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-Reductase Type 1 Knockout Mice Is Due to Impaired Cervical Ripening
Departments of Molecular Genetics (M.S.M., D.W.R.) and Obstetrics and Gynecology (A.P., R.A.W.) University of Texas Southwestern Medical Center Dallas, Texas 75235
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-reductase fail to deliver their young at term and thus manifest a
parturition defect. Using in vitro and in vivo
measurements we show here that rhythmic contractions of the uterus
occur normally in these mutant mice at the end of gestation. In
contrast, the cervix of the mutant animal fails to ripen at term as
judged by biomechanical, histological, and endocrinological assays.
Impaired metabolism of progesterone in the cervix of the mutant mice in
late gestation leads to an accumulation of this steroid in the tissue.
We conclude that a failure of cervical ripening underlies the
parturition defect in mice lacking steroid 5-reductase type 1
and that this enzyme normally plays an essential role in cervical
pro-gesterone catabolism at the end of pregnancy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In rats and mice, pregnancy is maintained by continued synthesis of
progesterone in the corpus luteum from fetal or maternal steroid
precursors. At term, progesterone synthesis decreases and catabolism
increases, producing a fall in serum progesterone (5). This process is
termed luteolysis (5). In sheep, the primary source of progesterone is
the placenta. Late in gestation, cortisol produced by the fetus acts on
placental trophoblast cells to induce synthesis of the enzyme steroid
17-hydroxylase, which diverts pregnenolone from the synthesis of
progesterone to the formation of estrogen precursors (6). This
cortisol-induced decline in serum progesterone is required for
successful parturition in sheep (4, 7).
In contrast to rodents and sheep, progesterone levels in women do not
decline in maternal or fetal blood before the onset of parturition (4, 8). However, several lines of evidence suggest that localized
progesterone withdrawal may initiate labor and delivery in human
pregnancy. First, artificially induced progesterone withdrawal results
in abortion or labor in pregnant women (9, 10). Second, removal of the
corpus luteum in early human pregnancy causes abortion (11). Third, in
some abnormal pregnancies (e.g. ectopic pregnancy and fetal
demise), a decrease in progesterone formation can precede the onset of
uterine contractions and the expulsion of uterine contents (12).
Fourth, the administration of antiprogestins to pregnant women causes
abortion and enhanced sensitivity of the uterus to oxytocin and
PGF2 (10). Thus, although the mechanisms of progesterone
synthesis during gestation and progesterone withdrawal at term vary
among species, this hormone appears to be essential for the maintenance
of most mammalian pregnancies (13).
The crucial role of progesterone in rodent pregnancy has been
documented in several strains of knockout mice. Null mutations in genes
encoding cyclooxygenase 1 (14), cytosolic phospholipase A2
(15), or the PGF2 receptor (16) result in delayed
parturition. Mutant animals do not undergo luteolysis at term, leading
to elevated progesterone levels and failure of parturition. A null
mutation in the murine gene encoding steroid 5-reductase type 1
(5R1-/-) also results in defective parturition (17). A
majority (
70%) of pregnant 5R1-/- mice fail to
undergo delivery at term. However, in contrast to knockout mice with
luteolysis defects, 5R1-/- animals experience normal
declines in serum progesterone at the end of pregnancy and this
decrease is associated with timely increases in the expression of genes
such as the oxytocin receptor (1). Thus, 5R1-/- mice
represent an unique model of defective parturition in which
progesterone withdrawal apparently occurs, but labor and delivery are
impaired.
In this study, the physiological mechanism of the parturition defect in
5R1-/- mice is elucidated. We show that while uterine
contractions develop in the knockout animals, proper cervical ripening
does not take place. Although circulating progesterone levels decline
at term, elevated progesterone levels persist within the cervix for
approximately 24 h longer than those in wild-type mice. We
conclude that dissociation of the normally linked processes of uterine
contraction and cervical ripening in these mutant animals leads to
dysfunctional labor and unsuccessful delivery.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RI Gene during Pregnancy-reductase type 1
mRNA levels increase at term in whole uterine/cervical tissues of
wild-type mice (17). To identify which cells express this mRNA during
late gestation (days 17–18), uterine and cervical tissues from
wild-type mice were subjected to in situ mRNA hybridization
(18) using radiolabeled antisense strand probes (Fig. 1
). Type 1 mRNA was detected
predominantly in the glandular epithelial cells of the uterine
endometrium (Fig. 1B). Little or no hybridization signal was observed
in stromal cells of the endometrium or in smooth muscle cells of the
myometrium. In the cervix, type 1 mRNA was localized to epithelial
cells lining the endocervical canal (Fig. 1D). No hybridization signal
was detected in the cervical stroma. Sense strand probes also failed to
produce a hybridization signal in uterine or cervical sections (data
not shown). Localization of 5-reductase type 1 mRNA in epithelial
cells of the cervix and endometrium during late gestation suggested
that parturition failure in the 5R1-/- mice might be
due to abnormalities in uterine function, cervical ripening, or
both.
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R1-/- (days 19–20) mice (19). As
summarized in Table 1, myometrial tissues
from mice of both genotypes developed spontaneous contractions in
vitro. The amplitude, frequency, and duration of the contractions
were similar in both sets of mice (Table 1). To ascertain the
responsiveness of the tissue to the uterotonin oxytocin, myometrial
strips were treated with increasing concentrations of this hormone
(0.02–20 nM), and active force generation was quantified
as a function of time. Myometrial tissues obtained from day 18–19
wild-type and 5R1-/- mice responded to oxytocin with
similar increases in both force amplitude and frequency of contraction
(Fig. 2). The EC50 for
oxytocin was 0.104 ± 0.038 nM in wild-type mice and
0.098 ± 0.040 nM in 5R1-/- mice.
Receptor-dependent (oxytocin, PGF2, and endothelin-1)
and receptor-independent (KCl) activations of contractions were also
similar in the uterine tissues of wild type and mutant mice (Table 1).
Taken together, these results suggested that myometrial contractility
in vitro was not impaired in 5R1-/- mice.
In addition, there appeared to be no difference in the coupling of the
oxytocin, PGF2, and endothelin-1 receptors to full
activation of the uterine contractile apparatus of term knockout
mice.
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R1-/- animals on
gestation day 16 (Fig. 3, A and B).
However, after the onset of spontaneous labor in wild-type mice on days
18–19, this quiescent pattern changed dramatically. Cyclic,
spontaneous increases in intrauterine pressure of approximately 10-min
duration occurred five or six times per hour, and frequent contractions
(1.3 ± 0.14/min) of short duration also occurred (Fig. 3A). As in
the wild-type animals, spontaneous uterine contractions also occurred
in 5R1-/- animals that did not deliver on day 19 (Fig. 3B). Peak amplitude, duration, and frequency of these contractions were
indistinguishable from those of wild-type animals in active labor
(Table 2).
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R1-/-
mice were of sufficient magnitude to achieve delivery, these parameters
were quantified after RU486-induced labor in knockout animals.
Administration of this steroid prevents the parturition defect in these
mutant mice (17). After treatment with RU486, all animals delivered
spontaneously on gestation day 19, 16–20 h after the drug was given.
The measured increases in intrauterine pressure were diminished
slightly in 5R1-/- animals treated with antiprogestin
compared with those in wild-type mice in labor (Table 2
). However, the
duration of individual contractions in RU486-induced labor was
prolonged. This anomalous uterine contractility pattern was consistent
with the observation that birthing of the first pup appeared to be
protracted in the RU486-treated knockout mice. Nevertheless, the
results indicated that nondelivering 5R1-/- mice on
day 19 exhibited uterine contractions of sufficient magnitude to bring
about delivery in the presence of RU486.
Assessment of Cervical Ripening
We next examined the biomechanical properties of intact
cervixes from nonpregnant and pregnant (days 15–20) wild-type and
5R1-/- mice. Cervixes were distended isometrically in
physiological saline solution in a water-jacketed tissue bath. The load
applied and the increase in diameter were recorded simultaneously, and
the results were translated into stress-strain curves (20). In
wild-type mice (open circles, Fig. 4), the slope of the stress-strain curve
was significantly increased in cervixes from nonpregnant animals
compared with that in pregnant animals at any time in late gestation
(8.06 ± 1.12 vs. 1.96 ± 0.2, days 15–19).
Additionally, the slopes of the stress-strain curves were similar to
each other on gestation days 15, 16, and 17 (Fig. 4). However, the
slope decreased significantly on day 18, indicating a marked increase
in cervical distensibility (compliance). On day 19, just before actual
delivery, the cervixes were so compliant that they could be stretched
to 14 mm before tearing, in contrast to the maximum stretch of 8–10 mm
in the day 15–18 tissues (Fig. 4).
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). However, the
marked increase in cervical compliance measured on day 18 in wild-type
tissue was not detected in the day 18 5R1-/- samples,
and little further increase in distensibility occurred on day 19 in the
knockout specimens (Fig. 4). Because only about 70% of the
5R1-/- mice exhibit a parturition defect (17),
approximately 3 of the 10 animals analyzed on day 19 would have
delivered spontaneously at term. The inclusion of the data generated
from these animals contributes to the slight change in slope observed
between days 18 and 19 in the knockout samples (from 2.6 ± 0.4 to
1.9 ± 0.3), and it increased the range of tension values measured
on these days (Fig. 4). In experiments not shown, cervical tension was
also measured on day 20 in knockout mice. The cervixes were even less
compliant on day 20 (slope = 3.7 ± 0.7; n =
12) than on day 19 (slope = 1.9 ± 0.3; n =
10; P < 0.05), suggesting that cervical ripening was
not simply delayed but, rather, prevented in the mutant mice. Taken
together, the data suggested that the parturition defect in the
5R1-/- mice was due to failed cervical ripening at
term.
Histological Analyses of Cervical Tissue
Normal cervical ripening is characterized by changes in basement
membrane organization and by an increase in the secretion of mucins
(21). To provide insight into the cause of the failure of cervical
ripening in the 5R1-/- mice, sections (5 µm) of
tissue from wild-type and mutant animals were mounted and assessed for
collagen organization and mucin content with Masson’s Trichrome and
Mayer’s mucicarmine stains, respectively. In wild-type cervixes,
Trichrome staining revealed cervical fibroblasts suspended in a loose
array of disordered collagen fibers on day 19 (Fig. 5A), which are morphometric features of
compliant tissue. In contrast, mutant cervixes exhibited
characteristics of inelastic tissue, i.e. a more dense,
compact, heavily stained matrix of collagen fibers (Fig. 5B). Both
Trichrome and mucicarmine staining indicated that epithelial cells of
wild-type (Fig. 5, C and E), but not 5R1-/-,
endocervixes (Fig. 5, D and F), contained abundant secretory vacuoles
laden with mucins on day 19. These heavily glycosylated proteins also
decorated the exterior of the wild-type, but not mutant, cells.
Epithelial cells of the vagina and decidua contained normal amounts of
mucin in animals of both genotypes (data not shown), indicating a
selective effect of the mutation on the endocervical epithelial
cell.
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R1-/- MiceR1-/- mice, we
administered supraphysiological doses (6.0 µg/day) of relaxin, a
polypeptide hormone known to promote cervical ripening (22), on
gestation days 16–19 and determined the consequences for birthing. As
expected, wild-type animals delivered on gestation days 19–20 with or
without relaxin treatment (Table 3). In
the absence of relaxin, 15 of 45 knockout mice delivered young by days
19–20. This rate of spontaneous delivery reflects the partial
penetrance of the parturition defect in the mutant mice (17). The
administration of exogenous relaxin increased the number of spontaneous
deliveries in 5R1-/- mice from 33% to 88% (Table 3).
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R1-/- mice (17). Nevertheless, the finding of
aberrant cervical ripening in the mutant mice coupled with the
expression of steroid 5-reductase type 1 in the cervixes of
wild-type mice (Fig. 1) suggested that a failure to catabolize
progesterone might underlie the defect in the mutant animals. To test
this hypothesis, the levels of progesterone were quantified by RIA in
cervical and uterine tissues as well as sera from wild-type and
5R1-/- mice on gestation days 17–19. As expected,
serum progesterone levels declined on day 18 in both wild-type and
mutant mice (Fig. 6). Although
progesterone concentrations tended to be higher in type 1 knockout mice
on day 18, the values did not reach statistical significance
(P 
0.21). Moreover, serum progesterone levels in
5R1-/- mice on day 19 were very low and virtually
identical to those in wild-type animals that had delivered (Fig. 6). In
contrast, levels of progesterone in the cervixes of mutant mice on day
18 were increased relative to those in wild-type animals
(P 
0.05), and these levels remained slightly
elevated on day 19 despite the large decrease in serum progesterone
(Fig. 6). In the uteri of wild-type mice, progesterone declined in
amount from approximately 20 ng/g tissue on gestation day 17 to less
than 5 ng/g tissue on day 19, whereas in mutant uteri, progesterone
remained elevated throughout this period (Fig. 6).
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-hydroxyprogesterone were
also measured in these samples. In the serum of wild-type mice, there
was a gradual rise of this inactive progesterone derivative from
gestation days 17–19 (Fig. 6), consistent with the decline in
circulating progesterone. In the knockout mouse, serum
20-hydroxyprogesterone did not show a gradual increase as term
approached. Only low levels of this metabolite were detected in the
cervixes of mice of both genotypes, and these did not change over time
(Fig. 6). In wild-type mice, uterine levels of
20-hydroxyprogesterone were also low and did not change during late
gestation. However, 20-hydroxyprogesterone amounts were markedly
elevated in the uteri of 5R1-/- animals (Fig. 6).
Effects of Ovariectomy on Parturition
The cervical data suggested that residual progesterone might
contribute to the abnormalities in ripening of this tissue in the
knockout mice. To further test this idea, 5R1-/-
animals were ovariectomized on day 18 to remove the biosynthetic source
of progesterone and then monitored through day 20. Whereas sham
operations did not increase the incidence of successful delivery in
knockout animals (one of six animals delivered; Table 3), six of eight
ovariectomized knockout animals delivered their litters (Table 3).
Ovariectomy on day 18 did not affect the normal onset of parturition in
wild-type mice (four of four animals delivered).
Progesterone Metabolism In Vitro
We next examined progesterone catabolism in the uterus and cervix
using in vitro assays. The data presented in Fig. 7 show that very little of this steroid
was metabolized when these tissues were isolated from nonpregnant mice
of either wild-type or 5R1-/- genotypes (lanes 6 and
7). This metabolic picture changed dramatically on day 18 of pregnancy
in wild-type mice, when progesterone was actively catabolized in the
uterus to 5-pregnan-3,20-dione by steroid 5-reductase, to
4-pregnen-20-ol-3-one by 20-hydroxysteroid dehydrogenase, and to
5-pregnan-3, 20-diol by the combined actions of these two
enzymes and 3-hydroxysteroid dehydrogenase (lane 2). In day 18
wild-type cervixes (lane 4), progesterone was converted to
5-pregnan-3,20-dione by 5-reduction and to one other unidentified
metabolite that is presumed to be 5-reduced based on its absence
from mutant tissue extracts (Fig. 6, lane 5).
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-ol-3-one by the 20-hydroxysteroid dehydrogenase as
expected (17, 23, 24); however, no 5-reduced progesterone
metabolites were detected (Fig. 6, compare lanes 2 and 3). In the day
18 mutant cervix, little breakdown of progesterone was observed (lane
5). These data suggested that both 20-hydroxysteroid dehydrogenase
and steroid 5-reductase catabolize progesterone in the uterus,
whereas the cervix relies predominantly on the latter enzyme. In
agreement with this interpretation, we were unable to detect
20-hydroxysteroid dehydrogenase mRNA in the wild-type cervix by RNA
blotting (data not shown), whereas 5-reductase type 1 mRNA was
detectable in the tissue, even with the relatively insensitive method
of in situ hybridization (Fig. 1). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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R1-/- mice. First, the 5-reductase type 1 gene is
normally induced in late gestation in the epithelial cells of the
cervix (Fig. 1). Second, the uterine contractile apparatus of
5R1-/- mice is indistinguishable from that of
wild-type animals (Figs. 2 and 3). Third, the compliance of the cervix
does not change in late gestation in the knockout mice (Fig. 4), nor
are histological changes characteristic of ripening detected in the
cervix at this time (Fig. 5). Fourth, supraphysiological levels of a
cervical ripening agent (relaxin) alleviate the parturition defect in
5R1-/- mice (Table 3). Fifth, although serum
progesterone levels decline in the mutant mice, levels of progesterone
within the cervix remain elevated at term (Fig. 6), perhaps due to the
dependence of this tissue on steroid 5-reductase type 1 for
progesterone catabolism and the absence of this pathway in the knockout
mice (Fig. 7).
Normal parturition requires a close collaboration at term between the
uterus and cervix. The onset of rhythmic contractions in the uterus
must be accompanied by dilation of the cervix to allow delivery of the
fetus through the birth canal. The failure of either cervical ripening
or adequate uterine contractions causes unsuccessful (or dysfunctional)
parturition (25). We show here that in the mouse, the unripened cervix
presents a formidable barrier to delivery that cannot be overcome by a
normally contracting uterus. The murine uterus is composed of three or
four layers of longitudinally arranged smooth muscle cells overlaid by
five or six layers of circumferentially arranged smooth muscle cells.
The contractile force generated per uterine contraction in both
laboring wild-type and 5R1-/- mice is 0.32 x
104 Newtons (N)/m2 (Fig. 3). This force is less
than that required to physically distend the cervix to 10 mm (the
average diameter of a day 19 pup) in term mutant animals (2.41 x
104 N/m2), but it is approximate to that
required to distend the softened cervix of day 19 wild-type mice
(0.53 x 104 N/m2). Thus, normal
contractions in the knockout mice are insufficient to breach the
unripened cervix.
One mechanism in rodents that promotes coordination between the uterus
and cervix at term involves the removal of circulating progesterone
(5). A decrease in the level of this steroid changes the patterns of
gene expression within these tissues, most likely by turning off
progesterone-dependent target genes and turning on
progesterone-repressed genes (1, 26, 27, 28, 29). In the case of
5R1-/- mice, progesterone levels decline normally at
term in the plasma, but the clearance of this hormone from both the
cervix and uterus is impaired (Fig. 6). The current data suggest that
the induction of steroid 5-reductase type 1 enzyme activity in these
tissues at term (Fig. 1) (17) normally serves to inactivate
progesterone by converting it to 5-pregnan-3,20-dione and other
metabolites (Fig. 7). In the absence of enzyme induction, levels of
progesterone within the cervix remain high and may prevent the changes
in gene expression required for cervical ripening.
Surprisingly, progesterone levels at the end of gestation are also
elevated in the 5R1-/- uterus (Fig. 6). Despite this
accumulation, rhythmic contractions are measured on day 19 in the
mutant mice (Fig. 3). These results suggest that this tissue is less
sensitive to residual progesterone than is the cervix. The experiments
shown in Figs. 6 and 7 and reported in previous studies (17, 23, 24)
reveal that the progesterone catabolic enzyme 20-hydroxysteroid
dehydrogenase is induced at term in the 5R1-/- uterus
and that 20-hydroxyprogesterone accumulates in the tissue. This
induction and the abnormal build-up of the metabolite may negate the
consequences of progesterone accumulation in the
5R1-/- uterus. The accumulation of
20-hydroxyprogesterone in the 5R1-/- uterus (Fig. 6) also suggests that 5-reduction of this steroid may facilitate
excretion from the tissue.
The correction of the parturition defect in seven of eight
5R1-/- animals (Table 3) by the administration of
relaxin suggests that residual progesterone in the cervixes of
untreated knockout mice may affect the expression of genes that
participate in the relaxin signaling pathway. The predominant source of
circulating relaxin in the mouse is the ovary (30). RNA blotting
indicates that relaxin expression in the murine ovary begins on days
9–10 of gestation, reaches a plateau on days 11–12, remains elevated
through day 18, and thereafter declines (our unpublished
observations). This temporal expression pattern was similar in
wild-type and 5R1-/- mice. Thus, we do not believe
that alterations in relaxin expression itself contribute to defective
cervical ripening in the knockout animals. It seems more likely that
expression of the relaxin receptor or a downstream component of the
signaling pathway may be adversely affected by residual progesterone in
the cervix and was compensated for by supraphysiological levels of
exogenous relaxin.
Histological analyses indicate that the expression of genes that
remodel the cervix before delivery is abnormal in the mutant mice (Fig. 5). The levels of collagen and other basement membrane components in
the cervixes of term 5R1-/- mice are characteristic of
inelastic tissue, and levels of mucin in the epithelial cells of the
endocervix are low. Progesterone regulates the expression of the
mucin-1 gene in the reproductive tract of the mouse (31, 32) and
affects the transcription of genes whose products contribute to tissue
remodeling, including interstitial collagenase (29) and matrix
metalloproteinase-9/gelatinase B (28). Thus, the aberrant expression of
one or more of these genes or related family members may adversely
affect cervical ripening.
We previously reported that administration of the 5-reduced
androgens dihydrotestosterone or 5-androstane-3,17ß-diol to
5R1-/- mice prevented the parturition defect (17). The
results described here suggest that the target tissue for 5-reduced
androgens is the cervix. These hormones may antagonize progesterone
action in the cervix or serve as ligands for a distinct receptor system
that performs this function. Alternatively, exogenous 5-reduced
androgens may replace one or more 5-reduced metabolites of
progesterone in this tissue. We are currently determining the effects
of 5-reduced androgens on cervical biology in wild-type and
5R1-/- mice using the assays developed in this
study.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
Animals were housed under a 12-h light cycle (lights on,
0600–1800 h) at 22 C. All mice were of mixed strain
(C57BL/6J//129SvEv) and were either wild type at the Srd5 1
locus on chromosome 13 or contained an induced null mutation in this
gene (deletion of the proximal promoter and exon 1) (17). Timed matings
were carried out by housing one male with three or four females in a
cage. Each day at 1200 h, females were evaluated for the presence
of vaginal plugs. Gestation day 0 was defined by the presence of a
plug. All studies were conducted in accordance with the standards of
humane animal care described in the NIH Guide for the Care and Use of
Laboratory Animals using protocols approved by an institutional animal
care and research advisory committee.
In Situ mRNA Hybridization
Transcripts of the steroid 5-reductase type 1 gene were
detected by in situ mRNA hybridization in 5-µm sections of
gestation day 18 uterine/cervical tissues using procedures described
previously (18). 33P-Radiolabeled RNA probes in sense and
antisense orientations were transcribed in vitro from a
complementary DNA fragment encoding amino acids 1–94 of the murine
type 1 gene. Exposure times were 21 days. After development, tissue
sections were photographed under lightfield and darkfield illumination
on a Nikon Eclipse 1000 microscope (Tokyo, Japan) equipped
with a low magnification darkfield illuminating condenser.
In Vitro Evaluation of Uterine Contractile
Properties
Uterine and cervical tissues were isolated from wild-type and
5R1-/- mice killed by cervical dislocation. Uterine
tissues were opened longitudinally along the mesenteric border, and the
endometrium was gently removed. Myometrial strips (1 x 3 x
0.5 mm) were cut parallel to the longitudinal or circular muscle fibers
and mounted for measurement of isometric force in water-jacketed muscle
baths that contained oxygenated (95% O2-5%
CO2) physiological salt solution [NaCl (120.5
mM), KCl (4.8 mM), MgCl2 (1.2
mM), CaCl2 (1.6 mM),
NaH2PO4 (1.2 mM),
NaHCO3 (20.4 mM), dextrose (10 mM),
and pyruvate (1.0 mM), pH 7.4, at 37 C]. Tissue strips
were stretched to an optimal length for maximal force development by
the application of 2 g of force. Contractile force was
recorded with a Grass model 7C polygraph and was stored digitally with
an A-D converter (Grass Polyview Systems, West Warwick, RI) on an IBM
PC computer. Contractile force in response to test agents was
determined by quantifying the area under the traces representing active
force development during a 10-min period before and after the addition
of compounds. Forces were normalized to cross-sectional area as
previously described (19).
In Vivo Evaluation of Uterine Contractile
Properties
Pregnant wild-type and 5R1-/- mice were
anesthetized with urethane (1.5 µg/kg, ip). A 21-gauge catheter was
inserted transabdominally into the uterus. The catheter was threaded
between the uterine wall and fetal membranes and was connected to a
Grass model PT300 pressure transducer. Contractile activity was
recorded on a Grass 7C polygraph. The transducer was calibrated before
and after each recording session. The integral of intrauterine pressure
above basal pressure was determined with a software program (Grass
Polyview Systems).
Evaluation of Cervical Ripening
Tensile properties of isolated cervical tissues were evaluated
using modifications of the method developed by Harkness and Harkness
(20). The excised cervix was mounted by means of two pins inserted
through the cervical canal. One pin was attached to a calibrated
mechanical drive, and the other pin was attached to a force transducer.
Tissues were incubated in a water-jacketed bath containing
physiological salt solution at 37 C bubbled with 95%
O2-5% CO2. Baseline cervical dilatation was
quantified by determining the difference in micrometer readings at 0
(pins juxtaposed) and the initiation of tension recorded by the
physiograph. Thereafter, the inner diameter of the cervix was increased
isometrically in 1-mm increments at 10-min intervals to effect cervical
distention. The amount of force required to distend the cervix and the
tension exerted by the stretched tissue after 10 min were recorded. The
diameter was increased until either forces exerted by the tissue
reached a plateau or the tissue tore. Force was plotted as a function
of cervical diameter. Cross-sectional area, stress
(force/cross-sectional area), and stretch modulus (change in
stress/change in length) were calculated. The slope of the linear
portion of the force-strain curve was computed as an index of tissue
stiffness and elasticity (Young’s modulus) under the assumptions that
increased amounts of force are required to distend a rigid or stiff
tissue (i.e. steep slope of the force-strain curve) and that
distention of an elastic material is associated with low forces
(decreased slope).
Steroid Hormone Measurements
Progesterone levels were measured in the sera and tissues of
mice on gestation days 17–19 (3–12 animals/time point). Blood was
drawn from the inferior vena cava, cells were removed by
centrifugation, and the resulting sera were stored at -20 C until
steroid analyses were performed. The uteri were opened longitudinally,
pups and placentae were removed, and uterine samples (50–150 mg) were
obtained. Cervixes (15–40 mg) were dissected free from uterine tissue
and vaginal epithelium. Dissected tissues were stored at -80 C before
steroid hormone measurement. For extraction, tissues were thawed, cut
in half along the long axis, weighed on a Cahn microbalance and
homogenized in 1 ml PBS at 4 C (Polytron homogenizer, Brinkmann Instruments, Inc., Westbury, NY). Steroids in the sera or tissue
homogenates were extracted into ether, separated by chromatography on
Sephadex LH-20, and subjected to RIA at the Oregon Regional Primate
Research Center (Beaverton, OR) by Dr. David Hess. PBS blanks did not
exceed 9 pg. The intraassay coefficient for progesterone was 8.9%, and
that for 20-hydroxyprogesterone was 10.1%.
Steroid Hormone Metabolism
Cervixes and uteri were dissected from nonpregnant wild-type
mice and from gestation day 18 wild-type and 5R1-/-
animals. Tissues were homogenized in 10 mM potassium
phosphate, 150 mM potassium chloride, 0.3 M
sucrose, and 1 mM EDTA. The protein concentration was
determined using commercially available reagents (Bio-Rad Laboratories, Inc., Hercules, CA). Progesterone metabolism in
the uterus and cervix was assessed by incubating tissue homogenates
(150 µg protein) in 0.1 M Tris-citrate buffer, pH 7.0,
containing 5 µM [14C]progesterone (New
England Nuclear Corp., Boston, MA) and 5 mM NADPH
(Sigma Chemical Co., St. Louis, MO) in a total volume of
0.5 ml for 1 h at 37 C. Steroids were extracted into 5 ml
methylene chloride and taken to dryness under a stream of nitrogen.
Steroids were dissolved in 20 µl chloroform-methanol (2:1, vol/vol),
spotted onto Silica Gel 150 TLC plates (4855–821,
Whatman, Clifton, NJ), and resolved by development in
chloroform-ethylacetate (3:1, vol/vol). Radiolabeled steroids were
visualized by exposing the plates to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) for 12–16 h. Nonradioactive standards
(Steraloids, Wilton, NH) were cochromatographed on the silica plates
and visualized by iodine staining.
Statistical Analyses
Results are expressed as the mean ± SEM. An
independent Student’s t test was used for statistical
comparisons between individual groups, and one-way ANOVA followed by
post-hoc tests using Fisher’s least significant difference
were used for comparisons between multiple groups. P
0.05 was considered significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the NIH (HD-11149 to R.A.W. and GM-43753 to D.W.R.), the Burroughs-Wellcome Foundation (Grant 0203 to M.S.M.), and the Perot Family Foundation (to D.W.R.).
Received for publication December 9, 1998. Revision received March 12, 1999. Accepted for publication March 23, 1999.
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