TY - JOUR
AU - Norman, Jane, E.
AB - Abstract Context: Uterine quiescence must be maintained until pregnancy reaches term. Premature activation of myometrial contractility leads to preterm labor and delivery. Objective: To scrutinize the potential of androgens to relax the myometrium and the mechanism of their action. Samples: A pregnancy-derived myometrial smooth muscle cell line (PHM1-41) and myometrial strips prepared from tissues obtained from pregnant women (lean, n = 9; obese, n = 6) undergoing elective cesarean section at term and from nonpregnant C57BL/6 mice (n=5) were each utilized. Design: The contraction of collagen-embedded PHM1–41s and the stretch-induced contraction of human and murine myometrial strips were assessed after incubation with Testosterone (T), dihydrotestosterone (DHT), and T conjugated to BSA. Intracellular calcium ([Ca2+]) and phosphorylated myosin light chain concentrations were quantified in PHM1–41s using a Fluo-4 Ca2+ assay and in-cell Westerns, respectively. Setting: University research institute. Results: DHT and T, but not T conjugated to BSA, impaired the contractile function of PHM1–41s and of human and murine myometrial strips. The response was rapid (observed within minutes), was sustainable for up to 48 hours, and was not abolished on knockdown of the androgen receptor. DHT (100 μm) reduced the amplitude of lean strip contraction to 2 ± 2% of the pretreatment value and T (100 μm) to 3.3 ± 1%. These values for obese strips were 15 ± 6.7% and 11± 6.7%, respectively. At the same doses, in murine strips, DHT reduced the amplitude to 4.8 ± 3% and T to 4.9 ± 3%. DHT (50 μm) pretreatment reduced the oxytocin-stimulated increase in [Ca2+] (P < .0001; n = 6) and phosphorylated myosin light chain (P < .05; n = 5) in PHM1–41s. Conclusion: Lipid-soluble androgens could be developed as tocolytic agents for the treatment of preterm labor. Preterm birth (PTB), defined as birth before 37 weeks of pregnancy, accounts for 5–18% of all recorded births worldwide (1). Importantly, PTB is associated with long-term neurodevelopmental outcomes and an increased risk for respiratory and gastrointestinal complications in the offspring (2). The major obstetric precursor leading to PTB is spontaneous preterm labor, the outcome of preterm onset of regular myometrial contractions. The first-line management of threatened PTB is initiation of tocolytic medications to suppress these contractions. Their mode of action is gene transcription-independent and involves rapid inhibition of key components in the contraction cascade, for example, the oxytocin (OXT) receptor (OXTR) and the various calcium (Ca2+) channels (3). Tocolytics reduce the availability of intracellular Ca2+ ([Ca2+]) and prevent the phosphorylation of myosin light chain (MLC) and, thereby, the synchronized contraction of the myometrium. The currently used short-term tocolytic agents, such as nifedipine, an L-type Ca2+ channel blocker, and OXTR antagonists, have high tocolytic efficacy in the short term, but their lack of longer-term effect limits their effect on perinatal mortality (4, 5). Conversely, magnesium sulfate, an inhibitor of MLC phosphorylation that is the most commonly used tocolytic in the United States, is associated with maternal side effects and has low tocolytic efficacy (6). Steroid hormones are currently the focus of much interest for PTB treatment and prevention. Prophylactic administration of vaginal progesterone (P) to pregnant women at high risk has been shown to reduce the rate of PTB by 50% (7). Our research group has previously demonstrated that exposure of spontaneously contracting myometrial strips to P resulted in a rapid (<30 minutes) reduction in the amplitude and integral of contraction, in line with the well-established role of P in the maintenance of pregnancy (8). In addition to P, one study reported that androgens in micromolar doses also relaxed human myometrial contractions ex vivo (9). We have recently reviewed all the evidence for a role of androgens in maintenance of pregnancy (10). Considering that: 1) tocolytics in current use delay delivery only by 24 hours to 7 days; 2) P supplementation prevents only one-third of all recurrent PTBs; and 3) androgens produced by the placenta could be involved in the maintenance of pregnancy, we hypothesized that androgens should be investigated as novel PTB therapeutic agents. However, there is limited evidence on the efficacy of androgens, and the mechanism of action of androgens in preventing uterine contractions is poorly understood. Herein, we sought to address the effects of androgens on myometrial contractions and explore how they interact with the contractile apparatus. Specifically, we aimed to: 1) deduce whether T, dihydrotestosterone (DHT; nonaromatizable metabolite of T), and the cell-surface impermeable T (T conjugated to BSA [TBSA]) inhibit the contraction of uterine myocytes in vitro and ex vivo in both human and mouse; and 2) test the hypothesis that androgens prevent uterine contractions via reduction in the concentration of [Ca2+] and, hence, reduction in the phosphorylation of MLC. Materials and Methods Human tissue Biopsies were obtained from the upper margin of the lower segment of myometrium from women undergoing elective cesarean section as previously described (11) at the Simpson's Centre for Reproductive Health at the Royal Infirmary of Edinburgh, following informed written consent. Ethics approval for recruitment of all pregnant women was granted by the West of Scotland Research Ethics Committee 4 (09/S0704/3) to the Edinburgh Reproductive Tissue BioBank. Biopsies were collected from lean (LN; 19 < body mass index <25 kg/m2) and obese (OB; body mass index >25 kg/m2) women delivering at term (>37 weeks gestation) before the onset of labor. Patients with twin pregnancies and pregnancy complications were excluded. The recovered biopsies were collected in ice-cold RPMI 1640 medium (Gibco), rinsed in PBS, and dissected into 2 × 2 × 15-mm strips parallel to the muscle fiber bundles. Mouse tissue Experimental procedures were licensed (PPL 60/4241; PIL 60/13875) under the UK Home Office Animals (Scientific Procedures) Act (1986). Murine uterine horns were harvested from 8-week-old nonpregnant C57BL/6 mice supplied by Charles River (London, UK) and prepared into uterine strips (1 cm long each). Human uterine myocytes Pregnant human myometrial 1–41 (PHM1–41) cells were obtained from a single late-term pregnant donor as previously described (12). PHM1–41s were cultured as detailed elsewhere (13, 14), with the exception that we used phenol red-free high-glucose DMEM (Lonza). A PHM1–41 cell line in which the androgen receptor (AR) had been silenced (hAR-PHM1–41s) was produced using microRNA lentivirus. A scramble microRNA lentivirus (in which the AR remained active) was used as a negative control (Scr-PHM1–41s), as detailed in the Supplemental Data and shown in Supplemental Figures 1 and 2 and Supplemental Tables 1 and 2. Experimental compounds DHT, T, nifedipine, and T3-(O-carboxymethyl)oxime:BSA (TBSA) were purchased from Sigma, and OXT was from Alliance Pharmaceuticals. DHT and T were reconstituted in ethanol (etOH), and nifedipine in dimethylsulfoxide (DMSO); OXT was diluted in distilled H2O. TBSA, with conjugation ratio T (30 molecules):BSA (1 molecule), was reconstituted in PBS. Anti-phosphorylated myosin light chain (PMLC) polyclonal antibody (Cell Signaling) was used in 1:50 dilution, anti-α-tubulin monoclonal antibody (Sigma) in 1:1000, and secondary antibodies 800CW and 680RD in 1:10 000 (Li-Cor Biosciences). Organ bath The assessment of myometrial contractility utilizing an organ bath is well established (8, 13, 15, 16). Briefly, human myometrial and mouse uterine strips were attached by silk suture (Mersilk 3-0; Ethicon Inc) to a force transducer (ML0186/10 Panlab; ADInstruments) and stretched under passive resting tension (20 mN) in Krebs buffer (115 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 1.2 mm NaH2PO4, 1.2 mm Na2SO4, 2.5 mm CaCl2, 25 mm NaHCO3, 10 mm glucose; pH 7.4) equilibrated with 95% O2–5% CO2 at 37°C. Strips were allowed an equilibration period of 2 hours to develop spontaneous rhythmic contractile activity before addition of DHT or T in cumulative concentrations (10 to 100 μm) or TBSA (0.5 μm equivalent to 100 μm dose of T). Each treatment was applied for 30 minutes for human tissue and 10 minutes for mouse. Equivalent doses of vehicle (etOH or PBS) were applied; the minimum and maximum concentrations of etOH used were 0.03 and 0.3%, respectively. At the end of each experiment, the strips were stimulated with KCl (55 mm) and washed with fresh Krebs buffer to verify tissue viability/recovery. Data were recorded with LabChart 7 acquisition software (ADInstruments). The average frequency, peak amplitude, and force integral (area under the curve [AUC]) after each treatment were calculated for each strip as a percentage of its pretreatment values. Gel contraction assay Cells were embedded in type I collagen in 24-well plates at 105 cells/well as previously described (13, 14). Briefly, the collagen/cell suspension was allowed to polymerize, and the gels were detached and incubated at 37°C for 24 and 48 hours with treatments prepared in 5% (v/v) charcoal-stripped fetal bovine serum (FBS) DMEM. Untreated or vehicle-treated cells developed a basal contraction, which manifested as a decrease in the gel area and was first evident 24 hours after detachment. The gels were photographed using a Leica MZ6 light microscope/camera at 0, 24, and 48 hours. Adobe Photoshop CS6 (Adobe Systems) was used to measure the gel area. The measurement (in pixels) for each gel area at 24 and 48 hours was reported as a percentage of the gel area at the 0-hour time point. The viability of cells in gels was assessed using the CellTitre 96 AQueousOne Solution Cell Proliferation Assay kit (Promega). In-cell Western blot analyses Due to the rapid oscillations between the phosphorylated and dephosphorylated states of MLC and in order to accurately capture the cell transient contractile state, we utilized in-cell Western analysis to quantify PMLC in PHM1–41s as described elsewhere (13, 17). Briefly, cells were seeded into black-wall/optically clear-bottom tissue culture treated 96-well plates (PerkinElmer) to a concentration of 1.8 × 104 cells/well in charcoal-stripped 5% (v/v) FBS DMEM. After the application of treatments, cells were fixed in 3.7% (v/v) formaldehyde (Sigma) and incubated with primary and secondary antibodies. The plate was scanned using the Li-Cor Odyssey Infrared Imaging System (Li-Cor Biosciences). The intensity of PMLC fluorescence was calculated relative to α-tubulin in the same well. Calcium assay The BDTM Calcium Assay Kit (BD Biosciences) was employed to measure [Ca2+] concentration in PHM1–41s. The assay was performed as described in Li et al (18). Briefly, PHM1–41s were seeded in white 96-well plates with a clear bottom (PerkinElmer) in charcoal stripped 5% (v/v) FBS DMEM at a density of 3 × 104 cells/well. After attachment, the cells were incubated with the Ca2+ indicator and then treated with DHT or vehicle (etOH). The plates were placed onto a fluorometric imaging plate reader (NOVOstar; BMG Labtech) with built-in injectors. Before the injection of a compound, the basal cellular fluorescence, which denoted the concentration of [Ca2+], was recorded for 20 seconds using the MARS Data Analysis Software (BMG Labtech). After injection, the changes in the fluorescence were recorded for 40 seconds. The readout was the highest fluorescence measurement recorded (peak) after injection, and that was compared between treatments. Statistics All analysis was conducted with GraphPad Prism version 6.0 (GraphPad Software, Inc). For human and mouse, “n” represents the number of individual patients or mice. For cell studies, “n” denotes the number of times the experiment was repeated, and the number of replicates per experiment is indicated in the figure legends. For statistical analysis, all percentage-presented data were arcsine-transformed. Data were analyzed as indicated in the figure legends and presented as the mean ± SEM; P < .05 was considered statistically significant. Results Androgens inhibit the contraction of uterine myocytes embedded in collagen gels We set out to explore the effect of lipid-soluble androgens DHT and T and of the cell-impermeable TBSA on the contraction of PHM1–41 cells. PHM1–41 cells were embedded in gels and incubated with vehicle (etOH), DHT, or T (1, 50, and 100 μm) or TBSA (0.5 μm) for 24 and 48 hours. Over time, vehicle gels developed a basal contraction resulting in a decrease in the gel area (Figure 1A). At 24 hours, the vehicle area was 77.2 ± 3.4% of the original (measured at 0 hours) (Figure 1B), and at 48 hours the area decreased to 65.2 ± 3.8% (Figure 1C). In contrast, the gel area of cells treated with DHT and T at 50 and 100 μm (Figure 1A), but not 1 μm, was significantly greater compared to the time-matched vehicle gel area, suggesting that both androgens prevented basal contraction. At 24 hours, the DHT (100 μm) gel area was 95.8 ± 1.2% (P < .0001 vs vehicle) of the area recorded at 0 hours (Figure 1B), and at 48 hours it was 82 ± 6.4% (P < .05 vs vehicle) (Figure 1C). For T (100 μm), these values were 94.9 ± 1.1% (P < .001 vs vehicle) at 24 hours (Figure 1B) and 87.72 ± 5.5% (P < .001 vs vehicle) at 48 hours (Figure 1C). TBSA treatment (Figure 1D) did not prevent basal contraction at 24 hours (Figure 1E) and 48 hours (Figure 1F), suggesting that the T-mediated inhibition of contraction is unlikely to be cell-surface receptor mediated. In addition, the finding that DHT (50 μm) prevented the basal contraction of PHM1–41s in which expression of the AR was silenced (hAR-PHM1–41s; Figure 1, G and H) suggested that AR is unlikely to be involved in the induction of relaxation by androgens. Finally, a viability assay ruled out the hypothesis that androgens at high micromolar doses induce cell death (Figure 1I). We conclude that long exposure (>24 hours) to lipid-soluble androgens can inhibit uterine smooth muscle contraction in vitro via an AR-independent mechanism that is likely to be mediated by penetration through the cell membrane. Figure 1. Open in new tabDownload slide DHT and T, but not TBSA, inhibited the contraction of human myometrial cells embedded in collagen gels. PHM1–41s were embedded in collagen gels in 24-well plates and incubated with vehicle, DHT, T, or TBSA for 24 and 48 hours. Over time, vehicle gels developed a basal contraction, which manifested as a decrease in the gel area (A). The gel area at each time point was measured and reported as a percentage of the original gel area. The post-treatment percentages of the original gel area were compared to those of vehicle. DHT and T (50 and 100 μm) incubation for 24 (B) and 48 (C) hours significantly inhibited the basal contraction of PHM1–41s. *, P < .05; **, P < .01; ***, P < .001, compared to vehicle (etOH). n = 7 (six replicates). TBSA treatment (D) at 0.5 μm (0.5 μm equivalent to 100 μm dose of T) did not inhibit the basal gel contraction after 24 (E) and 48 (F) hours of incubation. **, P < .01; ****, P < .0001; ns, nonsignificant compared to vehicle (etOH+PBS). n = 5 (six replicates). Silencing of AR in PHM1–41s did not prevent the effect of DHT (50 μm) on the basal contraction. The 24-hour (G) and 48-hour (H) incubation with DHT (50 μm) induced a significantly smaller reduction in the gel area of PHM1–41s, scramble miR-infected (Scr-PHM1–41s; negative control), and hAR miR-infected (hAR-PHM1–41s) cells (ie, with knockdown of the AR). ***, P < .001; ****, P < .0001 comparison between vehicle and DHT groups. a, not significant: comparison with wt-PHM1–41s vehicle; b, not significant: comparison with Scr-PHM1–41s vehicle; c, not significant: comparison with wt-PHM1–41s-DHT group; d, not significant: comparison with Scr-PHM1–41s-DHT group. n = 5 (six replicates). I, Viability of PHM1–41 cells after incubation with DHT and T (100 μm) for 48 hours. PHM1–41 cells were embedded in collagen gel and treated with DHT and T. Viability assay was performed on the gels 48 hours after treatment. Treatments with DHT and T did not affect viable cell number, which manifested as no change in cell metabolic activity. ns, nonsignificant compared to vehicle (etOH). n = 4. Cell viability data were analyzed using Kruskal-Wallis with Dunn's post hoc test. Gel contraction data were analyzed using one-way ANOVA with either Tukey's post hoc test (B, C, E, F) or Sidak's multiple comparison test (G, H). Figure 1. Open in new tabDownload slide DHT and T, but not TBSA, inhibited the contraction of human myometrial cells embedded in collagen gels. PHM1–41s were embedded in collagen gels in 24-well plates and incubated with vehicle, DHT, T, or TBSA for 24 and 48 hours. Over time, vehicle gels developed a basal contraction, which manifested as a decrease in the gel area (A). The gel area at each time point was measured and reported as a percentage of the original gel area. The post-treatment percentages of the original gel area were compared to those of vehicle. DHT and T (50 and 100 μm) incubation for 24 (B) and 48 (C) hours significantly inhibited the basal contraction of PHM1–41s. *, P < .05; **, P < .01; ***, P < .001, compared to vehicle (etOH). n = 7 (six replicates). TBSA treatment (D) at 0.5 μm (0.5 μm equivalent to 100 μm dose of T) did not inhibit the basal gel contraction after 24 (E) and 48 (F) hours of incubation. **, P < .01; ****, P < .0001; ns, nonsignificant compared to vehicle (etOH+PBS). n = 5 (six replicates). Silencing of AR in PHM1–41s did not prevent the effect of DHT (50 μm) on the basal contraction. The 24-hour (G) and 48-hour (H) incubation with DHT (50 μm) induced a significantly smaller reduction in the gel area of PHM1–41s, scramble miR-infected (Scr-PHM1–41s; negative control), and hAR miR-infected (hAR-PHM1–41s) cells (ie, with knockdown of the AR). ***, P < .001; ****, P < .0001 comparison between vehicle and DHT groups. a, not significant: comparison with wt-PHM1–41s vehicle; b, not significant: comparison with Scr-PHM1–41s vehicle; c, not significant: comparison with wt-PHM1–41s-DHT group; d, not significant: comparison with Scr-PHM1–41s-DHT group. n = 5 (six replicates). I, Viability of PHM1–41 cells after incubation with DHT and T (100 μm) for 48 hours. PHM1–41 cells were embedded in collagen gel and treated with DHT and T. Viability assay was performed on the gels 48 hours after treatment. Treatments with DHT and T did not affect viable cell number, which manifested as no change in cell metabolic activity. ns, nonsignificant compared to vehicle (etOH). n = 4. Cell viability data were analyzed using Kruskal-Wallis with Dunn's post hoc test. Gel contraction data were analyzed using one-way ANOVA with either Tukey's post hoc test (B, C, E, F) or Sidak's multiple comparison test (G, H). Androgens relax human and mouse uterine smooth muscle ex vivo We examined the effect of short-term (<6 hours) exposure of androgens on spontaneous contractions of LN and OB human (Figure 2) and mouse (Figure 3) myometrium. Cumulative doses of DHT and T were applied onto human myometrial (Figure 2A) and murine uterine strips (Figure 3A), all contracting in organ bath chambers. Progressive significant reductions in average amplitude and AUC were observed as the dose of T or DHT was increased from 10 to 100 μm for human (LN, Figure 2, B and D; OB, Figure 2, F and H) and murine (Figure 3, B and D) tissue. Only at the 100-μm dose, the frequency of contraction significantly decreased after treatment with DHT and T for LN (Figure 2C), OB (Figure 2G), and murine (Figure 3C) tissue. To inform future in vivo experiments, we calculated the IC50 values of DHT and T on amplitude and AUC of contraction (Table 1). The IC50 values were not significantly different between the OB and LN groups or between human and mouse tissue. Contractions of myometrial strips were not affected by TBSA (0.5 μm) in human (LN, Figure 2E; OB, Figure 2I) or mouse (Figure 3E). Figure 2. Open in new tabDownload slide DHT and T, but not TBSA, rapidly relaxed spontaneous contractions of myometrium obtained from LN and OB women undergoing elective cesarean section at term. A, Representative recordings show the effect of DHT, T, and TBSA on stretch-induced myometrial contractions of the LN group. Each contracting LN and OB myometrial strip was incubated with either cumulative doses (10–100 μm) of vehicle, DHT, or T, or with a single dose of TBSA (0.5 μm equivalent to 100 μm dose of T). Each dose was applied for 30 minutes. Concentration response curves were generated to show the effect of DHT, T, and vehicle on average amplitude, frequency, and AUC of LN (B–D) and OB (F–H) myometrial contraction. For LN, the amplitude (B) and AUC D of contraction decreased in a dose-dependent manner after either DHT or T; the decrease was significant at all doses tested. At the 100-μm dose of DHT, the amplitude of contraction was reduced to 2 ± 2% of the original value (B), and the AUC to 4.5 ± 2% (D). T (100 μm) also reduced the amplitude of contraction to 3.3 ± 1.3% (B) and the AUC to 15.8 ± 3.8% (D). The frequency (C) of contraction significantly decreased with the 100 μm dose of DHT and T (P < .0001 compared to vehicle). For the OB group, the amplitude (F) and the AUC (H) of contraction decreased in a dose-dependent manner after either DHT or T; the decrease was significant at all doses tested. At 100 μm, DHT reduced the amplitude to 15 ± 6% (F) and the AUC to 4.3 ± 2.7% (H). At the same dose, T reduced the amplitude to 11 ± 6.7% (F) and the AUC to 10 ± 5% (H). The frequency (G) of contraction significantly decreased only with the 100-μm dose of DHT and T (P < .01 compared to vehicle). Data were analyzed using one-way ANOVA with Tukey's post hoc test. TBSA did not relax LN (E) or OB (I) human myometrial contractions; the effect of TBSA on the AUC of contraction was no different from the effect induced by the vehicle (PBS). Data were analyzed with the two-tailed t test. ns, nonsignificant; LN, n = 5 women/1 strip per treatment; OB, n = 6 women/1 strip per treatment. Figure 2. Open in new tabDownload slide DHT and T, but not TBSA, rapidly relaxed spontaneous contractions of myometrium obtained from LN and OB women undergoing elective cesarean section at term. A, Representative recordings show the effect of DHT, T, and TBSA on stretch-induced myometrial contractions of the LN group. Each contracting LN and OB myometrial strip was incubated with either cumulative doses (10–100 μm) of vehicle, DHT, or T, or with a single dose of TBSA (0.5 μm equivalent to 100 μm dose of T). Each dose was applied for 30 minutes. Concentration response curves were generated to show the effect of DHT, T, and vehicle on average amplitude, frequency, and AUC of LN (B–D) and OB (F–H) myometrial contraction. For LN, the amplitude (B) and AUC D of contraction decreased in a dose-dependent manner after either DHT or T; the decrease was significant at all doses tested. At the 100-μm dose of DHT, the amplitude of contraction was reduced to 2 ± 2% of the original value (B), and the AUC to 4.5 ± 2% (D). T (100 μm) also reduced the amplitude of contraction to 3.3 ± 1.3% (B) and the AUC to 15.8 ± 3.8% (D). The frequency (C) of contraction significantly decreased with the 100 μm dose of DHT and T (P < .0001 compared to vehicle). For the OB group, the amplitude (F) and the AUC (H) of contraction decreased in a dose-dependent manner after either DHT or T; the decrease was significant at all doses tested. At 100 μm, DHT reduced the amplitude to 15 ± 6% (F) and the AUC to 4.3 ± 2.7% (H). At the same dose, T reduced the amplitude to 11 ± 6.7% (F) and the AUC to 10 ± 5% (H). The frequency (G) of contraction significantly decreased only with the 100-μm dose of DHT and T (P < .01 compared to vehicle). Data were analyzed using one-way ANOVA with Tukey's post hoc test. TBSA did not relax LN (E) or OB (I) human myometrial contractions; the effect of TBSA on the AUC of contraction was no different from the effect induced by the vehicle (PBS). Data were analyzed with the two-tailed t test. ns, nonsignificant; LN, n = 5 women/1 strip per treatment; OB, n = 6 women/1 strip per treatment. Figure 3. Open in new tabDownload slide DHT and T, but not TBSA, relaxed murine spontaneous uterine contractions. A, Representative recordings show the effect of DHT, T, and TBSA on stretch-induced contractions of uterine horn strips. Each contracting strip was incubated with either cumulative doses (10–100 μm) of vehicle, DHT or T, or with a single dose of TBSA (0.5 μm). Each dose was applied for 10 minutes. Concentration response curves were generated to show the effect of DHT, T, and vehicle on average amplitude (B), frequency (C), and AUC (D) of contraction. The amplitude (B) and AUC (C) of contraction were dose-dependently decreased; the decrease was significant at all doses tested. DHT (100 μm) reduced the amplitude to 4.8 ± 3% (B) and the AUC to 10.4 ± 5% (D). T (100 μm) reduced the amplitude to 4.9 ± 3% (B) and the AUC to 4.8 ± 2.9% (D). Only the 100-μm dose of DHT significantly decreased the frequency of contraction (P < .001 compared to vehicle). For T, the frequency was significantly reduced at both 80 μm (P < .001 compared to vehicle) and 100 μm (P < .0001 compared to vehicle) doses. Data were analyzed using one-way ANOVA with Tukey's post hoc test (n = 5 mice/1 strip per treatment). E, TBSA did not inhibit murine uterine horn strip contraction; the effect of TBSA on the AUC of contraction was no different from the effect induced by the vehicle (PBS). Data were analyzed with the two-tailed t test. ns, nonsignificant; n = 5 mice/1 strip per treatment. Figure 3. Open in new tabDownload slide DHT and T, but not TBSA, relaxed murine spontaneous uterine contractions. A, Representative recordings show the effect of DHT, T, and TBSA on stretch-induced contractions of uterine horn strips. Each contracting strip was incubated with either cumulative doses (10–100 μm) of vehicle, DHT or T, or with a single dose of TBSA (0.5 μm). Each dose was applied for 10 minutes. Concentration response curves were generated to show the effect of DHT, T, and vehicle on average amplitude (B), frequency (C), and AUC (D) of contraction. The amplitude (B) and AUC (C) of contraction were dose-dependently decreased; the decrease was significant at all doses tested. DHT (100 μm) reduced the amplitude to 4.8 ± 3% (B) and the AUC to 10.4 ± 5% (D). T (100 μm) reduced the amplitude to 4.9 ± 3% (B) and the AUC to 4.8 ± 2.9% (D). Only the 100-μm dose of DHT significantly decreased the frequency of contraction (P < .001 compared to vehicle). For T, the frequency was significantly reduced at both 80 μm (P < .001 compared to vehicle) and 100 μm (P < .0001 compared to vehicle) doses. Data were analyzed using one-way ANOVA with Tukey's post hoc test (n = 5 mice/1 strip per treatment). E, TBSA did not inhibit murine uterine horn strip contraction; the effect of TBSA on the AUC of contraction was no different from the effect induced by the vehicle (PBS). Data were analyzed with the two-tailed t test. ns, nonsignificant; n = 5 mice/1 strip per treatment. Table 1. DHT and T IC50 Values Generated From the Concentration Response Curves for Amplitude and AUC . Human LN, μm . Human OB, μm . Mouse, μm . IC50 . 95% CI . IC50 . 95% CI . IC50 . 95% CI . AUC     DHT 43 35–54 52 39–69 40 32–51     T 45 39–53 49 41–59 30 28–38 Amplitude     DHT 39 32–47 45 34–58 30.5 24–37     T 34 28–40 39 31–48 30.7 23–39 . Human LN, μm . Human OB, μm . Mouse, μm . IC50 . 95% CI . IC50 . 95% CI . IC50 . 95% CI . AUC     DHT 43 35–54 52 39–69 40 32–51     T 45 39–53 49 41–59 30 28–38 Amplitude     DHT 39 32–47 45 34–58 30.5 24–37     T 34 28–40 39 31–48 30.7 23–39 Abbreviation: CI, confidence interval. Open in new tab Table 1. DHT and T IC50 Values Generated From the Concentration Response Curves for Amplitude and AUC . Human LN, μm . Human OB, μm . Mouse, μm . IC50 . 95% CI . IC50 . 95% CI . IC50 . 95% CI . AUC     DHT 43 35–54 52 39–69 40 32–51     T 45 39–53 49 41–59 30 28–38 Amplitude     DHT 39 32–47 45 34–58 30.5 24–37     T 34 28–40 39 31–48 30.7 23–39 . Human LN, μm . Human OB, μm . Mouse, μm . IC50 . 95% CI . IC50 . 95% CI . IC50 . 95% CI . AUC     DHT 43 35–54 52 39–69 40 32–51     T 45 39–53 49 41–59 30 28–38 Amplitude     DHT 39 32–47 45 34–58 30.5 24–37     T 34 28–40 39 31–48 30.7 23–39 Abbreviation: CI, confidence interval. Open in new tab The organ bath studies combined with the gel contraction studies allowed the observation that lipid-soluble androgens induce a rapid but sustained inhibition of uterine contractions. Androgens inhibit MLC phosphorylation in uterine myocytes Elevation in [Ca2+] activates the Ca2+ sensor calmodulin, which binds to MLC kinase, activating MLC phosphorylation and subsequent contraction. We aimed to deduce whether DHT treatment prevented the phosphorylation of MLC (PMLC) in contracting PHM1–41s. OXT was utilized to stimulate contraction of collagen-embedded PHM1–41s. A 24- and 48-hour treatment with OXT enhanced contraction, which manifested as a decrease in gel area, with the area being smaller than that of vehicle (Figure 4A). After 24 hours (Figure 4B), the average vehicle gel area was 83.4 ± 6.9% of the original gel area (measured at 0 hours), and it was significantly different (P < .001) when compared to the time-matched OXT gel area (66 ± 1.9%). The cotreated OXT+DHT gel area was 82.8 ± 2.8% and significantly bigger than the OXT gel area (P < .01), demonstrating that DHT prevented the OXT-stimulated contraction (Figure 4B). The cotreated OXT+DHT gel area reduced to 78.2 ± 1.2% after 48 hours (Figure 4C) and was significantly different (P < .0001) from the time-matched OXT gel area (42.2 ± 3.7%). Figure 4. Open in new tabDownload slide DHT treatment prevented the phosphorylation of MLC stimulated by OXT in human myometrial cells. The effect of DHT pretreatment on OXT-stimulated contraction and OXT-stimulated MLC phosphorylation was investigated. PHM1–41s cells were embedded in collagen gels and incubated with vehicle (distilled H2O+etOH), OXT (100 nm), DHT (50 μm), or OXT+DHT for 24 hours and 48 hours (A). The gel area was measured and reported as a percentage of the original gel area (0 hour time point). The OXT gel area was significantly smaller when compared to the vehicle gel area; however, cotreatment with DHT+OXT prevented the OXT alone-induced effect on the gel area at 24 hours (B) and 48 hours (C). ***, P < .001; ****, P < .0001, comparison between OXT and vehicle. ##, P < .01; ####, P < .0001, comparison between OXT and OXT+ DHT. n = 5 (six replicates). D, PHM1–41s were seeded into 96-well plates and either directly exposed to acute (30 seconds) treatment with vehicle (distilled H2O) or OXT (100 nm), or initially pretreated (15 minutes) with vehicle (etOH) or DHT (50 μm) and then stimulated with acute OXT. E, The concentration of PMLC was significantly higher in the wells after acute OXT compared to the PMLC in the wells treated with the acute vehicle. ###, P < .001, n = 5 (6 replicates). The concentration of PMLC in the vehicle pretreated cells was higher compared to the concentration of PMLC in the DHT pretreated cells when both were exposed to acute OXT. *, P < .05. n = 5 (triplicate). F, PHM1–41 cells were either directly exposed to acute vehicle (distilled H2O) or OXT (100 nm) or first pretreated (15 minutes) with vehicle (DMSO) or nifedipine (50 μm) and then exposed to acute OXT. G, The concentration of PMLC in the DMSO pretreated cells was higher compared to the concentration of PMLC in the nifedipine pretreated cells when both were exposed to acute OXT. ####, P < .0001, comparison between acute OXT and acute vehicle; **, P < .01 comparison between nifedipine+OXT and vehicle+OXT. n = 5 (triplicate). Data were analyzed using one-way ANOVA with Tukey's post hoc test. Figure 4. Open in new tabDownload slide DHT treatment prevented the phosphorylation of MLC stimulated by OXT in human myometrial cells. The effect of DHT pretreatment on OXT-stimulated contraction and OXT-stimulated MLC phosphorylation was investigated. PHM1–41s cells were embedded in collagen gels and incubated with vehicle (distilled H2O+etOH), OXT (100 nm), DHT (50 μm), or OXT+DHT for 24 hours and 48 hours (A). The gel area was measured and reported as a percentage of the original gel area (0 hour time point). The OXT gel area was significantly smaller when compared to the vehicle gel area; however, cotreatment with DHT+OXT prevented the OXT alone-induced effect on the gel area at 24 hours (B) and 48 hours (C). ***, P < .001; ****, P < .0001, comparison between OXT and vehicle. ##, P < .01; ####, P < .0001, comparison between OXT and OXT+ DHT. n = 5 (six replicates). D, PHM1–41s were seeded into 96-well plates and either directly exposed to acute (30 seconds) treatment with vehicle (distilled H2O) or OXT (100 nm), or initially pretreated (15 minutes) with vehicle (etOH) or DHT (50 μm) and then stimulated with acute OXT. E, The concentration of PMLC was significantly higher in the wells after acute OXT compared to the PMLC in the wells treated with the acute vehicle. ###, P < .001, n = 5 (6 replicates). The concentration of PMLC in the vehicle pretreated cells was higher compared to the concentration of PMLC in the DHT pretreated cells when both were exposed to acute OXT. *, P < .05. n = 5 (triplicate). F, PHM1–41 cells were either directly exposed to acute vehicle (distilled H2O) or OXT (100 nm) or first pretreated (15 minutes) with vehicle (DMSO) or nifedipine (50 μm) and then exposed to acute OXT. G, The concentration of PMLC in the DMSO pretreated cells was higher compared to the concentration of PMLC in the nifedipine pretreated cells when both were exposed to acute OXT. ####, P < .0001, comparison between acute OXT and acute vehicle; **, P < .01 comparison between nifedipine+OXT and vehicle+OXT. n = 5 (triplicate). Data were analyzed using one-way ANOVA with Tukey's post hoc test. To determine whether the effect of DHT involved blockade of MLC phosphorylation, we assessed the impact of DHT pretreatment on PMLC concentration after acute (30 seconds) stimulation with OXT. Acute stimulation with OXT induced a dramatic increase (P < .001) in the concentration of fluorescently detected PMLC, and a short (15 minutes) preincubation with DHT, but not vehicle, significantly (P < .05) prevented the increase in PMLC after acute OXT (Figure 4E). Interestingly, preincubation with the Ca2+ channel blocker nifedipine, before acute OXT, also significantly (P < .01) prevented the increase in PMLC concentration (Figure 4G). We conclude that DHT inhibits PHM1–41s contraction via inhibition of MLC phosphorylation. The similarity between the actions of DHT and the L-type Ca2+ channel blocker nifedipine with regard to prevention of MLC phosphorylation contributes to the notion of an indirect effect of DHT on PMLC, potentially mediated via blockade of Ca2+ channels and subsequent decrease in [Ca2+]. Androgens inhibit Ca2+ flux in uterine myocytes We set to explore the hypothesis that DHT pretreatment would prevent the increase in [Ca2+] concentration in PHM1–41s. OXT was used to stimulate a rapid increase in [Ca2+] concentration. Addition of OXT to untreated PHM1–41s induced an immediate 2-fold increase above baseline (P < .0001) in the concentration of [Ca2+] (Figure 5B). The effect of OXT on [Ca2+] was examined after pretreatment with either DHT or vehicle. DHT pretreatment induced a dose-dependent reduction in the OXT-stimulated increase in [Ca2+], which was significant when compared to the OXT-stimulated increase in [Ca2+] in the vehicle pretreated cells (Figure 5, B–D). These data suggest that DHT blocks Ca2+ flux in uterine myocytes and impacts downstream MLC phosphorylation. Figure 5. Open in new tabDownload slide DHT treatment prevented the rapid increase in [Ca2+] concentration stimulated by OXT in human myometrial cells. A, Cells were seeded into 96-well plates and either not treated or treated with vehicle (etOH) or DHT (10 minutes) and then injected with OXT (10 nm). The injection of OXT to untreated wells rapidly increased the concentration of [Ca2+] above baseline (red plot). The DHT pretreatment (10 minutes) significantly reduced the response to OXT injection. OXT injection to vehicle (etOH) pretreated wells increased the concentration of [Ca2+] above baseline significantly more than to DHT (B, 300 nm; C, 800 nm; D, 50 μm) pretreated wells. *, P < .05; **, P < .01; ****, P < .0001, comparison between the groups vehicle (etOH)+OXT and DHT+OXT. ###, P < .001; ####, P < .0001, comparison between the groups vehicle (distilled H2O) and OXT. n = 6 (four replicates). Data were analyzed using one-way ANOVA with Tukey's post hoc test. Figure 5. Open in new tabDownload slide DHT treatment prevented the rapid increase in [Ca2+] concentration stimulated by OXT in human myometrial cells. A, Cells were seeded into 96-well plates and either not treated or treated with vehicle (etOH) or DHT (10 minutes) and then injected with OXT (10 nm). The injection of OXT to untreated wells rapidly increased the concentration of [Ca2+] above baseline (red plot). The DHT pretreatment (10 minutes) significantly reduced the response to OXT injection. OXT injection to vehicle (etOH) pretreated wells increased the concentration of [Ca2+] above baseline significantly more than to DHT (B, 300 nm; C, 800 nm; D, 50 μm) pretreated wells. *, P < .05; **, P < .01; ****, P < .0001, comparison between the groups vehicle (etOH)+OXT and DHT+OXT. ###, P < .001; ####, P < .0001, comparison between the groups vehicle (distilled H2O) and OXT. n = 6 (four replicates). Data were analyzed using one-way ANOVA with Tukey's post hoc test. Discussion A relaxant effect of androgens on smooth muscle contraction has been reported in different systems (19–23). Ten years ago, a single study demonstrated that various androgens, including DHT and T, relaxed human myometrial strips contracting under resting tension in organ bath chambers (9). The authors described the response as rapid (in minutes), transcription-independent (not prevented by protein synthesis inhibitors), achievable with pharmacological (micromolar) doses, and reversible. Herein we show for the first time that: 1) only lipid-soluble androgens (T and DHT) effectively relax OB and LN human and murine myometrial contractions; 2) the response is immediate (in minutes) but can be sustained for longer times (days) even in the presence of cell viability; 3) the mechanism of relaxation is a reduction in the availability of [Ca2+] concentration, which subsequently results in reduction of MLC phosphorylation in the uterine myocytes; and 4) the mechanism of relaxation is AR-independent. Other studies have reported the effects of sex hormones on [Ca2+] and PMLC concentrations in other cell types and tissues. For example, DHT treatment of Fura-2-loaded isolated rat vas deferens cells blunted the KCl-induced elevation in [Ca2+], whereas short incubation with estradiol inhibited the histamine-induced increase in [Ca2+] in Fura-2-loaded airway smooth muscle cells (24, 25). These findings are in line with the inhibitory effect of DHT on an OXT-stimulated increase in [Ca2+] concentration in Fura-4-loaded PHM1–41s in our study. Consistent with our finding that DHT blunted the effect of OXT on PMLC, incubation with estradiol and P in micromolar doses inhibited increases in PMLC in retinal epithelial and colon muscle cells (26, 27). It is reasonable to speculate that androgens restrict Ca2+ flux in uterine myocytes. Such an effect can be achieved either by physical interaction with Ca2+ channels or indirectly by interaction with molecules residing on the cell membrane, which are known to regulate Ca2+ channel activity (28). A physical interaction of androgens with Ca2+ channels has never been described, but there is some evidence to support an indirect effect of androgens on Ca2+ channels. The antagonism of OXT by DHT observed in our study might suggest that androgens interact with the mechanism by which OXTR signaling activates capacitive and noncapacitive Ca2+ entry in PHM1–41s (29). The binding of OXT to OXTR, a G protein-coupled receptor, activates transmembrane receptor-operated Ca2+ channels (ROCCs) to induce Ca2+ flux from the extracellular space into the cell but can also stimulate the inositol 1,4,5-triphosphate (IP3) cascade, which results in the activation of IP3 receptors on the sarcoplasmic reticulum and release of Ca2+ from the internal store into the cytoplasm (28, 30). Therefore, it is plausible that DHT blocked either the ROCC-associated pathway or the downstream activators of the IP3 pathway, which manifested as a decrease in total concentration of [Ca2+] in PHM1–41s. However, evidence from a coronary muscle study, where T failed to inhibit caffeine- and carbachol-induced (activators of IP3 pathway) Ca2+ release from the sarcoplasmic reticulum, suggests that androgens are likely to block the ROCC-associated Ca2+ flux rather than the IP3 pathway (31). We hypothesize two mechanisms by which androgens could decrease the ROCC-associated Ca2+ flux: 1) bind to a cell surface-associated binding protein that interacts with the OXTR and induce conformational changes to the receptor, which could result in impaired interaction of OXTR with the G protein; or 2) overload the plasma membrane and change membrane fluidity, which could prevent the OXTR from interacting with the G protein. Notably, if a membrane-initiated response were to mediate the effect of T in the myometrium, TBSA would be expected to inhibit the myometrial contractions in our study. However, TBSA did not induce relaxation, suggesting that the action of T is unlikely to be mediated via cell-surface receptors but requires penetration into, or through, the cell membrane. Therefore, it is possible that penetration of hydrophobic androgens into the negatively charged lipid bilayer altered the contractile function of PHM1–41s via impairment of cell membrane fluidity, which is known to affect active and passive transport of various molecules (32). The mechanism by which OXTR causes the opening of ROCCs is not clear (33); however, understanding this mechanism would help determine how androgens interact with the contractile cascades and inform whether they could be utilized as alternative tocolytics. It is noteworthy that the uterorelaxant effect of nifedipine comes to prominence within 20 minutes of administration to pregnant women presenting with preterm contractions, and the impact of a single dose can last up to 6 hours (34). The rapid response of myometrium to nifedipine resembles the immediate (in minutes) response to androgens observed in our study ex vivo in the term and possibly preterm (Supplemental Figure 3) myometrium. Adding to the similarity noted between the two responses, we showed that short incubations with DHT or nifedipine each reduced the OXT-stimulated PMLC in PHM1–41s, suggesting that both compounds can rapidly manipulate components of the contractile apparatus. With the aim of decreasing maternal and fetal side effects during tocolysis and extending pregnancy until term, there is growing interest in the discovery and validation of alternative tocolytics. The benefits and harms of supplemental P, which inhibits human myometrial contraction with similar IC50 values (16) to androgens in our study, are currently under investigation. Nifedipine, as well as other Ca2+ channel blockers, can cross the placenta and elicit adverse effects upon the fetus (3), but the placenta is known to possess mechanisms that inhibit the transport of androgens (35). In particular, the placenta can aromatize naive androgens, such as T, to estrogens to protect the fetus from virilization. A female fetus would only be in danger of virilization if the androgen were administered during the masculinization window, which is reported to exist during the first trimester of pregnancy (36). Conversely, animal studies have informed that maternal androgen excess is associated with the development of polycystic ovary syndrome in the offspring (37). However, in most of these studies, androgen excess was achieved by a daily administration of nonaromatizable DHT in high concentrations from midgestation up to term (38, 39). We believe it is unlikely that androgens will cause polycystic ovary syndrome in female offspring if given in native form for short periods to stop preterm-initiated contractions in the third trimester. Further basic understanding of the dose response and the mechanism of action of androgens on uterine contractions is required to inform the design of preclinical studies on androgens as tocolytic agents. Notably, the IC50 values generated here could help design experiments whereby administration of DHT or T to existing mouse models of PTB (40) could be used to investigate whether androgens can induce uterine relaxation. Such studies could contribute to the discovery of much needed novel PTB therapeutics. Acknowledgments The authors thank Professor B. Sanborn (Colorado State University, Fort Collins, Colorado) for her gift of PHM1–41 cells, Dr A. Henke (University of Edinburgh, Edinburgh, UK) for his gift of rat tail collagen, the Edinburgh Reproductive Tissues BioBank for providing myometrial samples, and Mr. Ronnie Grant for figure illustration. This work was supported by the Principal Career Development and Albert McKern studentships (to S.M.) and funding from Tommy's (J.E.N.) for consumables. P.T.K.S. was supported by MRC programme Grant G1100356/1. Disclosure Summary: The authors have nothing to disclose. J.E.N. has received funding from Tommy's the Baby Charity and the Medical Research Council to understand the physiology of parturition and to investigate mechanisms of uterine relaxation. 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TI - Androgen-Induced Relaxation of Uterine Myocytes Is Mediated by Blockade of Both Ca2+ Flux and MLC Phosphorylation
JF - The Journal of Clinical Endocrinology & Metabolism
DO - 10.1210/jc.2015-2851
DA - 2016-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/androgen-induced-relaxation-of-uterine-myocytes-is-mediated-by-LsrbCdLEWS
SP - 1055
VL - 101
IS - 3
DP - DeepDyve
ER -