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BOR - Papers in Press, published online ahead of print March 9, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036954
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BIOLOGY OF REPRODUCTION 73, 214–221 (2005)
DOI: 10.1095/biolreprod.104.036954
© 2005 by the Society for the Study of Reproduction, Inc.

Androgens Induce Relaxation of Contractile Activity in Pregnant Human Myometrium at Term: A Nongenomic Action on L-Type Calcium Channels1

Mercedes Perusquía 2 3, Erika Navarrete 3, Jaime Jasso-Kamel 3, and Luis M. Montaño 4,5 

Departamento de Biología Celular y Fisiología,3 Instituto de Investigaciones Biomédicas Departamento de Farmacología,4 Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México D.F., México Instituto Nacional de Enfermedades Respiratorias,5 14080 México D.F., México


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has long been accepted that progesterone regulates uterine contractile activity. However, little is known about the role of androgens in female physiology, and their importance and biological function on myometrial contractility so far have received limited attention. In this work, we examined the direct effect of androgens on the contractile activity of the isolated human myometrium. Myometrial biopsies were obtained, with consent, from pregnant women undergoing elective cesarean section at term. Each androgen tested (dehydroepiandrosterone, testosterone, 5{alpha}- and 5ß-dihydrotestosterone, androsterone, or androstanediol) caused a concentration-dependent inhibition of spontaneous contractile activity; a relaxing effect of these androgens was also observed on the contractions induced by high potassium (KCl) solution. Interestingly, nonpregnant myometrium was also sensitive to androgen-induced relaxation. 5ß-Dihydrotestosterone (5ß-DHT) was dramatically more potent than the other androgens in inducing myometrial relaxation in all preparations. Relaxation response to androgens had very rapid time courses and was affected by neither the specific antiandrogen (flutamide) nor inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D), implying that androgens act through a nongenomic mechanism. Importantly, 5ß-DHT significantly reduced the increase in intracellular calcium concentration associated with exposure to KCl in human myometrial smooth-muscle cells loaded with Fura-2-AM. The blockade of L-type calcium channels seems to be involved in the nongenomic relaxing action of androgens. These observations demonstrate that androgens may play a crucial role in maintaining pregnancy.

female reproductive tract, mechanisms of hormone action, pregnancy, steroid hormones, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well documented that progesterone plays an essential role in reproductive events associated with establishment and maintenance of pregnancy. In this respect, it is widely accepted that progesterone elicits relaxation of uterine contractility and is the main basis of quiescence in myometrium during pregnancy. This action was described by Csapo [1] as the progesterone block. Recent studies have shown that some 5-reduced metabolites of progesterone are also inhibitors of contractile activity in isolated uteri of animals [2] and humans [3, 4], some with more major relaxing potency than that induced by progesterone [4].

In addition to the relaxing effect induced by progestins, a few studies have examined the potential relaxing effect induced by male sex steroids (androgens) in myometrial tissue. An early study reported an inhibitory effect induced by testosterone in the isolated rabbit uterus [5]. Since then, little attention has been paid to the potential uterine-relaxing effect of other androgens. Studies in nonpregnant rats have revealed that testosterone and its 5-reduced metabolites, as well as their precursor, 3ß-hydroxy-{Delta}5 (dehydroepiandrosterone; DHEA), cause concentration-dependent uterine relaxation [6, 7]. Likewise, DHEA [8] and testosterone and its 5ß-dihydro and 3{alpha},5{alpha}-tetrahydro metabolites also have the ability to cause relaxation of the uterine contractions induced by oxytocin, serotonin, acetylcholine, calcium, or potassium [2, 9, 10]. Analysis of these data indicates that 5ß-reduced testosterone (5ß-dihydrotestosterone; 5ß-DHT) and 3{alpha},5{alpha}-C19-steroids, such as androsterone and androstanediol, are markedly more potent relaxants than the {Delta}4,-3ketosteroids (progesterone or testosterone). Although the available data have suggested that androgens cause an important relaxing effect in rat uterus, their role in women has been poorly explored and their biological effects during pregnancy are still unclear. Moreover, to our knowledge, no study has determined an inhibitory effect of androgens on the contractile activity of human myometrium, particularly during pregnancy.

On the other hand, it has been reported that DHEA, DHEA sulfate, androstenedione, testosterone, and 5{alpha}-dihydrotestosterone (5{alpha}-DHT) are the major androgens synthesized in women [11]. Although androgen biosynthesis in the materno-fetoplacental unit is not well characterized, maternal plasma concentrations of testosterone and its precursor androstenedione are significantly higher during pregnancy than in the nonpregnant state, and their values increase throughout pregnancy (reviewed by McClamrock and Adashi [12]).

Taken together, these findings suggest that testosterone and its metabolites may have a functional role on human myometrial contractility. We wondered whether androgens also exert a progestin-like relaxing effect on human myometrial activity that could contribute to uterine contractile modulation. Therefore, the present study was designed to analyze the potential relaxing action of androgens (DHEA, testosterone, 5{alpha}-DHT, 5ß-DHT, androsterone, and androstanediol) on myometrial contractile activity in humans. Accordingly, we investigated the sensitivity of nonpregnant myometrium and pregnant myometrium at term to androgens, the androgen molecular structure-relaxing-response relationship, and the role of L-type Ca2+ channels on this process. The overall results allowed us to elucidate the possible involvement of genomic and nongenomic pathways in the androgens' mode of action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs and Chemicals

The following compounds were purchased from Sigma (St. Louis, MO): cholesterol (3ß-hydroxy-5-cholesten), dehydroepiandrosterone (DHEA; 3ß-hydroxy-5-androsten-17-one), testosterone (T; 17ß-hydroxy-4-androsten-3-one), 5{alpha}-dihydrotestosterone (5{alpha}-DHT; 17ß-hydroxy-5{alpha}-androstan-3-one), 5ß-dihydrotestosterone (5ß-DHT; 17ß-hydroxy-5ß-androstan-3-one), androsterone (3{alpha},5{alpha}-androsterone; 3{alpha}-hydroxy-5{alpha}-androstan-17-one), androstanediol (3{alpha},5{alpha}-androstanediol; 5{alpha}-androstane-3{alpha},17ß-diol), and progesterone (P; 4-pregnen-3,20-dione); the androgen receptor antagonist (flutamide), the protein synthesis inhibitor (cycloheximide), the transcription inhibitor (actinomycin D), cysteine, L-glutamine, penicillin-streptomycin solution, Fura-2-AM, and ionomycin were also purchased from Sigma. Hanks solution and Lebovitz L-15 medium were obtained from Gibco BRL (Rockville, MD). Collagenases and neutral proteases mixture were provided by Roche (Indianapolis, IN). Androgens, antiandrogen, protein synthesis, and transcription inhibitors were dissolved in absolute ethanol and ionomycin and Fura-2-AM were dissolved in dimethyl sulfoxide, and solvent (ethanol or dimethyl sulfoxide) concentration never exceeded 0.1% v/v. Actinomycin D was kept in the dark until use to avoid light-induced degradation.

Uterine Samples

Written informed consent was obtained for removal of myometrial tissue from women undergoing elective (nonlabor) cesarean section at term (38–40 wk of gestation). None of the pregnant women included in this study had underlying disease; the indication for cesarean section was breech presentation, placenta previa, or maternal request. Biopsy specimens were taken from the midline of the upper edge of the lower segment incision and were placed immediately in cold physiological salt solution (Hartmann; Baxter Laboratories). The present study was approved by the Ethics Committee of Woman's Hospital, Ministry of Health of Mexico (Department of Medical Teaching RE:597) and was performed in accordance with the Declaration of Helsinki. The samples were transported to the laboratory and kept under refrigeration (4°C) for 24 and 48 h.

Tension Measurement in Myometrial Strips

The tissue samples were transferred to Krebs-Henseleit solution with the following composition (mM): NaHCO3 (25), NaCl (119), KCl (4.6), KH2PO4 (1.2), MgSO4 (1.2), CaCl2 (1.5), and glucose (12) and gassed continuously with 95% O2 in 5% CO2 to maintain pH 7.4 and constant temperature (37°C). The connective tissue was carefully removed and the myometrium dissected in strips parallel to the muscle fiber bundles. Prepared strips were approximately 10 mm long, 5 mm wide, and 5 mm thick. To record isometric tension, each strip was placed vertically in a temperature-controlled (37°C) organ bath containing 10 ml Krebs-Henseleit solution. One end of the strip was attached to a fixed support at the bottom of the chamber and the other end was connected to an isometric force transducer (FTO3C; Grass Instruments, Quincy, MA) with the help of stainless-steel wires. The organ bath solution was continuously bubbled with 5% CO2 in O2 (pH 7.4). Passive resting tension of 10 mN (1.0 g) was adjusted throughout the experiments. Isometric tension was recorded by a polygraph (79; Grass Instruments) and the spontaneous contractile activity was stabilized for approximately 90 min, which was stable for more than 5 h in previous trials. The data were acquired and analyzed by using PolyView 2.1 (Grass Instruments Division/Astro-Med, Inc., West Warwick, RI) data-acquisition software.

After tissue stabilization in Krebs-Henseleit solution, a 30-min control time was recorded. Immediately afterward, the effect of noncumulative concentrations (3, 10, 30, or 100 µM; only a single concentration was added in each strip) of DHEA, testosterone or its 5-reduced metabolites, 5{alpha}-DHT or 5ß-DHT, androsterone, and androstanediol on spontaneous contractions were then determined. Limitation of the solubility of these compounds prevented further exploration of concentrations higher than 100 µM. The progesterone concentration at maternal intervillous blood space is 4.7 µM [13]. Thus, it is important to consider that steroids at 100 µM in our system seem to be close to the placental progesterone concentration. The action of each androgen was recorded for 30 min. Only one treatment was made in each experiment, which means that the concentration-response curves to the different androgens were determined independently in samples from different donors; thus, each n refers to one patient. In a separate group of experiments, myometrial contractility was determined after exposure to vehicle (ethanol) alone.

Collaterally, in some experiments under the same experimental conditions, the effects of noncumulative concentrations (3, 10, 30, and 100 µM) of testosterone or 5ß-DHT on the spontaneous contractility of nonpregnant myometrial strips were studied; these samples were obtained (with ethical approval and provision of written informed consent) after abdominal hysterectomy from 16 patients of reproductive age with no obvious pathology.

In some pregnant samples, the effect of androgens was also studied on the contraction induced by high potassium (40 mM KCl) solution after replacing normal Krebs-Henseleit solution with an equimolar substitution of 40 mM KCl and 84 mM NaCl. The KCl-induced contraction was repeated three times before treatment with androgen. Each KCl stimulus was recorded for 60 min and then the tissues were washed with Krebs-Henseleit solution (repolarized). When the tone reached the baseline, tissues were allowed to equilibrate for 30 min and then 40 mM KCl was added. After a stable contractile tension was attained (~30 min), 100 µM of each androgen (highest concentration used; HC) was added, and the effect of each androgen was recorded, separately, for 30 min. Finally, the tissues were washed and a last contraction induced by KCl was observed for 60 min to check tissue recovery. Additionally, the effect elicited by each androgen was compared with a negative control, such as cholesterol, because no evidence of relaxation of a KCl-induced contraction was observed when this steroid was tested at 100 µM concentration.

In a series of experiments, the involvement of androgen receptor (AR) in the acute uterine relaxation to testosterone and its 5-reduced dihydro metabolites (5{alpha}- and 5ß-DHT) was examined on KCl-induced contraction pretreated with 10 µM flutamide (an AR antagonist) 30 min before observing the relaxing effect at 100 µM for 30 min. The comparison was made with the percent of inhibition induced by each androgen without any pretreatment. In other experiments, the myometrial tissues were incubated with the protein synthesis inhibitor cycloheximide (40 µM) or the transcription inhibitor actinomycin D (10 µM) for 30 min to determine the KCl-induced contraction. Testosterone or its dihydro metabolites (5{alpha}- and 5ß-DHT) were then added separately (at 100 µM each) and their effects (evaluated for a 30-min period) were compared in the presence and absence of those inhibitors. It is important to highlight that the concentrations of inhibitors employed in the present schedule are sufficient to abolish protein synthesis [14], transcription [15], and androgenic action at the receptor level [16]. In a separate group of experiments, the KCl-induced contraction was also exposed to progesterone at 100 µM; its effect was evaluated for a 30-min period and compared with the relaxing effect induced by each androgen at 100 µM. To examine the potential blocking effect of progesterone on androgen-induced relaxation, the effect of testosterone, 5{alpha}- or 5ß-DHT (each androgen separately added at 100 µM), was also studied on the KCl contraction pretreated, for 30 min, with 100 µM progesterone after addition of androgen. The androgen response was evaluated for 30 min and compared when progesterone was not present. In the same way, the possible blocking action of androgen on progesterone-induced relaxation was also analyzed; thus, the effect induced by progesterone alone was previously evaluated for 60 min and compared when the tissues were incubated for 60 min with 100 µM progesterone (for 30 min) and then 100 µM androgen (for 30 min). Finally, after all of the above experiments had been concluded, the tissues were washed out and a last contraction induced by KCl was observed for 60 min to check the tissue recovery.

Measurement of Intracellular Ca2+ in Human Myometrial Smooth Muscle Cells

Some samples of pregnant myometrium were carefully dissected of connective tissue; approximately 300 mg smooth muscle was minced, placed in 5 ml Hanks solution containing 2 mg cysteine and 0.05 U/ml papaine, and incubated for 10 min at 37°C. Tissue was washed with modified L-15 medium (containing 90% Leibovitz L-15 medium, 10% fetal calf serum, 2 mM L-glutamine, 15 mM glucose, 10 U/ml penicillin, 10 µg/ml streptomycin) to remove excess enzyme and then was put in nominally Ca2+-free solution containing 0.144 mg/ml of a highly purified collagenase and neutral proteases. After 10 min, the preparation was triturated by using a long-neck Pasteur pipette until single human myometrial smooth-muscle cells (HMSMC) were observed. The solution containing the dispersed cells was centrifuged, the supernatant was removed and discarded, and cells were resuspended in 5 ml of modified L-15. This last procedure was repeated. Then cells were loaded with 0.5 µM Fura-2-AM in low Ca2+ (0.02 mM) at room temperature (22–25°C). After 1 h, cells were allowed to settle into a 1.5-ml heated perfusion chamber with a glass cover in the bottom. This chamber was mounted on a Nikon inverted microscope (Diaphot 200; Tokyo, Japan) and cells adhered to the glass were continuously perfused at a rate of 2–2.5 ml/min with Krebs solution (37°C, equilibrated with 5% CO2 in O2, pH 7.4) containing 1.5 mM Ca2+.

HMSMC loaded with Fura-2-AM were exposed to alternating pulses of 340- and 380-nm excitation light, and emission light was collected at 510 nm using a microphotometer (Photon Technology International, Princeton, NJ). Background fluorescence was automatically subtracted and determined by removing the cell from the field before the experiments were started. The fluorescence acquisition rate was 0.5/sec. Intracellular Ca2+ concentration ([Ca2+]i) was calculated according to the formula of Grynkiewicz et al. [17]. The Kd of Fura-2-AM was assumed to be 224 nM. The mean 340/380 fluorescence ratios, Rmax and Rmin, were obtained by exposing the cells to 10 mM Ca2+ in the presence of 10 µM ionomycin and in Ca2+-free Krebs with 10 mM EGTA, respectively. Rmax was 6.06 and Rmin 0.39. The fluorescence ratio at 380-nm light excitation in Ca2+-free medium and Ca2+-saturated cells (ß) was 4.23. Recordings were stored in a microcomputer and analyzed using data acquisition and analysis software (Felix v1.21, PTI).

To evaluate the role of L-type Ca2+ channels in the relaxation induced by 5ß-DHT, single HMSMC were stimulated with 60 mM KCl (equimolar) and the response was considered as control. Afterward, cells were perfused 15 min with normal Krebs-Henseleit solution. Immediately, cells were incubated for 5 min, with different noncumulative concentrations of 5ß-DHT (100, 310, and 1000 nM) and stimulated again with 60 mM KCl for each concentration. Finally, cells were washed with Krebs-Henseleit solution and stimulated with 60 mM KCl to check their recovery.

Data Presentation and Statistical Analysis

Each experiment was performed on myometrial strips prepared from different patients. All data in the text and figures are expressed as mean ± SEM (n ≥ 6, where n = 1 represents one patient). The concentration for each substance is expressed as a final concentration in the organ bath. The effect of androgens on spontaneous or KCl-induced contraction was evaluated by comparing experimental responses with the control ones (set at 100%). The potency of each androgen was evaluated through inhibitory concentration 50 (IC50 = value for androgen concentration required to inhibit 50% of uterine contraction from the control). The IC50 was calculated by straight-line regression from every noncumulative concentration response curve. All values above 100 µM were marked as >100 µM and IC50 was expressed as median (range). To compare IC50 between groups, we used a nonparametric test, the two-sample Mann-Whitney rank sum test with Bonferroni correction using DHEA as the control group. Nonpaired Student t-test was used to compare the responses between two groups. For multiple comparisons, we used one-way ANOVA followed by Tukey test. In the experiments with single cells, we used paired Student t-tests. The accepted level of significance was P < 0.05 bimarginal.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Androgens on Spontaneous Myometrial Contractility

DHEA and testosterone and its 5-reduced metabolites (5{alpha}-DHT, 5ß-DHT, androsterone, and androstanediol) in the concentration range of 3.0–100 µM inhibited the spontaneous contractions of pregnant uteri at term in a concentration-dependent manner. The relaxing effect of androgens, at all concentrations tested, was significantly different (P < 0.001) when compared with that produced by 0.1% ethanol, (17.14 mM; a concentration identical to those used as solvent for androgen; this vehicle control relaxed no more than 1.8% ± 0.4%, n = 6).

All androgens tested induced a concentration-dependent relaxation. 5ß-DHT elicited the major inhibition on the spontaneous contractility in pregnant myometrium (Fig. 1A). As shown in Figure 2, the differences in IC50 and HC values obtained with different androgens were statistically significant for 5ß-DHT (P < 0.05 and P < 0.01, respectively) when compared with DHEA, while 5{alpha}-DHT and androstanediol were only different at the HC (P < 0.05 and P < 0.01, respectively). In addition, the analysis of covariance showed statistical differences between 5ß-DHT and androstanediol (P < 0.01; Fig. 2).



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  		FIG. 1. Concentration-response curves to androgens on spontaneous contractile activity of pregnant (A) and nonpregnant (B) human myometrium. Testosterone and 5ß-DHT curves in both pregnant and nonpregnant myometrium were not significantly different. Each point represents the mean ± SEM; n ≥ 6



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  		FIG. 2. Relaxing effect induced by androgens on spontaneous contractility of pregnant human myometrium at term. The values are mean (n = 6) ± SEM. The half-maximal relaxation (IC50) was calculated by straight-line regression from every noncumulative concentration-response curve. All values above 100 µM were marked as >100 µM and IC50 was expressed as median (range). Pearson's correlation coefficient (r) represents the fitness of the straight line. The highest concentration tested (HC) was 100 µM. a, The potency was calculated from IC50 by the formula: IC50 DHEA/IC50 metabolite, assuming a value of 1.0 to DHEA (precursor). * and {dagger}, P < 0.05; *, P < 0.01 compared with DHEA group

With respect to the IC50 and HC values, the order of potency was 5ß-DHT >> androsterone = DHEA = testosterone > 5{alpha}-DHT = androstanediol. Clearly, the data showed that 5ß-DHT was significantly more potent than its precursors, DHEA and testosterone, in inducing myometrial relaxation. In contrast, androstanediol had very little relaxing effect due to the fact that the contraction amplitude decreased and the frequency was slightly increased. The inhibitory effect of androgens was observed within 1 min after the uterine tissue was exposed to each androgen (Fig. 3A, left) and the spontaneous contractility was reversed after androgen was removed (washed out) from the tissue (data not shown).



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  		FIG. 3. A) Typical recordings of the contractile activity of isolated human myometrium at term. Relaxing effect of 5ß-DHT at 100 µM on the spontaneous contractility (left panel) and on the contraction induced by KCl (right panel); the solid black line indicates the incubation time with androgen; note the immediate relaxing effect of androgen. B) Relaxing effect induced by dehydroepiandrosterone (DHEA), testosterone (T), 5{alpha}-dihydrotestosterone (5{alpha}-DHT), 5ß-dihydrotestosterone (5ß-DHT), androsterone (3{alpha},5{alpha}-one), and androstanediol (3{alpha},5{alpha}-diol) on spontaneous and KCl-induced contraction in pregnant myometrium at term, when they were tested equimolarly at 100 µM. Statistical significances: *, P < 0.05, **, P < 0.005 as compared with spontaneous contraction. Androgen-induced relaxation on KCl contraction shows that 5ß-DHT (b) was different from all androgens (a, c, and d; P < 0.01); DHEA and 3{alpha},5{alpha}-one (a) were different from T (c; P < 0.01), 5{alpha}-DHT (d; P < 0.01), and 3{alpha},5{alpha}-diol (c; P < 0.01 and P < 0.05, respectively); and 3{alpha},5{alpha}-diol (c) is different from 5{alpha}-DHT (d; P < 0.05). Each bar represents the mean ± SEM; n = 6

To study the sensitivity of pregnant and nonpregnant uterus to androgen-induced myometrial relaxation, 5ß-DHT (which turned out to be the most potent relaxing androgen in pregnant uterus) and its precursor testosterone were tested on myometrial spontaneous contractility of nonpregnant women. As shown in Figure 1B, 5ß-DHT and testosterone also induced a rapid relaxing effect on nonpregnant myometrium, which was concentration dependent, and their IC50 values were 49.7 (44.7–53.3) and 96.0 (87.7–>100) µM, respectively. These curves (Fig. 1B) did not significantly differ from those obtained in pregnant uterus (Fig. 1A). After washout, the inhibitory effect was reversible, as is the case for their relaxing effect on spontaneous contractility of pregnant uterus (data not shown). Again, the relaxing effect of 5ß-DHT was dramatically more potent than that induced by testosterone (Fig. 1B). The relaxation induced by both 5ß-DHT and testosterone was significantly different (P < 0.001) vs. the vehicle control, 0.1% ethanol, which relaxed 1.2% ± 0.4%, n = 6.



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  		FIG. 5. Effect of 5ß-dihydrotestosterone (5ß-DHT) on the [Ca2+]i increase induced by 60 mM KCl in human myometrial smooth-muscle cells. A) Original recordings of [Ca2+]i changes induced by 60 mM KCl; the first trace was performed without androgen solvent (ethanol) and the remaining responses were in the presence of 0.1% ethanol (upper panel). The KCl response was notably reduced by the addition of 5ß-DHT (lower panel). The solid black line indicates the exposure time to KCl and the white line indicates the time for 5ß-DHT incubation at different concentrations. B) This effect was concentration dependent; statistical significance: *, P < 0.05; **, P < 0.01. Each bar represents the mean + SEM; n = 4

Effect of Androgens on KCl-Induced Myometrial Contraction

Addition of 40 mM KCl to the pregnant myometrial tissue caused a tonic contraction. Under these conditions, KCl-induced contraction was also inhibited by each androgen (DHEA, testosterone, 5{alpha}-DHT, 5ß-DHT, androsterone, or androstanediol) when they were tested equimolarly at 100 µM, with different efficacy over the first 30 min (see Fig. 3B); however, 5ß-DHT caused complete relaxation within ~40 min of its addition. The development of the relaxing effect in precontracted tissues started within a few seconds (~35 sec) after addition of each androgen; the tone of contractions decreased with time (Fig. 3A, right); after washout, the amplitude and tone of the next KCl-induced contraction was totally recovered (data not shown), and thus, the androgen action disappeared. As shown in Figure 3B, DHEA, androsterone, and androstanediol were more effective in relaxing the KCl-induced contraction than the spontaneous contraction, and the relaxing efficacy of 5ß-DHT was higher than that induced by the remaining androgens in both responses. The vehicle used with the androgens (0.1% ethanol) did not significantly affect (1.9% ± 0.9% of relaxation, n = 6, P > 0.05) the tone of KCl contraction, but the effect induced by each androgen tested was significantly different from the vehicle control (P < 0.001). Likewise, cholesterol (100 µM) did not relax the KCl-induced contraction and, therefore, was used as the negative control group. Thus, the effect induced by each androgen was significantly different from the effect of cholesterol (P < 0.001). The stable tension produced by KCl without treatment in the same tissue was used as control for statistical comparisons.

Furthermore, 40 µM cycloheximide or 10 µM actinomycin D did not block the relaxing effect induced by androgens at 100 µM on KCl-induced contraction in pregnant myometrium at term. Similarly, pretreatment with 10 µM flutamide had no effect on androgen-induced myometrial relaxation (Fig. 4). Progesterone at 100 µM elicited 25.6% ± 0.2% of relaxation (n = 6) over the first 30 min, which was significantly less (P < 0.05) than the percentage of relaxation induced by each androgen (Fig. 3B) at the same concentration (100 µM) and period (30 min). We also observed that addition of 100 µM progesterone did not block the relaxing effect induced by each androgen at 100 µM (Fig. 4). On the other hand, the effect induced by 100 µM progesterone over a 60-min period was 46.5% ± 0.5% of relaxation (n = 6) and was not blocked by testosterone, 5{alpha}-DHT, or 5ß-DHT (each one at 100 µM). On the contrary, progesterone-induced relaxation was significantly enhanced by adding testosterone (61.1% ± 2.5%; P < 0.05), 5{alpha}-DHT (65.1% ± 2.1%; P < 0.005), or 5ß-DHT (89.9% ± 0.7%; P < 0.0005).



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  		FIG. 4. Comparison of relaxing effect at 100 µM of testosterone, 5{alpha}-dihydrotestosterone (5{alpha}-DHT), or 5ß-dihydrotestosterone (5ß-DHT) on KCl-induced contraction in human pregnant myometrium at term, and in combination with 10 µM actinomycin D, 40 µM cycloheximide, 10 µM flutamide, or 100 µM progesterone. The effect of each androgen was not modified by the different treatments. Each bar represents the mean ± SEM; n = 6

5ß-DHT Inhibited the KCl-Induced Intracellular Ca2+ Concentration Increment in HMSMC

Stimulation of HMSMC with 60 mM KCl induced an increment of [Ca2+]i ({Delta} 211 ± 58 nM). Successive KCl stimulations produced similar responses (Fig. 5A). Incubation of the cells with 5ß-DHT (310, 1000 nM) produced a significant reduction (n = 4; P < 0.05 and P < 0.01, respectively) of the KCl-induced [Ca2+]i increase, and this reduction was higher at 1000 nM (~90%; Fig. 5, A and B). This effect on the KCl response was reversed when 5ß-DHT was removed from the cells. Addition of ethanol (0.1%) did not modify the KCl responses (Fig. 5A, upper panel).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that a wide series of androgens are uteroactive agents by inducing relaxation in pregnant and nonpregnant human myometrium. This effect is consistent with previous reports in the isolated rat uterus, which show that the same androgens are also inhibitors of the myometrial contractility (reviewed by Perusquía [2]). These findings provide evidence that androgens may have a biological role in the regulation of uterine contractility, establishing a progestinlike uterine-relaxing effect.

No difference was found in sensitivity to androgen between pregnant and nonpregnant myometrium, suggesting that the hormonal status in vivo might not be an important factor for the relaxing effect induced by androgens. In contrast, in a previous work, we observed that nonpregnant myometrium in vitro was significantly more sensitive to progesterone-induced relaxation [4]. The explanation might be that the potency of progesterone is reduced in the pregnant uterus at or near term, probably to control the onset of labor, but the reason for such differences is unclear.

It is important to emphasize that both nonpregnant and pregnant human myometrium are targets of androgens' relaxing action. Additionally, they are also capable of relaxing the KCl-induced contractions of pregnant myometrium at term. Myometrial relaxation induced by androgens was rapid and reversible after washouts, suggesting a nongenomic (membrane) mechanism rather than the classical steroid modulation of nuclear transcription. This proposal is strengthened by our findings that the relaxation response to androgens was not modified by AR antagonist or inhibitors of protein synthesis and transcription. With this background and, to the best of our knowledge, the present study reports, for the first time, a nongenomic relaxing action of androgens on human myometrial contractility, an effect that may contribute to the maintenance of pregnancy by inducing myometrial quiescence.

On the other hand, a rapid relaxation effect induced by androgens in epidydymis and seminal vesicles has also been reported [18]. Likewise, androgens also induce relaxation in nonreproductive smooth muscles, such as those from the gastrointestinal tract [19] and blood vessels [20– 22]. Interestingly, the vasorelaxing effect of androgens is correlated with the fact that 5-reduced androgens may elicit a vasodepressor response in vivo [23]. Consequently, androgen-induced relaxation is a generalized phenomenon on smooth-muscle contractility. In this context, we have confirmed that cholesterol is unable to induce myometrial relaxation, implying a particular relaxing effect of C19-steroids in smooth muscle.

The androgens tested in this study showed different potency to induce myometrial relaxation (Fig. 2). The major relaxing effect was induced by 5ß-DHT, while DHEA, androsterone, and testosterone were less active and equipotent among themselves, and 5{alpha}-DHT and androstanediol presented only a slight efficacy. Therefore, the order of potency observed deserves further consideration. In the first instance, the high relaxing potency of 5ß-DHT correlated with its strong vasorelaxant and vasodepressor effect [23– 25]. This 5ß-reduced androgen has also been the most potent steroid inducing relaxation in the guinea pig ileum [19] and on the contraction induced by different agents in the rat uterus [2, 20]. In line with this evidence, our results obtained with 5ß-DHT demonstrate that its potency is even more powerful than progesterone and some 5-reduced progestins in the human pregnant myometrium at term [4]. Even further, all androgens were more efficacious relaxants than progesterone on KCl-induced contraction as well as when their effect on spontaneous contraction was compared with progesterone in our previous results [4]. It is pertinent to note that 5ß-DHT, which has little or no affinity to intracellular AR and is totally devoid of androgenic properties [26], is several times more potent in inducing relaxation than is its 5{alpha}-epimer (5{alpha}-DHT), which has the best affinity to AR and, hence, with a high androgenic activity [26]. On this basis, it is tempting to suggest that the two dihydrometabolites of testosterone elicit different biological responses: 5{alpha}-DHT with high genomic-androgenic action and 5ß-DHT with high nongenomic-relaxing action. Accordingly, the acute relaxing effect of 5ß-DHT most likely is mediated via an AR-independent mechanism, and this line of evidence unequivocally demonstrates that the marked relaxing effect of 5ß-DHT is acting at a nongenomic level.

We also noted that the different structural conformation of each androgen could be important for inducing different relaxing potency. The change from {Delta}5,3ß-hydroxy structure of DHEA into {Delta}4,3-keto structure of testosterone did not modify the potency. However, it is important to consider the different structural conformation of the A-ring reduction of testosterone: in 5{alpha}-DHT, the C5 hydrogen is {alpha}-oriented and A/B-ring fusion is in trans, whereas, this hydrogen is ß-oriented in the 5ß-DHT and the A/B-ring fusion is in cis. In this context, we observed that the 5{alpha}/trans configuration was several times less potent than the 5ß/cis, configuration; particularly 5{alpha}-DHT and its subsequence 3{alpha}-hydroxylation (such as androstanediol), which had only a weak potency. Interestingly, the inclusion of a 17-keto group (such as androsterone) changes this pattern and makes the trans conformation (3{alpha}-hydroxy-5{alpha}) a little more effective. In summary, in the {Delta}5,3ß-hydroxy and {Delta}4,3-keto structure, and the trans ring fusion, the A-ring is planar with the steroid nucleus, but the A-ring bends 90° relative to the steroid nucleus when the fusion is in cis (see structures in Fig. 2). Therefore, the dramatic structural change of ß/cis configuration at C5 is a determining factor for an optimal myometrial relaxation. In this sense, it is likely that 5ß progestins are also potent relaxants of the human and rat myometrium [2–4]. This chemical structure-relaxing effect relationship has also been observed in rat uterus and vascular smooth muscle [2, 20], as well as on central nervous system excitability [27, 28]. Remarkably, it has been postulated that 5ß-DHT is the best steroid to induce a nongenomic-relaxing effect in smooth muscle; thus, this opens an opportunity for studying the role of 5ß metabolites as a new type of active steroid.

As previously implied, the uterine-relaxing properties of androgens can be explained by a nongenomic action; however, little is known about this issue. We have reported that androgens and progestins inhibited Ca2+-induced contractions in rat uterus depolarized by high KCl-Ca2+-free solution [9]. These results indicated that progestins and androgens inhibited contractile responses of the rat uterus to Ca2+ by blocking the voltage-operated Ca2+ channels (VOCCs).

The present study revealed that androgens also produce relaxation of the KCl-induced contraction of the human pregnant uteri. The simplest interpretation of the above findings is that androgen-induced uterine relaxation may involve a diminution of the Ca2+ influx to the myometrial smooth-muscle cells. Furthermore, KCl-induced contraction was completely inhibited by 5ß-DHT, which had a more sensitive relaxing effect than DHEA, androsterone, and androstanediol, implying a reduction of extracellular Ca2+ influx and also suggesting a preferential antagonism on VOCCs. In line with this proposal, we found that 5ß-DHT, at nanomolar concentrations (310 and 1000 nM), significantly reduced Ca2+ influx induced by KCl in uterine myocytes. This last observation provides strong evidence that, during the 5ß-DHT-induced myometrial relaxation, an antagonistic action upon L-type VOCCs is involved. However, androgens and progestins are capable of relaxing the contractile responses induced by several agonists in the rat uterus, for example: oxytocin, serotonin, acetylcholine, and prostaglandins [2], which suggests that sex steroids hormones are efficacious in blocking receptor-operated Ca2+ channels (ROCCs) and may indeed also have affinity for this type of calcium channel. This antagonistic action on VOCCs and ROCCs has also been reported for progesterone-and estrogen-induced relaxation on the contraction of human myometrium and intramyometrial arteries [29] and androgen-induced vasorelaxation [20, 21, 25, 30].

Regarding electrophysiological studies that examine Ca2+ antagonistic action of steroids in smooth-muscle cell, only one study shows that estrogens inhibit the Ca2+ currents from L-type VOCCs in myometrial cells from pregnant rats [31]. In vascular myocytes, 17ß-estradiol [32, 33], progesterone [34, 35], and testosterone [36] antagonize L-type VOCCs, decreasing Ca2+ currents and sarcoplasmic reticulum Ca2+ uptake in response to depolarizing stimuli. Another mechanism by which steroids may promote relaxation of smooth-muscle cells is membrane hyperpolarization as a consequence of K+ channel activation, and it has been suggested that this mechanism contributes, at least in part, to the relaxing effect of progesterone in pregnant human myometrial cells [37]. It is important to emphasize that this last finding was done under special experimental conditions because cells were incubated with high progesterone concentration (100 µM) for a long period (24 h), and thus, the effect can be considered genomic in origin.

On the other hand, the myometrial relaxation elicited by androgens suggests a possible interaction with ß2 adrenoceptors or GABAA receptors; however, this possibility appears not to be supported because their respective antagonists, propranolol and picrotoxin or bicuculline, did not block the relaxing effect of sex steroids in isolated rat myometrium [9, 10]. Thus, the available data suggest that the myometrial relaxation of steroids seems to be due to inhibition of Ca2+ influx through VOCCs and/or ROCCs. We concede that the different androgens could be interacting with several membrane proteins (ion channels and/or some membrane receptors) because these kinds of steroids also inhibit contractions elicited by different uterotonic agents [2]; in this respect, we cannot rule out that this relaxing effect may reflect changes in membrane physicochemical properties, altering its fluidity nonspecifically. Therefore, future systematic studies will have to be performed to define the nongenomic mechanism of myometrial relaxation that each steroid induces.

Even though the concentrations of androgens tested in isolated myometrial tissue were much higher than the reported plasma concentration of DHEA (4.2 ng/ml; 14.5 nM), testosterone (4.5 ng/ml; 15.6 nM), and 5{alpha}-DHT (0.1 ng/ml; 0.3 nM) in pregnant women at term [38, 39], in many instances, steroid concentrations required to elicit the nongenomic effects are in the micromolar (supraphysiologic) concentration range. Nevertheless, in single myometrial cells, we observed that the KCl-induced [Ca2+]i increase was diminished when nanomolar concentrations (310 to 1000 nM) of 5ß-DHT were used. This preparation may require much lower concentration of the androgen due to the fact that it was directly applied on a single myometrial cell. Additionally, in single cells, we are exclusively evaluating the effect of androgen on L-type VOCCs, while in the isolated-tissue preparation, the relaxing response is done through multiple cellular mechanisms and the androgen needs to cross some barriers in the serosa layer before it may act on myometrial cells. However, it is pertinent to point out that these androgens, at the micromolar concentration range employed in isolated tissues, are more effective uterine relaxants than Ritodrine, a ß2-adrenergic agonist widely used in obstetric practice for the prevention of premature labor. This compound, the sole FDA-approved agent for this use in the United States, requires a much higher concentration (1 mM) to achieve only 45% as much relaxation of myometrial contractile activity in pregnant women [40].

Further research is urgently needed to determine the range of normal 5ß-C19 steroid values in pregnant women. Although the origin of the androgen increase during pregnancy remains uncertain, the androgens are probably produced mainly by the maternal ovaries and less probably by the placenta. It has also been reported that androstenedione is metabolized by human uterine tissue to its 5{alpha}-reduced metabolites androstanedione and androsterone, as well as to testosterone, 5{alpha}-DHT, and androstanediol [41]. Furthermore, in the early 1960s, Benagiano and colleagues [42] reported that several fetal tissues have the ability to use testosterone and to convert into ring-A products; particularly, 5ß-reduced androgens (such as 5ß-DHT) are formed in the fetal liver. Therefore, all androgens tested in our study are produced in the materno-fetoplacental unit.

The role of androgens in female physiology is a topic of growing interest. Notably, the present data show that androgens possess a propregnancy property, the promotion of uterine quiescence during pregnancy, a new aspect in the regulation of functional processes in uterine smooth-muscle contractile activity. This action excludes the old concept that DHEA and/or testosterone might play a role as prohormones for other steroids (e.g., estrogens) and also that the 5ß-reduced metabolites of testosterone are just hormonally inactive excretion products. Consequently, androgens are biologically active on uterine contractile response. In addition, progesterone-induced myometrial relaxation was not blocked by androgens; on the contrary, both progesterone and androgens induced a greater relaxation. With this background, it seems reasonable to postulate that myometrial contractility might be modulated by progestins [4] accompanied by those androgens.

Interestingly, it has been reported that, during the first trimester of pregnancy, women with total testosterone plasma levels equivalent to those of nonpregnant women had pregnancies that subsequently terminated in abortion [38]. In this situation, female androgen insufficiency would not activate enough myometrial relaxation to prevent uterotonic responses.

Androgen replacement in pregnant women, particularly with androgenic components, should be carefully considered because of the additional risk that virilization of a female fetus may occur. However, 5ß-DHT is a potential component of androgen replacement therapy because this androgen induces nongenomic effects, such as myometrial relaxation, and is devoid of androgenic actions and is a nonaromatizable androgen without estrogenic side effects.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Hugo Bustos and the labor ward staff at ABC Hospital, Mexico, for providing the myometrial tissues for this study and Irma Rodríguez, Patricia López, and Joel García for their help with the collection of samples. We wish to acknowledge the diligent technical assistance of Edgar Flores during the performance of the intracellular Ca2+ recordings described in this report. This paper contains part of the M.Sc. Thesis of David Barrera.


   FOOTNOTES
 
1 Supported by the Dirección General de Apoyo al Personal Académico, Universidad Nacional Autónoma de México (PAPIIT; grant IN221102-2). Back

2 Correspondence: Mercedes Perusquía, Apartado Postal 70-492, CP 04511, México D.F., México. FAX: 5255 5622 3897; perusqui{at}servidor.unam.mx Back

Received: 6 October 2004.

First decision: 18 November 2004.

Accepted: 7 March 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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