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Estrogens Regulate Humans and Rabbit Epididymal Contractility Through the RhoA/Rho-kinase Pathway jsm_1282

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Benedetta Fibbi,* Sandra Filippi,† Annamaria Morelli,* Linda Vignozzi,* Enrico Silvestrini,* Aravinda Chavalmane,* Giulia De Vita,* Mirca Marini,‡ Mauro Gacci,§ Chiara Manieri,¶ Gabriella Barbara Vannelli,‡ and Mario Maggi* *Andrology Unit, Department of Clinical Physiopathology; †Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology; ‡Department of Anatomy, Histology and Forensic Medicine; §Department of Urology, University of Florence, Florence, Italy; ¶S.C.D.U. Endocrinology and Metabolic Disease, University of Turin, Turin, Italy DOI: 10.1111/j.1743-6109.2009.01282.x

ABSTRACT

Introduction. We have previously demonstrated that oxytocin (OT) and endothelin-1 (ET-1) peripherally regulate epididymal motility in an estrogen-dependent way. Because RhoA/Rho-kinase (ROCK) pathway is a contractile effector downstream to both OT and ET-1 receptors, we hypothesized an estrogenic modulation of OT- and ET-1-induced contraction through the up-regulation of RhoA/ROCK signaling. Aim. To evaluate the effect of changing endocrine milieu on RhoA/ROCK pathway in the epididymis. Methods. We induced a pharmacological hypogonadotropic hypogonadism in rabbits and replaced hypogonadal animals with different sex steroids (testosterone, T, or estradiol valerate, [E2v]). Effects of estrogen deprivation were also evaluated in rabbits chronically treated with the P450-aromatase inhibitor letrozole. An “in vitro” model of human epididymal smooth muscle cells was established and stimulated with sex hormones (72 hours). Protein and mRNA expression and functional activity of RhoA/ROCK signaling were studied by quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, western blot analysis, cell migration and by “in vitro” contractility studies using the ROCK inhibitor Y-27632. Main Outcome Measures. Effects of sex steroids on expression and functional activation of RhoA/ROCK signaling in rabbit epididymis and human epididymal smooth muscle cells. Results. The relaxant effect of Y-27632 on ET-1-pre-contracted epididymal strips was significantly reduced in hypogonadal rabbits, as well as in letrozole-treated animals. T supplementation normalized T plasma levels, but not Y-27632 epididymal strip sensitivity. E2v not only completely restored Y-27632 responsiveness but even amplified it, indicating an estrogenic up-regulation of RhoA/ROCK pathway. Accordingly, ROCK1 protein and gene expressions were strongly induced by E2v but not by T. The estrogen-induced up-regulation of RhoA/ROCK signaling was confirmed in human epididymal smooth muscle cells. Conclusions. Our results suggest that estrogens regulate epididymal motility by increasing RhoA/ROCK signaling, and therefore calcium sensitivity, which tunes up responsiveness to contractile factors. Fibbi B, Filippi S, Morelli A, Vignozzi L, Silvestrini E, Chavalmane A, De Vita G, Marini M, Gacci M, Manieri C, Vannelli GB, and Maggi M. Estrogens regulate humans and rabbit epididymal contractility through the Rhoa/Rho-kinase pathway. J Sex Med 2009;6:2173–2186. Key Words. Estrogens; Epididymis; RhoA/Rho-kinase Pathway

Introduction

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n humans, the emission phase of ejaculation consists of the integrated and time-coordinated activation of parasympathetic fibers, that regulates the secretion of seminal fluid from epithelial cells © 2009 International Society for Sexual Medicine

of the accessory sex glands, and of the sympathetic adrenergic fibers that induces the bladder neck closure and the strong contractile response of the epididymis and vas deferens. As a result, seminal fluid is propelled in the posterior urethra [1]. The release of stored and mature sperms from the J Sex Med 2009;6:2173–2186

2174 human epididymis, an elongated and convoluted duct representing the most proximal region to the testis, is the first step of this process. This propulsive activity is essentially promoted by the contraction of epididymal smooth muscle cells, which are located both in the proximal (caput and corpus) and distal (cauda) regions of the duct, although to a different extent. In the caput and in the corpus, contractile cells form a loose layer around duct, which allows a constant basal outflow by spontaneous peristaltic-like contractions, even in the absence of nervous stimuli [2]. In the cauda, they are organized in three distinct muscular layers able to generate the forceful propulsion during the emission phase [3]. In this region, sympathetic noradrenergic innervation is particularly abundant [4]. However, a crucial role in the peripheral regulation of human [5–7] and rabbit [7,8] epididymis motility is exerted by local, non-neuronal contractile factors, as oxytocin (OT) and endothelin-1 (ET-1). OT is a nonapeptide hormone, recently found to be involved in promoting orgasm-related male genital tract motility [1] and penile detumescence [9,10]. ET-1 is a potent constrictor peptide that regulates the smooth muscle tone of several districts, including vasculature [11] and corpora cavernosa [12]. Both OT and ET-1 promote epididymis contractility and sperm transport through a synergistic, estrogen-dependent autocrine/ paracrine loop [6,7,13,14]. In fact, epididymal epithelial cells in culture produce and secrete both OT and ET-1, which act on smooth muscle cells where the OT receptor (OTR) and the ETA subtype of ET-1 receptor are expressed. We hypothesized that OT release by posterior pituitary at orgasm evokes a nerve-independent contraction of the epididymis through a double mechanism: a direct effect on epididymal smooth muscle cells, and an estrogen-driven synergic cross-activation of these two local contractile systems [8,14]. It is well recognized that the epididymis synthesizes estrogens through a P450 aromatase (P450Ar) activity, which irreversibly converts androgens into estrogens [8,15–17]. Epididymis is also a target for estrogens, because its epithelial and smooth muscle cells express both a and b isoforms of the estrogen receptor (ER) [18,19]. In humans and larger mammals, one-third of the caput epididymis consists of efferent ducts, small tubule offshoots of the rete testis which form a series of coiled tubules between the rete testis and the epididymis, and are primarily involved in reabsorbing over 90% of the seminal fluid [20]. They highly express ERa and ERb and represent a J Sex Med 2009;6:2173–2186

Fibbi et al. major site for estrogen function in the male reproductive tract across numerous species, leading to a tight regulation of luminal fluid reabsorption and sperm concentration [20]. Another primary role of estrogens in the epididymis is the up-regulation of sensitivity to OT and ET-1 [8,14]. Indeed, in a rabbit model of hypogonadotropic hypogonadism, there was epididymal hypo-responsiveness to OT and ET-1, fully restored by estrogens, but not testosterone, supplementation [8,14]. Similarly, the blockage of endogenous estrogen synthesis by dosing letrozole, an inhibitor of P450Ar activity [21], reduced both OT- and ET-1-mediated epididymal contractility [8,14]. Finally, OTR expression is under estrogen control, with a still unidentified mechanism [8,22]. In fact, although in mouse, the OTR gene promoter contains a classical estrogen responsive element (ERE); in other animal species, including humans, only a nonfunctional half-palindromic ERE sequence has been detected [1]. Several Gq-protein coupled receptors, such as OTR [22] and ETA [23], are able to induce smooth muscle contraction via a transient phospholipase C (PLC)-dependent intracellular calcium rise. Thereafter, the maintenance of the contracted state is allowed by the activation of the calciumindependent, calcium-sensitizing RhoA/rhokinase (ROCK) pathway. RhoA is a tightly regulated small guanosine triphosphate hydrolase enzyme (GTPase) belonging to the Ras-related Rho family, implicated in the regulation of smooth muscle contraction [24], cytoskeletal organization [24], cell adhesion [25], motility [26], migration [26], proliferation [27], and transcription of smooth muscle-specific genes involved in smooth muscle cell commitment and function (“excitation-transcription coupling”) [28]. OT and ET-1 binding to their excitatory receptors induces RhoA activation, cycling from a cytoplasmic guanosine 5′diphosphate-bound complex to a GTPbound form, which translocates to the plasma membrane, thereby initiating signal transduction. The best-characterized downstream effectors of RhoA are the two isoforms of Rho-associated coiled-coil-containing protein kinase (ROCK1 or ROKb, and ROCK2 or ROKa), which share 65% homology in their amino acid sequence and 92% homology in their kinase domains [29]. They are able to increase calcium sensitivity of the contractile apparatus by maintaining the phosphorylated state of the myosin light chain, and thus the contractile tone, independently of intracellular calcium levels [29].

Estrogens and RhoA/ROCK Pathway in Epididymis Because ET-1- and OT-mediated contractile responses are induced by the activation of a common downstream effector signaling pathway, the RhoA/ROCK one, and both contractile agents positively regulate epididymal motility, we hypothesized that estrogens are able to increase epididymal contractile responsiveness through an up-regulation of RhoA/ROCK signaling. In the present study, we investigated the effect of changing endocrine milieu on the expression and functional activity of the RhoA/ROCK pathway in rabbit epididymis. The estrogens-induced activation of this calcium-sensitizing pathway was also evaluated in a cellular model of isolated human epididymal smooth muscle cells. Materials and Methods

Experimental Hypogonadism and Sex Steroid Replacement Sexually mature New Zealand white male rabbits (weighing ~3 kg; N = 35) were divided into the following five groups and treated as previously described [8,14]: (i) intact animals, not subjected to any pharmacological treatment (controls; N = 8); (ii) treated for 3 weeks with the P450Ar inhibitor letrozole (2.5 mg/kg daily; N = 5; Novartis, Basel, Switzerland) dissolved in drinking water; (iii) treated with a single injection of the long-acting gonadotropin-releasing hormone (GnRH) analog triptorelin pamoate (2.9 mg/kg; N = 8; Ipsen, Milan, Italy); (iv) treated with the GnRH analog plus testosterone (T; Bayer Schering; Berlin, Germany; 30 mg/kg weekly, N = 7; T supplementation was performed by using a mix of 110 mg of T enanthate and 25 mg of T propionate, corresponding to 100 mg of T); and (v) treated with the GnRH analog plus estradiol valerate (E2v; Bayer Schering; Berlin, Germany; 3.3 mg/kg weekly, N = 7). T and E2v supplementations were started after 2 weeks from triptorelin pamoate administration and were carried on for 6 weeks. After 2 months from triptorelin pamoate administration and after 1 week from the last supplementation of T/E2v, rabbits were killed, blood was drawn from the heart for sex steroid measurement and epididymides were collected for in vitro contractility studies, immunohistochemistry, and gene and protein expression analyses. Tissue Preparation Human epididymides were obtained at surgery from three male transsexual individuals undergo-

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ing bilateral orchiectomy for transgender reassignment and after informed consent. All individuals, aged between 25 and 35 years (mean 28.7 ⫾ 2.7 years), received at least 1 year of cyproterone acetate (50 mg/die) and estrogen (4 mg/die) treatment before surgical intervention. Pharmacological therapy was interrupted at least 20 days before surgery. Other human tissues (uterus and intestine) were collected during surgery for benign diseases. All tissue samples were obtained after the approval of the Hospital Committee for Investigation in Humans (protocol 6783-04; Azienda Ospedaliera Universitaria Careggi, Florence, Italy) and after receiving consent from the informed patients. For rabbit epididymis collection, untreated and treated animals were killed by a lethal dose of pentobarbital, then, epididymides were separated from testes and adherent fat. Tissue specimens were fresh-frozen for RNA preparation and western blot analysis. For immunohistochemistry, they were fixed in formalin. For in vitro contractility studies, they were immediately placed and maintained in cold Krebs solution (NaCl, 118 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; NaHCO3, 25 mM; CaCl2, 2.5 mM; glucose, 10 mM), until the beginning of the experiments.

In Vitro Contractility Studies Experiments were carried out as previously described [7,8,14]. Briefly, human or rabbit epididymis, at the border between corpus and cauda, was cut into three to four small strips (5 mm long, 3 mm wide, and 1 mm thick), which were vertically mounted under 700 mg resting tension in organ chambers containing 10 mL Krebs solution at 37°C, gassed with 95% O2 and 5% CO2 at pH 7.4. Changes in isometric tension were recorded on a chart polygraph (Battaglia Rangoni, Casalecchio di Reno, Bologne, Italy). To evaluate smooth muscle responsiveness to different constrictor agents, increasing concentrations of noradrenaline (NA; 10-9–10-5 M; Sigma-Aldrich, St. Louis, MO, USA), OT (10-11–10-5 M; SigmaAldrich), and ET-1 (10-10–10-6 M; Calbiochem, Merck, Darmstadt, Germany) were added to the bath; results were expressed as the percentage of increase in tonic tension induced by a selected dose of KCl (80 mM; Merck & Co Inc., Darmstadt, Germany) able to produce a stable tension. The in vitro effect of Y-27632 dihydrochloride (Y-27632; Tocris, Bristol, UK)—a well-established ROCK inhibitor—on NA-, OT-, and ET-1induced contraction (at a fixed concentration: NA J Sex Med 2009;6:2173–2186

2176 10 mM, OT 1 mM, ET-1 300 nM), was evaluated pre-incubating epididymal strips for 1 hour with a fixed dose (10-4 M) of the drug. To test the functional activity of RhoA/ROCK pathway on epididymal smooth muscle strips precontracted with ET-1 (100 nM), increasing concentrations (10-9–10-4 M) of Y-27632 were added to the bath at 7-minute intervals. The degree of contractile response induced by ET-1 was taken as 100%, and the dose-dependent relaxant effect of cumulative concentrations of the ROCK inhibitor was referred to this value.

Measurement of Testosterone and 17b-Estradiol Plasma levels of T and 17b-estradiol (17b-E2) were measured with an automated chemiluminescence system (Bayer Corp. Diagnostics, East Walpole, MA, USA) after appropriate extraction. For extraction, samples were mixed with four volumes of diethyl ether for 15 minutes, centrifuged for 5 minutes at 2,000 rpm, and the aqueous phase was frozen in dry ice. The organic phase was recovered, evaporated to dryness under a nitrogen stream, and reconstituted in the assay buffer. RNA Isolation and Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Total RNA was extracted from frozen tissues and cells using TRIZOL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and cDNA synthesis was performed as previously described [30]. Real-time RT-PCR for ROCK1 expression in rabbit tissues was performed using SYBR Green Realtime PCR Master Mix (Applied Biosystems, Foster City, CA, USA), as previously published [30]. Primers sequence (NCBI accession No. U42424) was sense 5′-CGG AAG TGA ACT CGG ATT GT-3′ and antisense 5′-TCC AAA TGC ACC TCT ACC AA-3′, covering a 197-base pair sequence. mRNA quantitative analysis in human epididymal cells and in human tissues was performed according to the fluorescent TaqMan methodology, as already reported [30]. PCR primers and probe for RhoA, ROCK1, ROCK2, P450Ar, ERa, and ERb were Assay-On-Demand gene expression products (assay no. Hs00357608_m1, Hs00178463_ m1, Hs00153074_m1, Hs00240671_m1, Hs01046818_m1, Hs01100358_m1, respectively) purchased from Applied Biosystems. The 18S ribosomal RNA subunit was chosen as the reference gene and was selected among the endogenous controls provided by Applied Biosystems. AmpliJ Sex Med 2009;6:2173–2186

Fibbi et al. fication and detection were performed with the ABI Prism 7,700 Sequence Detection System (Applied Biosystems). Each measurement was carried out in duplicate. Data analysis was based on the comparative cycle threshold method according to the manufacturer’s instructions (Applied Biosystems).

Immunohistochemistry Immunohistochemical studies were carried out as previously described [10]. Briefly, transversal sections from the corpus of rabbit epididymides (fixed in buffered formalin solution and embedded in paraffin) were incubated overnight at 4°C, with a mouse monoclonal ROCK1 primary antibody (1:1,000 dilution; sc-17794, Santa Cruz Biotechnology, CA, USA). Sections were rinsed in phosphate buffered saline (PBS), incubated with biotinylated secondary antibody, and then with streptavidin-biotin peroxidase complex (Ultravision large volume detection system anti-polyvalent, Lab-Vision, Fremont, CA, USA). The specificity of the anti-ROCK1 antibody was controlled by incubating the secondary antibody in the absence of the primary antibody. The reaction product was developed with the 3′,3′-diaminobenzidine tetrahydrochloride as chromogen (Sigma-Aldrich). Demonstration of peroxidase activity and controls for antiserum specificity were performed as previously described [10]. The slides were photographed using a Nikon Microphot-FXA microscope (Nikon, Tokyo, Japan). Cell Cultures Human epididymal smooth muscle cells were isolated from freshly delivered epididymis obtained at surgery, after informed consent, from a 22-yearold patient undergoing radical orchiectomy for a non-seminomatous germ cells tumor of the testis. Tissue was cut into small fragments and treated for 2 hours with 2 mg/mL bacterial collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA). Fragments were then extensively washed in PBS and cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium (1:1; Sigma-Aldrich), supplemented with 10% heatinactivated fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin (Sigma-Aldrich), and 100 mg/mL streptomycin (Sigma-Aldrich) in a fully humidified atmosphere of 95% air–5% CO2. Cells began to emerge within 1 week and were used within the ninth passage.

Estrogens and RhoA/ROCK Pathway in Epididymis

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Analysis For protein extraction from treated and untreated rabbit epididymides, frozen tissues were ground in liquid nitrogen and directly suspended in lysis buffer (25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid pH 7.4; 175 mM sodium chloride; 0.1% glycerol; 5 mM ethylenediaminetetraacetic acid; 0.1% Triton X-100; 50 mM sodium fluoride; 5 mM sodium orthovanadate), including protease inhibitors (Roche Diagnostics, Penzberg, Germany) and phosphatase inhibitor cocktail (Sigma-Aldrich); then, they were homogenized for protein analysis, as previously described [30]. For protein extraction from human cell cultures, subconfluent epididymal smooth muscle cells were starved for 24 hours in serum-free medium before exposure to a 72-hour treatment with 17b-E2 (1 nM; Sigma-Aldrich) or ET-1 (100 nM); they were then scraped into lysis buffer including protease inhibitors (Roche Diagnostics, Penzberg, Germany) and phosphatase inhibitor cocktail (Sigma-Aldrich), and proteins were extracted. Total protein was estimated by bicinchoninic acid assay (Pierce, Rockford, IL, USA). ROCK1 western blot assay was performed by resolving 30 mg of proteins from each sample by 7% SDS-PAGE, transferring them to a polyvinylidene difluoride membrane (ImmobilonP, Millipore Corporation, Billerica, MA, USA), and revealing ROCK1 specific band with an antiROCK1 primary antibody (1:1,000 in PBS and 5% nonfat dry milk for o/n), followed by peroxidaseconjugated IgG (1:3,000). Equal protein loading was verified by reprobing the membrane with a rabbit polyclonal anti-STAT1 antibody (1:1,000; Santa Cruz Biotechnology) and a mouse monoclonal a-tubulin antibody (1:1,000; Santa Cruz Biotechnology), as appropriate. ROCK activity was analyzed as previously described [30]. Briefly, cells were washed in PBS and scraped into lysis buffer; then, an equal amount of total proteins (20 mg) for each sample was resolved by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After o/n membrane incubation with a rabbit polyclonal P-myosin phosphatase target subunit 1 (MYPT-1; Thr-853) antibody (1:1,000; Upstate Biotechnology, Lake Placid, NY, USA), proteins were revealed using an anti-rabbit horseradish peroxydase-coniugated secondary antibody (1:10,000; Calbiochem). Equal protein loading was verified by reprobing the membrane with a goat

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polyclonal MYPT-1 antibody (1:500; Upstate Biotechnology). Immunoreactive bands were detected with enhanced chemiluminescence (ECL-Plus, GE Healthcare Ltd., Buckinghamshire, UK) and autoradiographed using hyperfilm (GE Helthcare). Densitometric analysis of band intensity was performed using Adobe Photoshop software (Version 8.0, Adobe Systems Incorporated, San Jose, CA USA). Densitometry data are expressed as percentage over control (mean ⫾ SEM) of ROCK1/Stat1 or P-MYPT-1/MYPT-1 ratio, as appropriate, and resulted from three different experiments.

Cell Chemotaxis Assay Cell chemotaxis assay was performed in a P48 multiwell Boyden chamber (Nuclepore Inc, Pleasanton, CA, USA) using polyvinyl-pyrrolidine-free polycarbonate filters with an 8-mm pore size coated with 20 mg/mL type I collagen (BD Biosciences, San Jose, CA, USA), as previously described [30]. Subconfluent epididymal smooth muscle cells were serum starved for 24 hours and then treated for 72 hours with 17b-E2 (1 nM) or with a single dose of T (1 nM; Sigma-Aldrich), the latter in the presence or absence of letrozole (10 mM). At the end of incubation, cells were washed and trypsinized, and 104 cells in 40 mL were seeded onto the upper wells of the chamber with or without C3 exoenzyme (1 mg/mL, Calbiochem) or Y-27632 (25 mM) and incubated at 37°C for 5 hours. Unstimulated cells in serum-free culture medium were used as controls for basal migration. The action of the different compounds on chemotaxis was tested using methanol-fixed cells stained with Diff-Quick (DADE Behring AG, Switzerland) and cell migration was measured by microscopic evaluation of the number of cells that moved across the filter into 10 random fields. Each experimental point was replicated at least five times. Results were obtained from three independent experiments. Results are expressed as percent of control response (mean ⫾ SEM), considering migration of control cells as 100%. Statistical Analysis Results are expressed as mean ⫾ SEM for N experiments. Statistical analysis was performed with one-way analysis of variance test followed by Tukey–Kramer post hoc analysis, and P < 0.05 was considered significant. Maximal effect and half maximal response inhibitory concentration (IC50) values were calculated using the computer program ALLFIT [31]. J Sex Med 2009;6:2173–2186

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Figure 1 (A) Effect of increasing concentrations of noradrenaline (NA; 10-9–10-5 M), oxytocin (OT; 10-11–10-5 M) and endothelin-1 (ET-1; 10-10–10-6 M) on rabbit epididymis contractility. Ordinate: contractile activity, expressed as a percentage of the maximal response obtained with KCl (80 mM); abscissa: molar concentration of the agonist. The relative EC50 values are reported in the text. (B) Effect of “in vivo” letrozole and “in vitro” Y-27632 treatment on NA-, OT-, or ET-1-mediated rabbit epididymis contractility. Epididymal strips responsiveness to NA (10 mM), OT (1 mM), and ET-1 (300 nM) in letrozole-treated rabbits (open bars) and in control animals (closed bars). NA-, OT-, and ET-1-induced contraction in epididymal strips pre-incubated (1 hour) with Y-27632 (10-4 M) added directly into the experimental bath (diagonally hatched bars). Ordinate: contractile activity, expressed as a percentage of the maximal response obtained with KCl (80 mM); abscissa: constrictor agents. *P < 0.05 and **P < 0.01 vs. its relative control. (C) Effect of changing endocrine milieu on the relaxant response to Y-27632. Ordinate: relaxant responses of ET-1-precontracted epididymal strips to increasing concentrations (10-9–10-4 M) of Y-27632 in control animals (closed circles), hypogonadal animals (closed squares), Testosterone (T)-supplemented hypogonadal animals (closed up-ward triangles), estradiol valerate (E2v)-supplemented hypogonadal animals (closed down-ward triangles), letrozole-treated animals (asterisk) expressed as a percentage of the maximal response obtained with 100 nM ET-1; abscissa: molar concentration of Y-27632. Emax and IC50 values are reported in the text. Results reported are the mean ⫾ standard error of the mean of at least seven different experiments. (D) Relaxant effect of increasing concentrations (10-11–10-4 M) of Y-27632 on human epididymal strips contraction. Tissues were obtained from male individuals who received for at least 1 year cyproterone acetate and estrogens therapy before undergoing surgery for transgender reassignment. Ordinate: contractile activity, expressed as a percentage of the maximal response obtained with ET-1 (100 nM); abscissa: molar concentration of Y-27632. Emax and IC50 values are reported in the text.

Results

Effect of Hormonal Milieu on RhoA/ROCK-mediated Epididymal Contractility Figure 1A shows the response of rabbit epididymal strips to increasing concentrations of the major J Sex Med 2009;6:2173–2186

contractile agents, NA, OT, and ET-1. NA (10-9– 10-5 M), OT (10-11–10-5 M), and ET-1 (10-10– 10-6 M) induced a dose-dependent increase in contractility, although with different EC50s (NA: OT: 5.4 ⫾ 4.0 ¥ 10-8 M; 3.5 ⫾ 1.3 ¥ 10-7 M; -8 ET-1: 4.9 ⫾ 1.2 ¥ 10 M). In epididymal strips

Estrogens and RhoA/ROCK Pathway in Epididymis Table 1 Sex steroid plasma levels in different experimental animal groups

a) b) c) d) e)

Control (N = 8) Letrozole (N = 5) GnRH analog (N = 8) GnRH analog + T (N = 7) GnRH analog + E2v (N = 7)

T (nmol/L)

17-bE2 (pmol/L)

10.1 ⫾ 1.7 11.7 ⫾ 3.5 1.9 ⫾ 0.4** 19.6 ⫾ 6.0 1.0 ⫾ 0.5**

170.1 ⫾ 10.0 111.9 ⫾ 10.6** 142.3 ⫾ 0.7* 178.5 ⫾ 15.6 273.4 ⫾ 35.5**

Blood for testosterone (T) and 17b-estradiol (17b-E2) measurements was drawn after 3 weeks of treatment with the P450 aromatase inhibitor letrozole, dissolved in the drinking water (2.5 mg/kg/die, group b) or 2 months after a single administration of the long-acting gonadotropin-releasing hormone (GnRH) analog triptorelin pamoate (2.9 mg/kg, groups c, d, and e) and 1 week after the last injection of T (30 mg/kg/week, group d) or E2v (3.3 mg/kg/week, group e). N = number of animals. *P < 0.05 and **P < 0.01 vs. group a.

from rabbits chronically (3 weeks) treated with the P450Ar inhibitor letrozole (2.5 mg/Kg/day), the contractile response to all the compounds (NA 10 mM, OT 1 mM, ET-1 300 nM) was significantly reduced, when compared with untreated animals (Figure 1B). Letrozole administration significantly decreased circulating 17-bE2 (control: 170.1 ⫾ 10 pmol/L, letrozole: 111.9 ⫾ 10.6 pmol/L, P < 0.01; see Table 1) without affecting T (control: 10.1 ⫾ 1.7 nmol/L; letrozole: 11.7 ⫾ 3.5 nmol/L; see Table 1). In vitro pre-incubation for 1 hour of epididymal strips from untreated rabbits with the selective ROCK inhibitor Y-27632 (10-4 M) also significantly impaired the contractile response to NA, OT, and ET-1 (Figure 1B). The most dramatic effects of letrozole in vivo and Y-27632 in vitro were observed on OT- and ET-1-induced contractions. Hence, all the subsequent experiments were performed in ET-1-precontracted epididymal strips. In these preparations from intact animals, increasing concentrations (10-9–10-4 M) of Y-27632 dosedependently induced relaxation (Figure 1C), with Emax = 45.9% ⫾ 2.1%. Letrozole-induced estrogen deprivation (see Table 1) significantly reduced the relaxant effect of Y-27632, as compared with controls (Emax = 69.5% ⫾ 1.8%, P < 0.0001, Figure 1C), suggesting that endogenous estradiol is involved in the regulation of the RhoA/ROCK pathway. To further investigate the effect of changing endocrine milieu on RhoA/ROCK pathway, we used an animal model of hypogonadotropic hypogonadism, according to a previously described protocol [8,14]. Rabbits were treated for 2 months with the GnRH analog triptorelin pamoate (2.9 mg/kg), and replaced with different sex steroids (T, 30 mg/kg weekly or E2v, 3.3 mg/kg weekly), as previously described [8,14]. After an 8-week treatment with the GnRH analog, T and

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17b-E2 levels were significantly reduced (P < 0.01 and P < 0.05 vs. control, respectively; see Table 1). In hypogonadal rabbits, the relaxant response to Y-27632 was almost completely abolished (Emax = 91.2% ⫾ 1.7%, P < 0.0001 vs. control), and only partially restored in the T-supplemented group (Emax = 66.1% ⫾ 1.8%, P < 0.0001 vs. control and vs. untreated hypogonadism). Epididymal responsiveness to Y-27632 was not only completely restored, but also amplified by estrogen replacement (Emax = 17.2% ⫾ 2.4%, P < 0.0001 vs. control). Different hormonal treatment did not significantly affect Y-27632 IC50s (shared IC50 = 5.6⫾1.2 ¥ 10-8 M). The relaxant effect of Y-27632 was also confirmed in ET-1 precontracted human epididymal strips from male transsexual individuals who received cyproterone acetate and estrogen treatment for at least 1 year before undergoing transgender reassignment surgery (Emax = 19.2% ⫾ 10.7% and IC50 = 7.0 ⫾ 7.5 ¥ 10-7 M, see Figure 1D).

Effect of Hormonal Milieu on ROCK1 Expression in Rabbit Epididymis Localization of ROCK1 protein in transversal sections of epididymal tissues was studied by immunohistochemistry (see Figure 2), using a specific antibody against the expected 160 kDa band in epididymal lysates (see Figure 3A). By using this antibody, an intense positive ROCK1 staining was observed in the smooth muscle component of epididymal ducts and in the vessels for each animal group, although to a different extent (Figure 2). Immunopositivity appeared markedly reduced in hypogonadal animals (Figure 2B), as compared with controls (Figure 2A). ROCK1 specific staining was partially restored by T supplementation (Figure 2C) and completely restored by estrogens (Figure 2D). Similar results were obtained in two other separate experiments. The specificity of the ROCK1 immunostaining was confirmed by a representative negative control image performed in the absence of the primary antibody (Figure 2E). Quantitative analysis of ROCK1 protein changes was derived from western studies and showed in Figure 3A. ROCK1 was significantly reduced in epididymal extracts from GnRH analog-treated animals (P < 0.05 vs. control rabbits). T supplementation restored ROCK1 expression up to the control level, although estrogens administration dramatically up-regulated it (P < 0.01 vs. control). A quantitative analysis of mRNA expression by quantitative RT-PCR (qRT-PCR) essentially confirmed western findings (Figure 3B). J Sex Med 2009;6:2173–2186

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Effect of Estrogens on RhoA/ROCK Pathway in Human Epididymal Smooth Muscle Cells The estrogen-induced activation of RhoA/ROCK pathway observed in rabbit epididymis was also tested in primary cultures of human epididymal smooth muscle cells. ERa and ERb mRNA expression in epididymal cells was demonstrated by qRT-PCR and compared with human estrogendependent tissues (epididymis, uterus, and intestine; Figure 4E). As illustrated in Figure 5A, a 72-hourh treatment with 17b-E2 (1 nM) was able to significantly increase ROCK1 protein expression (P < 0.05 vs. control). Estrogen-mediated ROCK1 up-regulation was also confirmed by qRT-PCR (P < 0.05 vs. control), where P450Ar gene up-regulation was used as positive control (P < 0.05 vs. control) [32]. In contrast, 17b-E2 J Sex Med 2009;6:2173–2186

Figure 2 Effect of hormonal milieu on rho-kinase 1 (ROCK1) immunolocalization in transversal sections of rabbit epididymis corpus (magnification 80x). (A) In control animals specific ROCK1 staining is present in smooth muscle cells surrounding epididymal duct and the vessels; (B) in hypogonadal animals ROCK1 immunopositivity is dramatically reduced; (C) ROCK1 staining is partially restored by testosterone (T) supplementation in hypogonadal animals; (D) estradiol valerate (E2v) supplementation fully restored ROCK1 immunopositivity in the smooth muscle component of epididymal ducts, in the fibromuscolar stroma and in the vessels; (E) representative negative control image, performed incubating the section with the secondary IgG in the absence of the primary anti-ROCK1 antibody.

treatment did not change RhoA and ROCK2 mRNA expression (Figure 5B). To investigate on estrogen-induced functional activation of RhoA/ROCK signaling, we measured ROCK activity by evaluating phosphorylation of MYPT-1, the regulatory subunit of myosin light chain phosphatise [33]. Both 17b-E2 and ET-1, a well-known activator of RhoA/ROCK pathway in smooth muscle cells, enhanced MYPT-1 phosphorylation at residue Thr-853 (Figure 6A; P < 0.05 and P < 0.01 vs. control, respectively). Estrogenic dependency of RhoA/ROCK signaling was further validated by analysis of cell migration in a P48 multi-well Boyden chamber (Figure 6B). In fact, cell migration is a RhoA/ROCK-dependent cellular process [25]. A 72-hour-pretreatment of epididymal smooth muscle cells with 1 nM T or 17b-E2 was able to promote their basal migration

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Figure 3 Effect of hormonal milieu on rho-kinase 1 (ROCK1) expression in rabbit epididymis. (A) Western blot analysis of ROCK1 protein expression in epididymal samples obtained from control (N = 8; open bars), hypogonadal (N = 8; closed bars), testosterone (T)-supplemented hypogonadal (N = 7; diagonally hatched bars) and estradiol valerate (E2v)supplemented hypogonadal (N = 7; horizontally hatched bars) rabbits. The upper panel shows the densitometric evaluation of ROCK1 protein positivity calculated after normalization of band intensity over a-tubulin positivity and expressed as percentage over the control taken as 100%. Data are expressed as mean ⫾ standard error of the mean. Lower panel: representative western blots. Molecular weight markers (kDa) are indicated to the left of the blots. *P < 0.05 and **P < 0.01 vs. control rabbits. (B) ROCK1 mRNA expression in epididymal samples obtained from control (N = 6; open bars), hypogonadal (N = 6; closed bars), T-supplemented hypogonadal (N = 6; diagonally hatched bars) and E2v-supplemented hypogonadal (N = 6; horizontally hatched bars) rabbits, obtained by real-time reverse trasnscriptase-polymerase chain reaction. Results are expressed as mean ⫾ standard error of the mean of arbitrary units calculated according to the comparative Ct method and using the 18S ribosomal RNA subunit expression as housekeeping gene for normalization, and are reported as percentages of expression of control rabbits. **P < 0.01 vs. control animals.

(P < 0.05 and P < 0.0001 vs. control, respectively). The effect of 17b-E2 was significantly higher than that one of T (P < 0.0001), which was completely abolished by simultaneous letrozole incubation (P < 0.0005 vs. T). Inhibition of RhoA/ROCK pathway by 5-hour treatment with C3 exoenzyme (a selective inhibitor of RhoA through ADPribosylation; 1 mg/mL) or Y-27632 (25 mM) completely abrogated 17b-E2-induced migration (P < 0.0001 vs. 17b-E2), that was reduced it even below the basal level (P < 0.005). Discussion

Our results provide a possible explanation of the previously observed estrogen-driven regulation of epididymal motility [1,8,14]. We essentially found that estrogens up-regulate the calcium-sensitizing RhoA/ROCK pathway in smooth muscle epididymal cells. Calcium sensitization is a process

whereby there is a greater contractile force generated from a given increase in cytosolic calcium, and RhoA/ROCK signaling represents one of the most important modulator of this process, through an increase in myosin light chain phosphatase enzymatic activity [33]. We found that epididymal smooth muscle cells express ROCK-1, and this expression is sex steroid-regulated. In fact, we described an estrogen-induced up-regulation of ROCK1 gene and protein expression, which was associated to an increased kinase activity, as demonstrated by the estrogen-dependent increased ROCK-dependent phosphorylation of MYPT-1, the regulatory subunit of myosin light chain phosphatase. MYPT1 phosphorylation determines conformational changes in the catalytic domain of the enzyme, resulting in its inhibition and thereby in the enhanced and sustained actomyosin crossbridging with subsequent force production [33]. The RhoA/ROCK-promoted remodeling of the J Sex Med 2009;6:2173–2186

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Figure 4 (A–D) Characterization of primary human epididymal smooth muscle cells by immunohistochemical staining with specific antibodies for the smooth muscle marker desmin (A) and a-smooth muscle actin (B) and for the other markers vimentin (C) and cytokeratin (D). For the two smooth muscle-related markers, the percentage of positive cells (calculated by counting the number of stained cells over the total number in 10 fields for each slide) is 86.9 ⫾ 1.7 and 77.7 ⫾ 2.4, respectively. Data are expressed as mean ⫾ standard error of the mean. Original magnification: 20x. (E) Estrogen receptor a (ERa; closed bars) and ERb (open bars) mRNA expression in human epididymal smooth muscle and several human tissues (epididymis, uterus and intestine; N = 3), obtained by quantitative reverse transcriptase-polymerase chain reaction. Results are reported as mean ⫾ standard error of the mean of arbitrary units calculated according to the comparative Ct method and normalized over 18S ribosomal RNA subunit expression.

actin cytoskeleton also supports the stimulation of epididymal cells pro-migratory properties by estradiol that we described. Estrogens pro-contractile properties are well established in the female genital tract. In fact, in several species, including humans, they are primarily involved in the physiological control of myometrial spontaneous activity [34] and in the control of myometrial transition from a relaxed state—during pregnancy—to a highly contractile state—at parturition [35–38]. Several studies demonstrated that uterotonic agents increase myometrial contraction not only by increasing intracellular calcium, but also by improving calcium sensitivity of myometrial force production via the RhoA/ROCK pathway [39–42]. An estrogen-driven up-regulation of ROCK expression in the murine [42] and human [43] pregnant J Sex Med 2009;6:2173–2186

myometrium has been described. During pregnancy, uterine muscle is relatively refractory to ROCK inhibition. In fact, in OT pre-contracted strips, Y-27632 was less effective [43]. This scenario was completely reverted at parturition. A rather similar change was observed in this study. Sensitivity to Y-27632 was dramatically affected by the estrogens’ milieu in epididymis. Decreasing estrogens by letrozole or GnRH analog resulted in a lower sensitivity to the relaxant effect of Y-27632, which was reverted by E2v, but not T, supplementation. In our view, the regulation of epididymal and uterine motility appears very similar and both express OTR and ETA in their smooth muscle component [7,13,34,44]. A recent study shows that estradiol is able to promote breast cancer cell invasion and metastatization through RhoA/ROCK signaling [45].

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Figure 5 Effect of a 72-hour treatment with 1 nM 17b-estradiol (17b-E2) on rho-kinase (ROCK) expression in human epididymal smooth muscle cells. (A) Western blot analysis of ROCK1 protein expression. The upper panel shows the densitometric evaluation of ROCK1 protein positivity calculated after normalization of band intensity over Stat1 positivity and expressed as percentage over untreated cells (open bars) taken as 100%. Data are expressed as mean ⫾ standard error of the mean from three different experiments. A representative ROCK1 blotting is shown in the lower panel. *P < 0.05 vs. control. (B) RhoA, ROCK1, ROCK2, and P450 aromatase (P450Ar) mRNA expression in untreated (open bars) and 17b-E2-treated (closed bars) cells, obtained by real-time reverse transcriptase-polymerase chain reaction. Results are reported as mean ⫾ standard error of the mean of arbitrary units calculated according to the comparative Ct method and normalized over 18S ribosomal RNA subunit expression from 3 separate experiments. *P < 0.05 vs. control.

Our results are in agreement with this finding, showing an estrogen-dependent RhoA/ROCKinduced regulation of epididymal cell motility. Interestingly, we observed also an up-regulation of P450Ar mRNA expression in epididymal smooth muscle cells following estradiol treatment. This finding is in agreement with results recently obtained in human endometrial smooth muscle cells [32], suggesting that estrogens overdraw their epididymal actions through an inductive loop, which involves up regulation of their conversion from androgens. In the adult male, fertility and sexual functions are androgen-dependent. However, it is well accepted that also estrogens, the “female hormones,” are able to affect proliferation, differentiation, and function of the male reproductive system, allowing the maintenance of its structural and functional integrity [20,46]. Although estrogens levels are relatively low in male blood, they are extraordinarily abundant in semen and rete testis fluids, where their concentration is even higher than female blood [47,48]. Epididymis and efferent ductules of the testis express high amounts of ERa and ERb [18,19,49]. Estrogens

binding allows epithelial reabsorption of water, ions, and proteins from the ductal lumen, in order to concentrate sperm and improve spermatozoa survival and maturation during epididymal storage [20]. Accordingly, human ERa and P450Ar gene mutations decrease sperm counts and result in poor sperm viability [20]. In addition, tamoxifen, which acts as an estrogen agonist in several districts including epididymis [8], has been used to increase sperm counts in idiopathic oligospermic men, and it has been recently proposed by a World Health Organization working committee as the first line of treatment for idiopathic oligospermia [50]. Along with its supposed central effect in stimulating gonadotropin release, tamoxifen may also act at peripheral level, stimulating epididymal sensitivity to contractile agents through RhoA/ROCK up-regulation, although this finding needs appropriate experimental confirmation. In conclusion, our data confirm that epididymis is a male target for estrogens, which contribute to the maintenance of its structural and functional integrity [20,46]. We originally demonstrate that estrogens regulate epididymal motility tuning up J Sex Med 2009;6:2173–2186

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therefore for the first phase of ejaculation process. OT and ET-1 are two of the best-characterized factors that have been demonstrated to create an autocrine/paracrine contractile loop in the epididymis [1]. Mutual, feed forward, interactions between these contractile agents is supposed to support the autonomous peristaltic movements of the epididymis that favor sperm progression throughout the duct [1]. We now provide evidence that estrogens (and not androgens) are essential for maintaining epididymal sensitivity to these locally produced contractile peptides, and even to noradrenaline, by up regulating the calcium-sensitizing pathway of RhoA/ROCK.

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Corresponding Author: Mario Maggi, MD, v.le Pieraccini 6, 50139, Florence (Italy). Tel: +39554271415; Fax: +39554271371; E-mail: [email protected]. IT

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Conflict of Interest: None declared. e st o le ste tro ro zo ne le + 17 bes tra di ol 17 bes tr C3 adio l+ 17 be Y- stra 27 d i 63 ol 2 +

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Figure 6 Effect of a 72 h-treatment with sex steroids on rho-kinase (ROCK) activity and on migration in human epididymal smooth muscle cells. (A) Cells were treated with endothelin-1 (ET-1; 100 nM; positive control; horizontally hatched bars) or 17b-estradiol (17b-E2, 1 nM; closed bars), then the western blot analysis of myosin phosphatase target subunit 1 (MYPT-1) phosphorylation was performed with a specific anti-phospho-MYPT-1 (Thr-853) antibody. The upper panel shows the densitometric analysis of P-MYPT-1 protein positivity normalized over non-phosphorylated MYPT-1 expression. Data, expressed as percentage over untreated cells (open bars) taken as 100%, are reported as mean ⫾ standard error of the mean from three different experiments. Representative P-MYPT-1 and MYPT-1 blottings are shown in the lower panel. *P < 0.05 and **P < 0.01 vs. untreated cells. (B) Cells were treated with 1 nM testosterone (T, diagonally hatched bars), 1 nM T with the addition of 10 mM letrozole (horizontally hatched bars) or 1 nM 17b-E2 (closed bars), then their basal migration was evaluated in a P48 multi-well Boyden chamber. The effect of C3 exoenzyme (1 mg/mL; vertically hatched bars) and Y-27632 (25 mM; grey bars) on 17b-E2-pretreated cells migration was also tested. Results are expressed as percentage over untreated cells (open bars) taken as 100% and reported as mean ⫾ standard error of the mean from three different experiments. *P < 0.05, **P < 0.005 and ***P < 0.0001 vs. control; °P < 0.0005 vs. T; ∧P < 0.0001 vs. 17b-E2.

contractile hormones and local peptide responsiveness by increasing RhoA/ROCK signaling, and therefore calcium sensitivity. This calcium sensitization might be crucial for epididymal motility and J Sex Med 2009;6:2173–2186

Statement of Authorship

Category 1 (a) Conception and Design Benedetta Fibbi; Sandra Filippi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi (b) Acquisition of Data Benedetta Fibbi; Sandra Filippi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi (c) Analysis and Interpretation of Data Benedetta Fibbi; Sandra Filippi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi

Category 2 (a) Drafting the Article Benedetta Fibbi; Sandra Filippi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi (b) Revising It for Intellectual Content Benedetta Fibbi; Sandra Filippi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi

Estrogens and RhoA/ROCK Pathway in Epididymis

Category 3 (a) Final Approval of the Completed Article Benedetta Fibbi; Sandra Filifpi; Annamaria Morelli; Linda Vignozzi; Enrico Silvestrini; Aravinda Chavalmane; Giulia de Vita; Mirca Marini; Mauro Gacci; Chiara Manieri; Gabriella Barbara Vannelli; Mario Maggi References

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