Differential Effects of Testosterone and Estradiol on Clitoral Function: An Experimental Study in Rats

Introduction: Female sexual response is a complex phenomenon in which psychological, neurologic, and vascular mechanisms and hormonal factors interact. During the arousal phase, they cooperate to increase genital blood flow, thus inducing engorgement of the clitoris and lubrication of the vagina. Regulation of vascular and non-vascular smooth muscle tone is the crucial event in the erectile process. Preclinical studies have suggested that nitric oxide (NO) is the main vasodilator neurotransmitter modulating, through the second messenger cyclic guanosine monophosphate (cGMP), clitoral flow vessels. Aim: To investigate the effects of sexual steroid hormones on pro-erectile and relaxant (mediated by NO and cGMP) and anti-erectile and contractile (mediated by ras homolog gene family member A [RhoA] and Rho-associated protein kinase [ROCK]) mechanisms in the clitoris using a validated animal model of female ovariectomized Sprague-Dawley rats. Methods: Subgroups of ovariectomized rats were treated with 17 beta-estradiol, progesterone, testosterone, or testosterone and letrozole for 6 weeks. The experimental groups were compared with a control group of intact rats. Main Outcome Measures: Sex steroids plasma levels were assessed and in vitro contractility studies were carried out in order to investigate the effect of ovariectomy and in vivo treatments on clitoris smooth muscle activity. Smooth muscle cells (SMCs) from rat clitoral biopsies were isolated and characterized. RhoA activity was determined in SMCs cell cultures. RNA from tissues and cells was analyzed by quantitative real-time RT-PCR. Results: Using real-time polymerase chain reaction, testosterone treatment upregulated the expression of NO-mediated pathway genes (endothelial and neuronal NO synthase, guanylate cyclase soluble subunit-alpha(3), guanylate cyclase soluble subunit-beta(3), cGMP-dependent protein kinase 1, and phosphodiesterase type 5). Conversely, estrogen replacement upregulated the expression of calcium-sensitizing RhoA-ROCK pathway genes. In vitro contractility studies were performed on phenylephrine pre-contracted clitoris strips. Ovariectomy resulted in a decreased responsiveness to Y-27632, a ROCK inhibitor, which was fully restored by 17 beta-estradiol supplementation. To further examine the effect of 17 beta-estradiol on the RhoA-ROCK pathway, smooth muscle cells were isolated from rat clitoris and their migration capacity was evaluated. Conclusion: Collectively, these data demonstrate that testosterone improves the relaxation of vascular smooth muscle cells through the NO-cGMP pathway, and that testosterone and 17 beta-estradiol are necessary to maintain a functional contractile and relaxant machinery in the clitoris. This new concept might provide support for the concomitant use of estrogen and testosterone during the treatment of sexual arousal disorders related to hormonal imbalance or insufficiency. Copyright (C) 2016, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.