Direct functional effects of hormones on bladder contractility remain understudied. Recently [1], we described reversible anti-contractile effects of progesterone on methacholine (MeCh) responses in the male rat bladder. While previous studies in the pig bladder neck [2] suggest direct relaxatory effects to progesterone to be similar in males and females, its anti-contractile properties to MeCh (i.e. progesterone’s ability to impair a contractile cholinergic response) remains incompletely studied, which would be important in further understanding its functional role. Furthermore, previously proposed mechanisms involving the progesterone receptor (PR) or the progesterone receptor membrane component 1 (PGRMC1), require further investigation. 

Therefore, this study investigates the mechanisms underlying progesterone’s direct anti-contractile effects on urinary bladder smooth muscle, focusing on possible sex-related differences.

Sprague-Dawley rats (~300g; n=20, 10 of each sex) were euthanized and the urinary bladders collected under local ethics committee approval. Three full-thickness bladder strips were prepared per bladder, from the area above the trigone near the ureters, and mounted in organ baths (20mL, Biopac systems Inc., Goleta, CA; MP35, SS63L force transducer). Functional responses were recorded and analyzed using the BSL Analysis 4.1 software.

Viability of the preparations was assessed by high-potassium Krebs solution (124 mM). Contractile responses to MeCh were recorded and then repeated 10 min after progesterone treatment (5x10-4 M for 10 min before wash). MeCh administrations were finally repeated once more after 90 min recovery.

Separately, mifepristone (10-4 M; PR antagonist) or AG-205 (10-5 M; PGRMC1 modulator) were added to preparations from the abovementioned bladders, equilibrated for 30 min, after which progesterone (5x10-4 M) was added for 10 min before wash. After an additional 10 min the MeCh series were repeated as described above.

MeCh was dissolved in Milli-Q water and administered cumulatively (100 µL). Progesterone, mifepristone, and AG-205 were dissolved in DMSO and added at volumes of 500 µL, 100 µL and 100 µL, respectively. Effects of these DMSO concentrations on contractility have been previously excluded [1].

Statistical significance was determined by 2-way ANOVA with Dunnett’s multiple comparison test, computed using GraphPad Prism 11.0.0.

MeCh evoked concentration-dependent contractions that reached a maximum of 90.4±4.6% in males and 94.8±4.8% in females (relative to high-potassium response; n=8-10; ns). Compared to male control strips, progesterone treatment statistically significantly reduced contractions at 10-6-10-3 M MeCh, resulting in maximal responses of 47.1±10.1% in males and 57.2±8.4% in females (p<0.05). After a 90-min resting period, MeCh responses were fully restored in both sexes, reaching 94.9±11.6% and 98.7±9.6% in males and females, respectively (n.s; Fig.1).

Notably, administration of the PR antagonist mifepristone (n=3-5 for each sex) or the PGRMC1 modulator AG-205 (n=5 for each sex) before progesterone treatment failed to maintain the contractility to MeCh. Antagonist application before progesterone consistently resulted in similar or, if anything, lower responses than in vehicle+progesterone treated preparations. Relative contractions at the highest MeCh concentration were only 26.7±4.3% (female) and 23.3±7.1% (male) after AG-205 and progesterone, and 4.3±2.1% (male) and 23.0±14.3% (female) after mifepristone and progesterone. Thus, no functional receptor blockade could be shown.

Anti-contractile effects of a rather high, pharmacological, concentration of progesterone are robust after washing but reversible over 90 min. The impact of progesterone treatment does not differ between the sexes. Nonetheless, antagonists of the suggested targets PR and PGRMC1 failed to mitigate this effect, suggesting other pathways to be of importance in progesterone mediated urinary bladder effects in the rat.

This study demonstrates that similar, reversible, anti-contractile responses to progesterone were seen in male and female rat bladders, but could not be shown to be mediated by PR or PGRMC1.

Ferizovic A, Winder M, Sato M, Aronsson P. 293 - Anti-contractile effects and recovery time after progesterone in the rat urinary bladder. Continence. 2025;15:102217.
Fernandes VS, Ribeiro ASF, Martínez-Sáenz A, Blaha I, Serrano-Margüello D, Recio P, et al. Underlying mechanisms involved in progesterone-induced relaxation to the pig bladder neck. European Journal of Pharmacology. 2014;723:246-52.