The Parturition Defect in Steroid 5{alpha}-Reductase Type 1 Knockout Mice Is Due to Impaired Cervical Ripening

Mala S. Mahendroo, Amina Porter, David W. Russell and R. Ann Word

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Successful delivery of the fetus (parturition) depends on coordinate interactions between the uterus and cervix. A majority (70%) of mice deficient in the type 1 isozyme of steroid 5{alpha}-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{alpha}-reductase type 1 and that this enzyme normally plays an essential role in cervical pro-gesterone catabolism at the end of pregnancy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Parturition involves orderly and carefully timed biological events in both the uterus and the cervix that culminate in delivery of the fetus. In the uterus, parturition is marked by the onset of rhythmic contractions of the myometrium, while remodeling of the cervix leads to an increase in tissue elasticity. Although the endocrinological events that maintain pregnancy are complex, experimental and clinical studies in a variety of species indicate that progesterone withdrawal leads to a change in gene expression that triggers parturition and the end of pregnancy (1, 2, 3, 4).

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{alpha}-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{alpha} (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{alpha} 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{alpha}-reductase type 1 (5{alpha}R1-/-) also results in defective parturition (17). A majority (~70%) of pregnant 5{alpha}R1-/- mice fail to undergo delivery at term. However, in contrast to knockout mice with luteolysis defects, 5{alpha}R1-/- 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, 5{alpha}R1-/- 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 5{alpha}R1-/- 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell-Type Specific Expression of 5{alpha}RI Gene during Pregnancy
Previous studies demonstrated that steroid 5{alpha}-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. 1Go). Type 1 mRNA was detected predominantly in the glandular epithelial cells of the uterine endometrium (Fig. 1BGo). 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. 1DGo). 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{alpha}-reductase type 1 mRNA in epithelial cells of the cervix and endometrium during late gestation suggested that parturition failure in the 5{alpha}R1-/- mice might be due to abnormalities in uterine function, cervical ripening, or both.



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Figure 1. Identification of Cells Expressing Steroid 5{alpha}-Reductase Type 1 mRNA

Uterine and cervical tissues from gestation day 18 wild-type mice were isolated, and pups were gently removed from the uterine lumen. Formalin-fixed tissues were serially sectioned (5 µm) onto polylysine slides for in situ mRNA hybridization analyses using antisense strand probes complementary to a portion of the murine steroid 5{alpha}-reductase type 1 mRNA. Exposure times were 21 days. Hybridized sections were stained with hematoxylin and eosin and photographed using lightfield and darkfield optics on a Nikon E1000 microscope at 10-fold magnification. A, Lightfield photograph of a day 18 uterine cross-section. The tissue was everted during dissection and fixation, thus placing the endometrium on the outside and the myometrium on the inside of this specimen. B, Darkfield photograph of A, antisense probe. C, Lightfield photograph of a day 18 cervical cross-section. D, Darkfield photograph of C, antisense probe. En, Endometrium; My, myometrium; Ep, epithelium; St, stroma.

 
Assessment of Uterine Function In Vitro
To determine which of these essential physiological processes was disrupted in the mutant mice, we measured the frequency, duration, and force amplitude of spontaneous and agonist-induced myometrial contractions in uterine smooth muscle strips from wild-type (days 18–19) and 5{alpha}R1-/- (days 19–20) mice (19). As summarized in Table 1Go, 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 1Go). 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 5{alpha}R1-/- mice responded to oxytocin with similar increases in both force amplitude and frequency of contraction (Fig. 2Go). The EC50 for oxytocin was 0.104 ± 0.038 nM in wild-type mice and 0.098 ± 0.040 nM in 5{alpha}R1-/- mice. Receptor-dependent (oxytocin, PGF2{alpha}, and endothelin-1) and receptor-independent (KCl) activations of contractions were also similar in the uterine tissues of wild type and mutant mice (Table 1Go). Taken together, these results suggested that myometrial contractility in vitro was not impaired in 5{alpha}R1-/- mice. In addition, there appeared to be no difference in the coupling of the oxytocin, PGF2{alpha}, and endothelin-1 receptors to full activation of the uterine contractile apparatus of term knockout mice.


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Table 1. Quantification of in Vitro Uterine Contractility in Wild-Type and 5{alpha}R1-/- Mice

The variability in area measurement reflects inconsistencies in the duration of the fused contraction in animals of both genotypes.

 


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Figure 2. Effect of Oxytocin on Force of Contraction in Myometrial Tissues from Wild-Type and 5{alpha}R1-/- Mice

Oxytocin was added to myometrial strips in increments of 0.5–1 log unit every 10 min to obtain cumulative, final concentrations ranging from 2 x 10-12 to 2 x 10-8 M. Contractile response represents the computed force of contraction generated during a 10-min period after the addition of oxytocin to the tissue bath. Data (mean ± SEM) were obtained from nine strips from four wild-type animals (days 18–19; open circles) and 16 strips from six 5{alpha}R1-/- animals (days 19–20; closed circles).

 
Assessment of Uterine Function In Vivo
Myometrial contractions were next monitored in vivo by intrauterine pressure catheters. Spontaneous uterine contractions were infrequent in wild-type and 5{alpha}R1-/- animals on gestation day 16 (Fig. 3Go, 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. 3AGo). As in the wild-type animals, spontaneous uterine contractions also occurred in 5{alpha}R1-/- animals that did not deliver on day 19 (Fig. 3BGo). Peak amplitude, duration, and frequency of these contractions were indistinguishable from those of wild-type animals in active labor (Table 2Go).



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Figure 3. Assessment of In Vivo Uterine Contractility

Representative tracings of intrauterine pressure in pregnant wild-type (A) and 5{alpha}R1-/- (B) mice. Increases in intrauterine pressure were absent in both wild-type and knockout animals on gestation day 16 (A and B, left). Spontaneous, cyclic increases in intrauterine pressure were recorded in wild-type animals after the onset of labor (A, right). Similar increases were observed in nondelivering 5{alpha}R1-/- animals on day 19 (B, right).

 

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Table 2. Quantification of in Vivo Uterine Contractility in Wild-Type and 5{alpha}R1-/- Mice

 
To test the hypothesis that contractions in the 5{alpha}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 5{alpha}R1-/- animals treated with antiprogestin compared with those in wild-type mice in labor (Table 2Go). 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 5{alpha}R1-/- 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 5{alpha}R1-/- 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. 4Go), 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. 4Go). 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. 4Go).



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Figure 4. Biomechanical Properties of Cervical Tissues from Wild-Type and 5{alpha}R1-/- Mice

Stress (force per cross-sectional area) generated by the cervix is plotted as a function of increases in cervical diameter. Data from nonpregnant animals were obtained from four wild-type mice (open circles) and five 5{alpha}R1-/- mice (closed circles). Data obtained from gestation day 16–18 tissues are presented as the mean ± SEM of three to five animals in each group. Data from day 19 represent six wild-type (open circles) and 10 knockout (closed circles) animals.

 
In cervixes isolated from knockout mice, the slopes of the stress-strain curves were not significantly different from those of wild-type tissues from nonpregnant mice or from pregnant animals on gestation days 15–17 (closed circles, Fig. 4Go). However, the marked increase in cervical compliance measured on day 18 in wild-type tissue was not detected in the day 18 5{alpha}R1-/- samples, and little further increase in distensibility occurred on day 19 in the knockout specimens (Fig. 4Go). Because only about 70% of the 5{alpha}R1-/- 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. 4Go). 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 5{alpha}R1-/- 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 5{alpha}R1-/- 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. 5AGo), 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. 5BGo). Both Trichrome and mucicarmine staining indicated that epithelial cells of wild-type (Fig. 5Go, C and E), but not 5{alpha}R1-/-, endocervixes (Fig. 5Go, 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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Figure 5. Cervical Histology at Term

A, C, and E, Cross-sections of gestation day 19 wild-type cervixes. B, D, and F, Cross-sections of gestation day 19 cervixes from 5{alpha}R1-/- mice. Cervical sections were assessed for collagen (blue) using Masson’s trichrome stain (A–D) and for mucins (pink stain) using Mayer’s mucicarmine (E and F). All sections were photographed at x10 magnification.

 
Relaxin Prevents Parturition Defect in 5{alpha}R1-/- Mice
To obtain endocrine evidence to support the hypothesis of abnormal cervical ripening in the 5{alpha}R1-/- 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 3Go). 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 5{alpha}R1-/- mice from 33% to 88% (Table 3Go).


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Table 3. Effects of Relaxin and Ovariectomy on Delivery Rates in Wild-Type and 5{alpha}R1-/- Mice

 
Tissue Steroid Hormone Levels
Serum progesterone levels decline normally at term in 5{alpha}R1-/- mice (17). Nevertheless, the finding of aberrant cervical ripening in the mutant mice coupled with the expression of steroid 5{alpha}-reductase type 1 in the cervixes of wild-type mice (Fig. 1Go) 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 5{alpha}R1-/- mice on gestation days 17–19. As expected, serum progesterone levels declined on day 18 in both wild-type and mutant mice (Fig. 6Go). 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 5{alpha}R1-/- mice on day 19 were very low and virtually identical to those in wild-type animals that had delivered (Fig. 6Go). 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. 6Go). 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. 6Go).



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Figure 6. Progesterone and 20{alpha}-Hydroxyprogesterone Levels in Sera and Tissues

Sera and cervical and uterine tissues were collected from wild-type (open bars) and 5{alpha}R1-/- (solid bars) mice on gestation days 17–19. Samples were obtained from 7–10 animals/genotype·gestation day. Steroids were extracted, and levels of progesterone (P, upper panels) and 20{alpha}-hydroxyprogesterone (20{alpha}-OHP, lower panels) were measured in duplicate by RIA. Bars indicate the mean hormone concentrations ± SEM. *, P <= 0.05 compared with wild-type value.

 
Levels of the progesterone metabolite 20{alpha}-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. 6Go), consistent with the decline in circulating progesterone. In the knockout mouse, serum 20{alpha}-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. 6Go). In wild-type mice, uterine levels of 20{alpha}-hydroxyprogesterone were also low and did not change during late gestation. However, 20{alpha}-hydroxyprogesterone amounts were markedly elevated in the uteri of 5{alpha}R1-/- animals (Fig. 6Go).

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, 5{alpha}R1-/- 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 3Go), six of eight ovariectomized knockout animals delivered their litters (Table 3Go). 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. 7Go show that very little of this steroid was metabolized when these tissues were isolated from nonpregnant mice of either wild-type or 5{alpha}R1-/- 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{alpha}-pregnan-3,20-dione by steroid 5{alpha}-reductase, to 4-pregnen-20{alpha}-ol-3-one by 20{alpha}-hydroxysteroid dehydrogenase, and to 5{alpha}-pregnan-3{alpha}, 20{alpha}-diol by the combined actions of these two enzymes and 3{alpha}-hydroxysteroid dehydrogenase (lane 2). In day 18 wild-type cervixes (lane 4), progesterone was converted to 5{alpha}-pregnan-3,20-dione by 5{alpha}-reduction and to one other unidentified metabolite that is presumed to be 5{alpha}-reduced based on its absence from mutant tissue extracts (Fig. 6Go, lane 5).



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Figure 7. Progesterone Metabolism in Uterus and Cervix

Uterine and cervical tissue homogenates (150 µg protein) were incubated with [14C]progesterone (5 µM) for 60 min at 37 C and pH 7.0. Thereafter, steroids were extracted and separated by TLC. The migration patterns of progesterone and various metabolite standards are indicated on the right and left of the autoradiogram. Genotypes marked + are wild-type, and those marked - are 5{alpha}R1-/-. The tissue labeled + is a positive control (1827 cell line expressing human steroid 5{alpha}-reductase type 1) (33 ). Ut, Uterus; Cv, cervix; NP, nonpregnant.

 
In uteri from day 18 knockout mice, progesterone was converted to 4-pregnen-20{alpha}-ol-3-one by the 20{alpha}-hydroxysteroid dehydrogenase as expected (17, 23, 24); however, no 5{alpha}-reduced progesterone metabolites were detected (Fig. 6Go, 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{alpha}-hydroxysteroid dehydrogenase and steroid 5{alpha}-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{alpha}-hydroxysteroid dehydrogenase mRNA in the wild-type cervix by RNA blotting (data not shown), whereas 5{alpha}-reductase type 1 mRNA was detectable in the tissue, even with the relatively insensitive method of in situ hybridization (Fig. 1Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several lines of evidence presented here suggest that a failure of cervical ripening underlies the parturition defect observed in 5{alpha}R1-/- mice. First, the 5{alpha}-reductase type 1 gene is normally induced in late gestation in the epithelial cells of the cervix (Fig. 1Go). Second, the uterine contractile apparatus of 5{alpha}R1-/- mice is indistinguishable from that of wild-type animals (Figs. 2Go and 3Go). Third, the compliance of the cervix does not change in late gestation in the knockout mice (Fig. 4Go), nor are histological changes characteristic of ripening detected in the cervix at this time (Fig. 5Go). Fourth, supraphysiological levels of a cervical ripening agent (relaxin) alleviate the parturition defect in 5{alpha}R1-/- mice (Table 3Go). Fifth, although serum progesterone levels decline in the mutant mice, levels of progesterone within the cervix remain elevated at term (Fig. 6Go), perhaps due to the dependence of this tissue on steroid 5{alpha}-reductase type 1 for progesterone catabolism and the absence of this pathway in the knockout mice (Fig. 7Go).

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 5{alpha}R1-/- mice is 0.32 x 104 Newtons (N)/m2 (Fig. 3Go). 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 5{alpha}R1-/- 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. 6Go). The current data suggest that the induction of steroid 5{alpha}-reductase type 1 enzyme activity in these tissues at term (Fig. 1Go) (17) normally serves to inactivate progesterone by converting it to 5{alpha}-pregnan-3,20-dione and other metabolites (Fig. 7Go). 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 5{alpha}R1-/- uterus (Fig. 6Go). Despite this accumulation, rhythmic contractions are measured on day 19 in the mutant mice (Fig. 3Go). These results suggest that this tissue is less sensitive to residual progesterone than is the cervix. The experiments shown in Figs. 6Go and 7Go and reported in previous studies (17, 23, 24) reveal that the progesterone catabolic enzyme 20{alpha}-hydroxysteroid dehydrogenase is induced at term in the 5{alpha}R1-/- uterus and that 20{alpha}-hydroxyprogesterone accumulates in the tissue. This induction and the abnormal build-up of the metabolite may negate the consequences of progesterone accumulation in the 5{alpha}R1-/- uterus. The accumulation of 20{alpha}-hydroxyprogesterone in the 5{alpha}R1-/- uterus (Fig. 6Go) also suggests that 5{alpha}-reduction of this steroid may facilitate excretion from the tissue.

The correction of the parturition defect in seven of eight 5{alpha}R1-/- animals (Table 3Go) 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 5{alpha}R1-/- 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. 5Go). The levels of collagen and other basement membrane components in the cervixes of term 5{alpha}R1-/- 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{alpha}-reduced androgens dihydrotestosterone or 5{alpha}-androstane-3{alpha},17ß-diol to 5{alpha}R1-/- mice prevented the parturition defect (17). The results described here suggest that the target tissue for 5{alpha}-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{alpha}-reduced androgens may replace one or more 5{alpha}-reduced metabolites of progesterone in this tissue. We are currently determining the effects of 5{alpha}-reduced androgens on cervical biology in wild-type and 5{alpha}R1-/- mice using the assays developed in this study.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant human relaxin was obtained from Connective Therapeutics (Palo Alto, CA). RU486 (mifepristone) was purchased from Roussel-UCLAF (Romainville, France).

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{alpha}-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 5{alpha}R1-/- 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 5{alpha}R1-/- 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{alpha}-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 5{alpha}R1-/- 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.


    ACKNOWLEDGMENTS
 
We thank Jean Wilson and John Porter for critical reading of the manuscript, Gundula Girschick and Linette Casey for help with steroid analysis, and Kevin Anderson and Kristine M. Cala for expert technical assistance.


    FOOTNOTES
 
Address requests for reprints to: R. Ann Word, M.D., Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9032. E-mail: aword{at}mednet.swmed.edu

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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 MATERIALS AND METHODS
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