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Regular Article |
-Reductase Type 1 in Mice1
a Department of Obstetrics and Gynecology, Divisions of Reproductive Endocrinology and Urogynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
ABSTRACT
A null mutation in the murine gene encoding steroid 5
-reductase type 1 (5R1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5R1 increase significantly at term in normal pregnant animals, indicates that 5R1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5R1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17ß-estradiol (E2), progesterone (P4 ), or E2+P4 for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5R1 specific activity, and Northern blot analysis of 5R1 mRNA expression. The 5R1 enzyme activity was significantly increased in animals treated with E2+P4. However, activity was much less in uterine tissues from E2+P4-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5R1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5R1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5R1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5R1 is regulated by E2+P4 in uterine tissues. Whereas E2 alone is insufficient to induce enzyme activity, E2 may be required to increase P4 receptors and, thereby, mediate the effects of P4 on 5R1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5R1 is regulated by both hormonal and fetal-derived signaling pathways.
female reproductive tract, parturition, pregnancy, progesterone, uterus
INTRODUCTION
Parturition has been characterized as the withdrawal of factor(s) implicit in the maintenance of pregnancy [1–3]. The withdrawal of progesterone (P4) that accompanies parturition in almost all animal species is an example of retreat from pregnancy maintenance. Although parturition in women is not associated with decreasing levels of P4 in blood, parturition in women and all animal species likely involves withdrawal from endocrinologic factors that inhibit uterine contractions and maintain cervical competency. The recent development of a colony of mice bearing a null allele in the 5-reductase type 1 gene (Srd5a1) provides further insight regarding possible mechanisms that may mediate P4 withdrawal at the tissue level [4]. Mice deficient in 5-reductase type 1 (5R1) fail to deliver their young at the expected time of parturition; pregnancy often continues until all pups die and undergo resorption or the mother succumbs to complications of sepsis. The mechanism of delayed parturition in these Srd5a1-deficient mice is not the absence of P4 withdrawal in blood or the absence of uterine contractions at the expected time of parturition but, rather, failure to metabolize P4 in uterine and cervical tissues at term [4]. These studies in 5R1-deficient animals indicate that, in mice, successful parturition is dependent not only on P4 withdrawal in blood (by way of luteolysis) but also by local tissue metabolism of P4 by steroid 5R1. In addition, it has been suggested that steroid 5R1 may be required to generate a 5-reduced steroid necessary for initiation of labor [5].
Steroid 5-reductases utilize NADPH to catalyze the conversion of testosterone to dihydrotestosterone and the conversion of other steroids with a 3-oxo-
4,5 configuration (e.g., P4, hydrocortisone, aldosterone, deoxycorticosterone) to 5-reduced metabolites. The conversion of testosterone to 5-dihydrotestosterone results in generation of a more potent androgen with increased affinity for the androgen receptor. On the other hand, 5-reduction of other steroid hormones leads to generation of metabolites with low affinity for their respective nuclear steroid hormone receptors [6]. Thus, 5-reductases may exhibit anabolic or catabolic roles depending on their tissue distribution and the steroid hormone receptor expression in those tissues.
In mice, the expression of 5R1 mRNA is low in uteri of nonpregnant animals, but expression increases dramatically in the days before parturition, peaking on Days 17–18 [5]. The enzyme is expressed in glandular epithelial cells of gestational endometrium and cervix [4]. These cells also express high levels of P4 receptors during pregnancy [7], and the time course of 5R1 mRNA expression parallels that of estrogen and P4. Although the stimulus for this increase in uterine 5R1 is unknown, we considered the possibility that sex-steroid hormones of pregnancy (e.g., estrogen, P4) or uterine occupancy (placenta or fetal factors or stretch [8]) may regulate the expression of 5R1 in target tissues of P4 action in the female reproductive tract. To investigate these possibilities, we treated ovariectomized mice with estrogen, P4, or both, and we utilized a unilateral pregnancy model in which gravid and nongravid uterine horns were exposed to the normal systemic hormonal milieu of pregnancy. We found that estrogen plus P4 induce 5R1 gene expression in uterine tissues. However, the prepartum up-regulation of 5R1 was greatly attenuated in the nongravid horn of unilateral pregnant animals. Thus, 5R1 is regulated by both fetal occupancy and steroid hormones, like other genes involved in parturition.
MATERIALS AND METHODS
Hormonal Treatment of Ovariectomized Mice
Adult female ICR mice (3–4 mo of age; Harlan, Indianapolis, IN) were housed under a 12L:12D cycle (lights-on, 0600–1800 h) at 22°C with free access to water and standard mice laboratory chow. Ovariectomy was conducted under anesthesia using aseptic conditions with a single ventral incision. After 2 wk, ovariectomized animals were divided into four groups (nine animals per group). Control animals (group 1) received vehicle alone. Group 2 received 17ß-estradiol (E2; 50 µg/kg per day), and group 3 received P4 (1 mg/day). Group 4 received combination treatment (E2+P4). Steroids were solubilized in corn oil, and injections were administered s.c. in 50-µl volumes daily for 3 days. Animals were killed on Day 4. Uterine horns were carefully separated under microscopy and trimmed of excess connective tissue and fat. The cervix and uterine horns were frozen separately in liquid N2 and stored at -80°.
Unilateral Ligation Studies
Unilateral tubal interruption was conducted under anesthesia (avertin, 25 µl/g) by ligation of either the right or left fallopian tube with 8-0 silk suture. Two weeks after ligation, timed matings were carried out by housing one male with three to four females per cage. Each day, females were evaluated for vaginal plugs. Gestational Day 0 was defined by the presence of a plug. Placental discs, fetal pups, and cervix were dissected from uterine tissue at various times in gestation (Days 12–18, n = 26) utilizing dissection microscopy. Uterine tissues were snap-frozen in liquid N2 and stored at -80°C until used. 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.
Enzyme Activity
Tissues were homogenized in buffer that contained 10 mM potassium phosphate, 150 mM KCl, 0.3 M sucrose, and 1 mM EDTA (pH 7.0) with three short pulses of a Brinkman polytron. The concentration of protein in tissue homogenates was determined by the method of Bradford using bovine albumin as a standard. 5-Reductase enzyme activity was determined by incubating tissue homogenates (150 µg of protein) in 0.1 M Tris-citrate buffer (pH 7.0) containing 1.4 µM [14C]testosterone (NEN Research Products, Boston, MA) and 5 mM NADPH (Sigma, St. Louis, MO) for 60 min in a 37°C water bath. The reaction was quenched with 5 ml of dichloromethane. Extraction tubes were then vortexed and centrifuged at 2500 rpm for 10 min. Steroids were taken to dryness under a stream of nitrogen and dissolved in 50 µl of chloroform/methanol (2:1, v/v), spotted onto Silica Gel 150 thin-layer chromatography plates (product no. 4855–821; Whatman, Clifton, NJ), and resolved by development in chloroform:ethylacetate (3:1, v/v). Radiolabeled steroids (testosterone, Rf 0.38; dihydrotestosterone, Rf 0.52; and 5-androstane-3-ol-diol, Rf 0.34) were visualized by exposing the plates to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 12–16 h and quantified using a PhosphorImager. Nonradioactive standards (Steraloids, Wilton, NH) were cochromatographed on the silica plates and visualized by iodine staining. Specific activity was quantified as the amount of 5-reduced products formed per minute per milligram of protein.
Northern Blot Analysis
Total RNA was extracted from tissues using Ultraspec (Biotecx, Houston, TX) according to the manufacturer's directions. Poly(A)+ RNA was isolated from total RNA using an mRNA purification kit (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). Poly(A)+ RNA (10 µg) was size-fractionated by electrophoresis through a 1.4% (w/v) agarose gel, transferred to nylon filters (Biotrans; ICN Corp., Cleveland, OH) by capillary blotting, and hybridized to a 32P-radiolabeled probe for 5R1. The radiolabeled probe was generated by asymmetric polymerase chain reaction using [32P]deoxycytosine triphosphate (3000 Ci/mmol) and murine 5R1 cDNA as template (10 ng; kindly provided by Dr. David Russell, University of Texas Southwestern Medical Center, Dallas, TX) and oligonucleotide primers corresponding to base pairs (bp) 114–136 (80 nM, 5'-GCTCTTCCTACGGCCGCTACTCC-3', forward primer) and bp 373–350 (800 nM, 5'-GAAGGCCAAGACAAAGGTGAACAG-3', reverse primer). Prehybridization was conducted at 65°C for 2 h. Thereafter, the membrane was hybridized at 65°C overnight in 5x SSPE (single strength: 150 mM NaCL, 10 mM NaH2PO4, and 1 mM Na/EDTA), 50% (v/v) formamide, 5x Denhardt solution, 1% (w/v) SDS, and 7% (w/v) dextran sulfate. The membrane was washed in 2x SSC (single strength: 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS for 20 min at 25°C, then four washes of 15 min each in 1x SSC and 0.1% SDS at 65°C, followed by autoradiographic exposure to x-ray film. Thereafter, the membrane was stripped and reprobed with a 32P-radiolabeled probe for cyclophilin (with lower specific activity).
Statistical Analysis
Results are expressed as the mean ± SEM. Statistical comparisons between multiple groups were conducted using either an ANOVA followed by Student-Neuman-Keuls post-hoc testing for normally distributed data or a Kruskal-Wallis ANOVA on Ranks followed by post-hoc Dunnett testing for unequally distributed data. Paired comparisons of 5R1 activity within animals were made by a two-way ANOVA. A P value of less than 0.05 was considered to be significant.
RESULTS
Effect of Estrogen and P4 on Expression of 5R1 in Uterine Tissues
The 5R1 enzymatic activity was low in uterine tissues from control ovariectomized mice, and this level was not increased by treatment with either E2 or P4 alone (Fig. 1). Enzyme activity was significantly increased in uterine tissues from animals treated with E2+P4. In cervical tissues, enzyme activity was more variable than in corresponding uterine horns (Fig. 1B). However, as with uterine tissue levels, E2+P4 increased 5R1 activity in the cervix (P < 0.05) (Fig. 1B). The variability in expression of enzyme activity in the cervix was probably due to difficulty in precise recognition of the junction between uterine horns and cervix. Therefore, further experiments were conducted with uterine tissues.
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The effect of E2 and P4 on 5R1 mRNA expression was determined by Northern blot analysis using pooled uterine horns from each treatment group (Fig. 2). The cDNA probe for 5R1 detected a strong signal at 2.6 kilobases (kb) and a less intense signal at approximately 1.5 kb in uterine tissues; mRNA for 5R1 was not detected in cardiac tissues (negative control). Low levels of 5R1 mRNA were detected in uterine tissues from ovariectomized control animals and nonpregnant cycling mice. Treatment with E2 or P4 alone resulted in modest increases in the tissue levels of 5R1 mRNA, but these increased levels of mRNA were not accompanied by increased enzyme activity (as determined by thin-layer chromatography) (Fig. 1A). Treatment with E2+P4, however, produced 12-fold increases in 5R1 mRNA.
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Effect of Fetal Occupancy on Expression of 5R1 in Uterine Tissues
Although E2+P4 significantly increased the levels of enzyme activity in ovariectomized mice, the level of expression was considerably less than that in uterine tissues from pregnant animals at term (Figs. 3 and 4). A unilateral pregnant mouse model was utilized to explore the possibility that factors related to fetal occupancy may influence the expression of 5R1 during gestation. The 5R1 enzyme activity was determined in unilateral pregnant mice as a function of gestational age (Fig. 3). No significant increase was found in 5R1 activity in the nongravid horn from Day 12 to Day 18, and these levels were similar to those of uterine tissues from nonpregnant mice treated with E2+P4 (Fig. 4). In contrast, uterine 5R1 activity increased eightfold from Day 12 to Day 18 in the gravid horn (Fig. 3). In cervical tissues, 5R1 activity also increased, from 4.3 ± 1.8 on Days 12–13 (n = 6) to 15.3 ± 2.2 on Day 18 (n = 6, P 
0.003). This temporal pattern of 5R1 paralleled marked increases in uterine diameter (from 2 mm to 9 mm) (Fig. 3, right).
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Placental Effects of 5R1 Activity
In unilateral pregnant animals, fetal occupancy was necessary for gestational-dependent increases in uterine 5R1 activity. We considered the possibility that placental-derived substances may influence enzyme activity during gestation. Uterine tissue overlying the placenta was microdissected from the nonplacental (distended) uterine tissue, and 5-reductase activity was quantified (Fig. 5). Enzyme activity was significantly increased in the distended nonplacental region of the uterus compared with the uterine tissues adjacent to the placenta. These results indicate that increased 5R1 activity in the gravid horn of unilateral pregnant animals is not due to the influence of the placenta.
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DISCUSSION
In rodents, 5R1 is widely distributed among a variety of tissues, including brain, liver, adrenal, kidney, nongenital skin, ovary, cervix, and uterus [9]. 5-Reductase type 2 is expressed in kidney, male adrenal, and androgen-dependent structures (e.g., prostate, epididymis, seminal vesicles) [9]. Expression of 5-reductase type 2 in androgen-dependent tissues is essential for sexual differentiation of the external male genitalia [10]. Induction of 5-reductase type 2 by testosterone results in the conversion of testosterone to dihydrotestosterone. Dihydrotestosterone not only binds to the androgen receptor but also increases gene expression of 5-reductase type 2 by an autoregulatory mechanism, whereby increased testicular androgen production at puberty induces gene expression of 5R1 and 5-reductase type 2 in target tissues of androgen action [11]. Gene expression is further induced by dihydrotestosterone administration, thereby supporting an additional level of autoregulation. The regulation of steroid 5R1, however, is not well characterized.
The wide distribution of 5R1 within the female reproductive organs in mice signals its significance in female reproductive function. The principal substrate for 5R1 in female reproductive tissues, however, may be P4 rather than testosterone. Androgen levels are significantly lower in females compared with males, and P4 levels fluctuate with a cyclic variation. Furthermore, 5-reductase is also known to have a higher affinity for P4 than testosterone. Thus, in female mice, 5R1 not only metabolizes androgens but also P4 to elicit physiologic responses for normal reproductive function in nonpregnant and pregnant states.
The findings presented herein provide evidence that E2 and P4 regulate expression of 5R1 in the female reproductive tract. Although in some respects regulation of the type 1 enzyme by P4 is similar to regulation of the type 2 enzyme by testosterone, significant differences exist, because 5-dihydroprogesterone is not biologically active with respect to binding to the P4 receptor. Thus, at first glance, it seems paradoxical that P4 may induce its own catabolism in P4-responsive tissues. However, metabolism of P4 by the sequential action of 5-reductase and 3-hydroxysteroid dehydrogenase leads to the formation of 5,3-hydroxy-dihydroprogesterone (allopregnanolone), which is a biologically active neurosteroid [12–14]. 5-Reduced pregnanolones act predominantly through nongenomic mechanisms to alter cellular function. For example, allopregnanolone directly activates and potentiates GABA-A receptor-activated membrane currents in the brain [15, 16]. Allopregnanolone has also been shown to inhibit action potentials in the brain and to synergize with other neurotransmitters to inhibit action potentials [17]. The GABA receptor subunits are highly expressed in the female reproductive tract [18–20]. These results, together with the demonstration of marked increases in uterine 3-hydroxysteroid dehydrogenase during late gestation [5], indicate that P4 induction of 5R1 may result not only in P4 catabolism but also in generation of bioactive 5-reduced pregnanolones or androgens. These steroids may affect the activity of uterine or cervical GABA receptors or other cell membrane proteins [21] to facilitate uterine contractions and cervical ripening. Further studies are necessary to test this hypothesis. Nevertheless, the increased expression of 5R1 in uterine tissues on Day 14 is insufficient to initiate parturition in the presence of continued P4 production by the corpus luteum. In the absence of 5R1, however, P4 withdrawal in blood alone is insufficient to induce parturition in mice [4]. Thus, it appears that both are necessary for successful labor and delivery.
In the present studies, both P4 and fetal occupancy were necessary to obtain maximal levels of 5R1 in uterine tissues. Treatment of castrated females with E2+P4 resulted in 8- to 10-fold increases in enzyme activity and a similar response in mRNA levels. Numerous studies have documented that, physiologically, estrogen is required to induce P4 receptor gene expression in P4-responsive tissues. Thus, sensitivity to P4 is indirectly under the control of estrogen.
The observations that estrogen plus P4 only partially induces uterine 5R1 expression in nonpregnant rats and that 5R1 is significantly decreased in the nongravid horn relative to the gravid horn of unilateral pregnant mice suggest that the systemic hormonal changes of late pregnancy are insufficient for maximal induction of 5R1 expression. Although it is possible that fetal or placental factors affect 5R1 expression in a paracrine manner, 5-reductase activity in uterine tissues adjacent to the placenta was lower than that in nonplacental uterine tissues. The massive distention of the uterus caused by the rapidly growing fetus provides one explanation for the marked increase in 5R1 activity during pregnancy.
Previous studies have established the importance of stretch in increasing transcription of certain genes believed to be important in the initiation of parturition [22–25]. During normal gestation, growth of the conceptus results in uterine distention. Uterine smooth muscle cells undergo cellular hypertrophy to accommodate the increase in uterine diameter. Endometrial epithelial cells, however, proliferate to line the increased lumenal surface area caused by fetal occupancy. Indeed, it was established as early as 1969 [8] that this endometrial hyperplastic response is mediated by uterine distention rather than by fetal-derived products. In unilateral pregnant mice on Day 18, the lumenal surface area is significantly increased in the gravid horn compared with the empty horn. Thus, increased gene expression of steroid 5R1 in the uterus during pregnancy likely is mediated not only by estrogen and P4 but also by stretch-induced increases in the size and number of endometrial epithelial cells, which are known to express 5R1 mRNA [4]. Although E2 treatment alone induced uterine enlargement and endometrial glandular proliferation, 5R1 enzyme activity was not increased. Thus, growth of endometrial epithelial cells in the absence of P4 was not sufficient to induce 5R1 gene expression.
During pregnancy, many proteins are regulated in opposing directions by estrogen and P4 in reproductive tissues. For example, oxytocin receptors [26], connexin 43 [27], cGMP-dependent protein kinase [28], and interstitial collagenase [29] are up-regulated by E2 and down-regulated by P4. In most species at term, P4 withdrawal, together with increasing levels of estrogen, leads to increased expression of these genes, thereby providing effective contractions of labor and cervical ripening. The induction of 5R1 at term by the combined actions of estrogen plus P4 plus uterine distention would, eventually, create an environment favorable for labor by removing the opposing actions of P4 on estrogen-dependent genes (i.e., connexin 43, oxytocin receptors, estrogen receptors, and cervical metalloproteases).
In summary, the mechanisms that lead to the coordinated expression of genes that relieve inhibition of myometrial contractility and promote cervical ripening during parturition have yet to be determined. The elucidation of the involvement of steroid 5R1 in parturition has increased our understanding of this process. The data reported herein indicate that 5R1 is under tight regulation by coordinated interactions between mechanical and hormonal factors. Regulation of 5R1 gene expression in the murine uterus is analogous to that of 5-reductase type 2 in the prostate. Both genes are regulated by their substrate hormones (P4 or testosterone). During gestation, 5R1 in uterine tissues is also regulated by fetal occupancy. The induction of 5R1 by both hormonal and mechanical signals at term provides an important tissue-specific branch point for the coordinated expression of several parturition-related genes.
ACKNOWLEDGMENTS
We thank Xiang Hong Li, Patrick Keller, and Jesse Smith for technical assistance. The generous gift of the murine 5R1 cDNA probe from Dr. David Russell and the insightful discussions with Dr. Mala Mahendroo and Dr. Stefan Andersson are acknowledged.
FOOTNOTES
First decision: 10 January 2001.
1 Supported by NIH P01-11149. D.M. was supported by the Ortho McNeil ACOG-Ortho Academic Training Fellowship in Obstetrics and Gynecology and NRSA 5-T32-HD07190. 
2 Correspondence: R. Ann Word, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9032. FAX: 214 648 8066; ruth.word{at}utsouthwestern.edu
Accepted: June 14, 2001.
Received: November 17, 2000.
REFERENCES
-reductase type 1 knockout mice is due to impaired cervical ripening. Mol Endocrinol 1999; 13:981-992-Reduced androgens play a key role in murine parturition. Mol Endocrinol 1996; 10:380-392[Abstract]-reductase: two genes/two enzymes. Annu Rev Biochem 1994; 63:25-61[Medline]-reductase isozyme expression. J Clin Invest 1993; 92:903-910-reductase 2 deficiency. Endocr Rev 1993; 14:577-593[Abstract]-reductase, an enzyme essential for male sexual differentiation. J Biol Chem 1989; 264:16249-16255-hydroxy-5-pregnan-20-one and carbachol in the control of neuronal excitability in hippocampal slices of female rats in defined phases of the estrus. Acta Physiol Scand 1998; 162:77-88[CrossRef][Medline]This article has been cited by other articles:
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  S. Andersson, D. Minjarez, N. P. Yost, and R. A. Word Estrogen and Progesterone Metabolism in the Cervix during Pregnancy and Parturition J. Clin. Endocrinol. Metab., June 1, 2008; 93(6): 2366 - 2374. [Abstract] [Full Text] [PDF]
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 J. P Wiebe Progesterone metabolites in breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 717 - 738. [Abstract] [Full Text] [PDF]
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 S. M. Yellon, A. M. Mackler, and M. A. Kirby The Role of Leukocyte Traffic and Activation in Parturition Reproductive Sciences, September 1, 2003; 10(6): 323 - 338. [Abstract] [PDF]
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