregnant sheep myometrium. We validated the TSP1 antiserum, by Western blot, before performing immunocytochemistry, showing that the antiserum specifically hybridized to proteins with expected molecular weights. Determination of the precise distribution of TSP1 in pregnant sheep myometrium is essential for a complete understanding of TSP1’s action in its target tissue. Specific immunostaining for TSP1 was mainly associated with fibroblasts in the pregnant sheep myometrium. However, myometrial cells also contained TSP1 localized by immunostaining. To further determine which cell type is responsible for producing TSP1, in situ hybridization was performed. Using in situ hybridization, TSP1 mRNA was identified mainly in fibroblasts; however, some myometrial cells also contained TSP1 mRNA, indicating that both fibroblasts and myometrial cells are involved in producing TSP1.
In conclusion: 1) our data provide the first evidence that changes in TSP1 mRNA and protein are associated with myometrial activation during both STL and BPL in sheep; 2) both myometrial fibroblasts and smooth muscle cells are responsible for producing TSP1; and 3) SSH is a powerful technique that enables us to study differentially regulated genes during labor.
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Acknowledgments |
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Footnotes |
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Received May 19, 1998.
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