Article Figures & Data
Figures
- Fig. 1.
Structures of CatSper activators PROG, PGE1, and l-sirenin.
- Fig. 2.
Structures of CatSper antagonists MPA, LNG, and ALDO.
- Fig. 3.
Structures of T-type calcium channel blockers mibefradil, NNC55-0396, and ML218.
- Fig. 4.
The T-type CCBs mibefradil and ML218 inhibit PROG-, PGE1-, l-sirenin-, and potassium-induced calcium influx in human sperm. (A) Representative FLIPR traces showing concentration-dependent reduction of PROG-mediated increase in [Ca2+]i by mibefradil (upper) and ML218 (lower). (B–E) Potencies of mibefradil and ML218 for inhibiting (B) PROG-, (C) PGE1-, (D) l-sirenin-, or (E) high K+/high pH-induced calcium influx. Ligand-induced activation used EC80 concentrations of activator (30 nM PROG, 10 nM PGE1, 3 µM l-sirenin). Buffer containing 140 mM K+, pH 8.2 was added to elicit alkalinization/depolarization calcium influx (E). The data in B–E are plotted as the mean ±SEM and expressed as a percent of the response produced by each activator alone. IC50 and n values are in Table 1.
- Fig. 5.
PROG analogs have a broad range of potencies for eliciting calcium influx in human sperm. (A) Representative FLIPR traces showing the concentration-dependent increase in calcium influx produced by PROG. (B) Concentration-response curves comparing potencies of parent compound PROG and C17- and/or C6-modified analogs: 1, PROG; 4, MPA; 20, 6α-methylprogesterone; 25, 17α-hydroxyprogesterone; 29, 17α-acetoxyprogesterone; 30, 17α-hydroxy-6α-methylprogesterone. Data are plotted as mean ±SEM and expressed as a percent of the response produced by a saturating concentration of PROG (3 µM). EC50, Emax, and n values are in Tables 2 and 3.
- Fig. 6.
Several clinically used progestins, antiprogestins, and androgens elicit calcium influx in human sperm. Concentration-response curves for 31, drospirenone; 32, ulipristal; 33, mifepristone; 41, 7β,11β-dimethylandrolone. Data are plotted as mean ±SEM and expressed as a percent of the response produced by a saturating concentration of PROG (3 µM). EC50, Emax, and n values are in Table 3.
- Fig. 7.
The steroids MPA, LNG, and ALDO inhibit PROG-, PGE1-, and l-sirenin-induced calcium influx in human sperm. (A) Representative FLIPR traces showing concentration-dependent reduction of PROG-mediated increase in [Ca2+]i by MPA (upper) and LNG (lower). FLIPR traces for inhibition of PGE1- and l-sirenin-induced calcium influx by MPA and LNG are in Supplemental Figs. 7 and 8, respectively, and traces for ALDO antagonism of all three activators are in Supplemental Fig. 9. (B–E) Potencies of MPA, LNG, or ALDO for inhibiting (B) PROG-, (C) PGE1-, (D) l-sirenin-, or (E) high K+/high pH-induced calcium influx. Ligand-induced activation used EC80 concentrations of activator (30 nM PROG, 10 nM PGE1, 3 µM l-sirenin). Buffer containing 140 mM K+, pH 8.2 was added to elicit alkalinization/depolarization calcium influx (E). The data in B–E are plotted as the mean ±SEM and expressed as a percent of the response produced by each activator alone. IC50 and n values are in Table 4.
- Fig. 8.
T-type CCBs produce an insurmountable block of PROG-induced calcium influx in human sperm. (A) Mibefradil and (B) ML218 reduce the PROG Emax in a concentration-dependent manner with little change in EC50 values, indicating an insurmountable inhibition. Emax, EC50, slopes, and n values are in Table 5. The data are plotted as the mean ±SEM and expressed as a percent of the response produced by a saturating concentration of PROG (3 µM).
- Fig. 9.
MPA, LNG, and ALDO produce a surmountable inhibition of PROG-induced calcium influx in human sperm. Increasing concentrations of (A) MPA, (B) LNG, and (C) ALDO increase the observed EC50 values for PROG with little change in Emax values (left panels). The same data are plotted in the Schild analyses (right panels) indicating surmountable inhibition. Emax, EC50, and n values are in Table 5, and pKB values are in Table 6. The data are plotted as the mean ±SEM and expressed as a percent of the response produced by a saturating concentration of PROG (3 µM).
- Fig. 10.
T-type CCBs inhibit total, progressive, and hyperactivated motility, whereas steroidal CatSper antagonists selectivity inhibit hyperactivated motility in human sperm. A. Total (solid) and progressive (checkered) motility was monitored by CASA in the presence of increasing concentrations of inhibitor. The steroidal antagonists LNG, ALDO, and MPA show little or no effect on total and progressive motility, whereas MBF and ML218 completely block these motility parameters. Asterisks indicate differences from vehicle control. B. HAM was induced with 100 nM PROG under capacitating conditions. The steroidal CatSper antagonist MPA reduced the percentage of sperm cells displaying HAM to the level of the vehicle control, whereas LNG and ALDO were less potent. The T-type CCBs MBF and ML218 reduced the percentage of sperm cells displaying HAM to zero. Asterisks indicate differences from the PROG only control. In both A and B, data are presented as mean ±SD and are expressed as a percent of the total sperm cell population analyzed. Statistical significance: * p < 0.05, ** p < 0.005 and **** p < 0.0001.
- Fig. 11.
SAR of CatSper activation by progesterone analogs.
Tables
- TABLE 1
Potencies of mibefradil and ML218 to inhibit calcium influx produced by PROG, PGE1, l-sirenin, and high K+/high pH in human sperm
Opener Blocker pIC50 n PROG mibefradil 5.17 ± 0.24 7 ML218 5.02 ± 0.08 4 PGE1 mibefradil 5.08 ± 0.31 8 ML218 4.87 ± 0.15 3 l-sirenin mibefradil 5.09 ± 0.34 5 ML218 4.85 ± 0.17 3 high K+/high pH mibefradil 4.76 ± 0.09 8 ML218 4.99 ± 0.16 7 Inhibitors were evaluated in the presence of an EC80 concentration of each activator (30 nM PROG, 10 nM PGE1, 3 µM l-sirenin). Buffer containing 140 mM K+, pH 8.2 was added to elicit alkalinization/depolarization calcium influx. pIC50 values are expressed as the mean ±SD with the number of independent experiments indicated by n.
- TABLE 2
Potencies for PGE1, l-sirenin, PROG, and PROG analogs to elicit calcium influx in human sperm
# Steroid Steroid Alone + 30 µM Mibefradil pEC50 Emax, % n pEC50 Emax, % n Activator 1 PROG 8.43 ± 0.21 111 7 8.22 ± 0.15 42 3 2 PGE1 8.40 ± 0.24 101 8 8.33 ± 0.43 22 5 3 l-sirenin 6.02 ± 0.26 102 3 5.48 ± 0.23 22 3 A Ring or A–B Ring Fusion 10 3α,5α-pregnanolone 6.56 ± 0.21 85 5 6.40 ± 0.19 29 3 11 3α,5β-pregnanolone 6.26 ± 0.25 102 3 6.47 ± 0.22 33 3 12 3β,5α-pregnanolone 7.16 ± 0.11 99 4 7.08 ± 0.31 38 4 13 3β,5β-pregnanolone 6.89 ± 0.20 92 4 7.05 ± 0.23 37 4 14 5α-dihydroprogesterone 6.99 ± 0.05 87 3 6.95 ± 0.12 34 3 15 5β-dihydroprogesterone 6.87 ± 0.17 95 3 6.67 ± 0.35 20 3 16 5α,6α-epoxypregnan-3,20-dionea 6.08 ± 0.37 83 3 5.96 ± 0.19 12 3 17 5β,6β-epoxypregnan-3,20-dionea 6.38 ± 0.07 84 3 — <10 3 18 2,2-dimethylprogesteronea 6.31 ± 0.16 99 3 — <10 3 B Ring 19 6β-methylprogesteronea 6.58 ± 0.12 90 3 — <10 3 20 6α-methylprogesteronea 6.16 ± 0.14 96 3 6.62 ± 0.20 18 3 21 6β-hydroxyprogesterone 6.91 ± 0.10 116 3 6.72 ± 0.09 35 3 22 5α-hydroxy-6β-methylpregnan-3,20-dionea 5.30 ± 0.55 95 3 — <10 3 C Ring 23 11β-hydroxyprogesterone 7.73 ± 0.13 90 5 7.54 ± 0.55 52 4 24 11α-hydroxyprogesterone 6.90 ± 0.08 100 3 6.93 ± 0.45 32 4 D Ring 25 17α-hydroxyprogesterone 7.98 ± 0.35 115 5 8.02 ± 0.36 67 3 26 3α,5β-THDOC 5.80 ± 0.26 100 3 5.98 ± 0.39 18 4 27 3α,5α-THDOC 6.05 ± 0.21 96 6 6.20 ± 0.26 37 4 28 16α-hydroxyprogesterone 6.77 ± 0.18 106 3 6.71 ± 0.27 47 3 29 17α-acetoxyprogesterone <5 31 8 <5 37 3 30 17α-hydroxy-6α-methylprogesterone 6.33 ± 0.10 96 3 — <10 3 The potency (EC50 values, mean ±SD) and maximum response (Emax) produced by each compound was determined in the absence and presence of 30 µM mibefradil. Emax is expressed as a percent of the response produced by a saturating concentration of PROG (3 µM). The number of independent experiments indicated by n. Fitted parameters were not calculated for compounds producing Emax < 10%.
↵aSynthesized compound (see Supplemental Schemes 1–3 and Synthetic Procedures for synthesis and characterization).
- TABLE 3
Potencies for clinically used progestins, antiprogestins, endogenous steroid hormones and androgens to elicit calcium influx in human sperm
# Steroid Steroid Alone +30 µM Mibefradil pEC50 Emax, % n pEC50 Emax, % n Progestins and Anti-progestins 4 MPA <5 17 3 — <10 3 5 LNG <5 15 6 — <10 2 31 drospirenone 6.28 ± 0.22 115 5 — <10 4 32 ulipristal 5.45 ± 0.22 106 3 <5 12 5 33 mifepristone (RU-486) 5.33 ± 0.30 109 4 — <10 5 34 ulipristal acetate <5 27 3 — <10 3 35 segesterone acetate <5 57 4 — <10 4 Endogenous 6 ALDO — <10 3 — <10 1 36 testosterone 6.66 ± 0.22 114 3 <5 17 4 37 17β-estradiol 6.53 ± 0.25 94 3 <5 37 1 Androgens 38 11β-methyl-19-nortestosterone 6.37 ± 0.32 85 6 6.27 ± 0.25 15 3 39 7α-methyl-19-nortestosterone 6.25 ± 0.37 86 7 6.29 26 2 40 7α,11β-dimethylandrolone 6.06 ± 0.49 80 9 <5 11 3 41 7β,11β-dimethylandrolone 5.41 ± 0.24 42 5 <5 26 3 The potency (EC50 values, mean ±SD) and maximum response (Emax) produced by each compound was determined in the absence and presence of 30 µM mibefradil. Emax is expressed as a percent of the response produced by a saturating concentration of PROG (3 µM). The number of independent experiments indicated by n. Fitted parameters were not calculated for compounds producing Emax < 10%.
- TABLE 4
Potencies of MPA, LNG, and ALDO to inhibit calcium influx produced by PROG, PGE1, l-sirenin, and high K+/high pH in human sperm
Opener Antagonist pIC50 n PROG MPA 5.18 ± 0.39 12 LNG 4.49 ± 0.23 9 ALDO 4.48 ± 0.16 10 PGE1 MPA 5.01 ± 0.26 11 LNG 4.52 ± 0.11 6 ALDO 4.24 ± 0.20 10 l-Sirenin MPA 5.21 ± 0.33 7 LNG 4.09 ± 0.05 5 ALDO 4.21 ± 0.16 9 high K+/high pH MPA <5 4 LNG <5 4 ALDO <5 4 Inhibitors were evaluated in the presence of an EC80 concentration of each activator (30 nM PROG, 10 nM PGE1, 3 µM l-sirenin). Buffer containing 140 mM K+, pH 8.2 was added to elicit alkalinization/depolarization calcium influx. IC50 values are expressed as the mean ±SD with the number of independent experiments indicated by n.
- TABLE 5
T-type CCBs mibefradil and ML218 reduce the maximal response, whereas MPA, LNG, and ALDO reduce the potency of PROG-, PGE1-, and l-sirenin-induced calcium influx in human sperm
Inhibitor Progesterone PGE1 l-Sirenin Conc., µM pEC50 Emax, % n pEC50 Emax, % n pEC50 Emax, % n MBF 1 8.71 ± 0.29 90 3 8.16 ± 0.08 87 3 5.71 ± 0.01 87 2 10 8.14 ± 0.20 69 4 8.53 ± 0.37 53 4 5.57 ± 0.41 58 4 30 8.31 ± 0.42 23 4 8.23 ± 0.51 41 3 5.37 ± 0.51 20 4 100 8.26 ± 0.24 16 3 — <10 5 — <10 3 ML218 1 8.12 ± 0.13 83 3 8.20 ± 0.10 73 3 ND 10 7.77 ± 0.35 42 3 8.01 ± 0.05 64 3 5.61 ± 0.45 38 2 30 — <10 3 7.86 ± 0.04 15 3 — 4 2 100 — <10 3 — <10 2 — 2 2 MPA 10 7.69 ± 0.40 106 3 7.83 ± 0.43 111 3 5.56 ± 0.15 108 3 30 7.51 ± 0.38 99 3 7.34 ± 0.15 105 3 5.24 ± 0.58 100a 3 100 6.64 ± 0.07 100a 3 6.96 ± 0.03 100a 3 4.71 ± 0.21 100a 3 LNG 10 7.58 ± 0.16 110 8 7.78 ± 0.30 96 4 5.16 ± 0.08 95 4 30 7.06 ± 0.26 100a 4 7.48 ± 0.18 106 4 4.83 ± 0.16 102 4 100 6.64 ± 0.06 100a 4 7.29 ± 0.22 117 4 4.52 ± 0.03 100a 3 ALDO 10 7.44 ± 0.14 114 4 7.88 ± 0.22 107 4 5.28 ± 0.28 104 4 30 7.09 ± 0.36 106 4 7.82 ± 0.31 99 4 5.10 ± 0.24 109 4 100 6.59 ± 0.16 100a 4 7.44 ± 0.31 100a 4 4.51 ± 0.21 100a 4 EC50 values are expressed as the mean ±SD, and Emax values are the percent of a saturating concentration of PROG (3 µM). The number of independent experiments is indicated by n. EC50, Emax, and n values for PROG, PGE1, and l-sirenin in the absence of inhibitor are in Table 2. Fitted parameters were not calculated for conditions producing Emax < 10.
↵a Constrained value due to rightward shift of dose response.
- TABLE 6
Dissociation constants for steroidal antagonists of activator-induced calcium influx in human sperm
Opener Antagonist Schild Leff-Dougall pKB PROG MPA 6.19 ± 1.75 6.16 ± 0.39 LNG 6.80 ± 0.10 5.47 ± 0.23 ALDO 5.38 ± 0.27 5.46 ± 0.16 PGE1 MPA 5.38 ± 0.59 6.00 ± 0.26 LNG 3.36 ± 0.41 5.50 ± 0.11 ALDO 3.14 ± 0.95 5.22 ± 0.20 l-Sirenin MPA 5.22 ± 0.33 5.20 ± 0.20 LNG 4.25 ± 0.23 5.07 ± 0.05 ALDO 4.82 ± 1.03 5.19 ± 0.16 The Schild pKB values (mean ± standard error) were obtained using the EC50 values corresponding to the pEC50 values listed in Table 5 derived from 3–8 independent experiments. For all Schild analyses, the slopes of the regression lines had values of 1.0 within the 95% CI. The Leff-Dougall pKB values (mean ± SD) were obtained using the IC50 values corresponding to the pIC50 values listed in Table 4 derived from 5–12 independent experiments.
Data Supplement
- Supplemental Data -
Supplemental General Synthesis Information
Supplemental Scheme 1. Synthesis of 2,2-dimethylprogesterone
Supplemental Scheme 2. Synthesis of 6alpha-methylprogesterone
Supplemental Scheme 3. Sythesis of 5alpha, 6alpha- and 5 beta, 6beta-epoxyprogesterone
Supplemental Figure 1. Representative FLIPR traces showing inhibition of PGE1-, l-sirenin- and elevated K+ - induced calcium influx by mibefradil
Supplemental Figure 2. Representative FLIPR traces showing inhibition of PGE1-, l-sirenin- and elevated K+ - induced calcium influx by MIL218
Supplemental Figure 3. FLIPR traces demonstrating that ML218 did not elicit calcium influx in human sperm cells
Supplemental Figure 4. Chemical structures of compouonds in Table 2
Supplemental Figure 5. Ketalized steroids have little or no activity in the calcium influx assay in human sperm
Supplemental Figure 6. Chemical structures of compounds in Table 3
Supplemental Figure 7. MPA inhibits PGE1- and l-sirenin- induced calcium influx in human sperm.
Supplemental Figure 8. LNG inhibits PGE1- and l-sirenin- induced calcium influx in human sperm.
Supplemental Figure 9. ALDO inhibits PROG-, PGE1- and l-sirenin-induced calcium influx in human sperm.
Supplemental Figure 10. T-type CCBs produce an insurmountable block, but MPA, LNG, and ALDO produce a surmountable inhibition of PGE1-induced calcium influx in human sperm.
Supplemental Figure 11. T-type CCBs produce an insurmountable block, but MPA, LNG, and ALDO produce a surmountable inhibition of l-sirenin-induced calcium influx in human sperm.
Supplemental Figure 12. T-type CCBs and steroid antagonists do not block the calcium influx produced by the calcium ionophore A23187.
Supplemental Figure 13. T-type CCBs and steroidal CatSper antagonists inhibit PGE1-induced hyperactivated motility (HAM) in human sperm.
1H, 13C, DEPT, NOESY NMR spectra
- Supplemental Data -
