International Journal of Impotence Research (2003) 15, 397–404 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 www.nature.com/ijir Sex hormones differentially regulate nitric oxide synthase and arginase activities in the proximal and distal rabbit vagina

AM Traish1,2, NN Kim2, Y-H Huang2, K Min2, R Munarriz2 and I Goldstein2

1Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA; and 2Department of Urology, Institute for Sexual Medicine, Boston University School of Medicine, Boston, MA, USA

Nitric oxide synthase (NOS) and arginase have been shown to regulate nitric oxide (NO) production reciprocally in genital tissues. In animal models, NO is an important regulator of vaginal blood flow and vaginal wall contractility. In this study, we investigated the modulation of NOS and arginase activities by and in the proximal and distal rabbit vagina. In intact control animals, total NOS activity was higher in the proximal (528 7 78 pmol/mg protein) than the distal (391 7 44 pmol/mg protein) vagina. However, arginase activity was higher in the distal (206 7 8 nmol/mg protein) than the proximal (64 7 5 nmol/mg protein) vagina. Ovariectomy enhanced NOS activity in the proximal but not distal vagina with concomitant decrease in arginase activity in both the proximal and distal vagina. In ovariectomized rabbits, replacement with 5a- (DHT) or D5- (Adiol) increased NOS activity beyond that observed in ovariectomized rabbits receiving vehicle. In contrast, DHT and Adiol treatment reduced arginase activity more than that of the ovariectomized rabbits receiving vehicle. exhibited inconsistent effects on NOS and arginase activity in the distal and proximal vagina. replacement in ovariectomized animals reduced NOS activity in the proximal vagina down to levels that were comparable to intact control animals. However, estradiol positively modulated arginase activity in the distal vagina. Western blot analyses indicated that in the proximal vagina, neural NOS protein levels paralleled the changes observed in enzyme activity. These observations suggest that steroid hormones differentially regulate NOS and arginase activities of the proximal and distal regions of the vagina. Although treatment reduced total NOS activity in proximal vagina, estrogens are known to enhance vaginal blood flow. This paradoxical observation may be explained by differential regulation of n-NOS and e-NOS in the proximal and distal vagina. We suggest that changes in vaginal blood flow and compliance may depend on the endocrine status and the levels of circulating androgens and estrogens. International Journal of Impotence Research (2003) 15, 397–404. doi:10.1038/sj.ijir.3901097

Keywords: vagina; androgens; estrogens; nitric oxide synthases; arginase; female genital sexual arousal

Introduction remains poorly understood.5–14 In menopause, reduced circulating estrogens may be associated with vaginal dryness, irritation, genital pain, dimin- Female genital sexual arousal is a physiological ished sexual desire, decreased genital sensation process modulated by the hormonal milieu and and difficulty achieving orgasm.15,16 Further, neurovascular input, resulting in increased genital decreased pelvic blood flow resulting in decreased blood flow, increased vaginal lubrication and length, congestion in genital tissue during sexual arousal, 1–4 and decreased vaginal luminal pressure. Sex increased clitoral fibrosis, thinning of vaginal steroid hormones play an important role in repro- epithelial layers and decrease in vaginal submucosal ductive physiology. However, the role of estrogens vasculature are attributed in part to loss of estro- and androgens in female genital sexual arousal gens.7,12,14,17–19 Estrogen replacement in women has been reported to increase pelvic blood flow and restore the structural and functional integrity of vaginal tissue.12–14,18 *Correspondence: AM Traish, PhD, Boston University Although androgens are critical for development, School of Medicine, 700 Albany Street, Room W607, growth and maintenance of secondary sex charac- Boston, MA 02118, USA. 5,6 E-mail: [email protected] teristics, their role in female genital sexual arousal Received 20 March 2003; revised 16 June 2003; accepted remains controversial. Treatment of ovariectomized 22 July 2003 animals with testosterone or 5-a-dihydrotestoster- Nitric oxide synthase and arginase in the vagina AM Traish et al 398 one (DHT) increased vaginal weight and mucifica- at a rate of 200 mg/day and androgens or progester- tion in the absence of estrogen replacement.20,21 one at a rate of 100 mg/day. For ovariectomized (DHEA), applied topically animals undergoing replacement with E+TorE+P, to the vagina of ovariectomized rats, showed two pumps were implanted. In this study, our significant effects on vaginal epithelium.22 The objective was to determine the effects of ovariect- effects of DHEA were attributed to conversion of omy and replacement with a single dose of sex DHEA to estrogens by vaginal cells. These observa- on vaginal NOS and arginase. To tions suggest that androgens modulate vaginal this end, we did not attempt to investigate a range of physiology. dose–responses to steroid hormones. However, we The nitric oxide synthase (NOS)/cGMP pathway chose doses of hormones estimated to provide has been implicated in regulating vaginal smooth plasma hormone concentrations between 20 and muscle contractility and blood flow.23,24 We have 50 nM in the rabbit. recently reported on the physiological role of NO in modulating vaginal blood flow25 employing inhibi- tors of NOS and guanylyl cyclase. We demonstrated that the NO/cGMPathway modulates pelvic nerve- Vaginal tissue dissection mediated blood flow in the vagina. Furthermore, the activity of NOS has been shown to be modulated by The uterovaginal tract was removed en bloc, the estrogens26–30 and androgens.31–34 NOS activity may vaginal tissue was dissected, cleaned of mesenteric also be regulated by limiting the availability of the tissue and cut open longitudinally. A squamo- substrate L-arginine by the enzyme arginase, which 35 columnar junction located between the upper and metabolizes L-arginine to urea and orni-thine. If lower vagina was identified and used as a divide arginase activity is regulated by steroid hormones, it between upper (proximal) and lower (distal) re- would suggest that steroid hormones could also 37 gions. Proximal and distal vaginal tissues were regulate NOS in an indirect manner. A recent study frozen in liquid nitrogen and pulverized. The tissue by Cama et al36 showed that inhibition of arginase powder from each group of animals was pooled and improved vaginal blood flow in the animal model. stored at À701C for biochemical assays. Thus, NOS and arginase could serve as biochemical markers for assessing androgenic or estrogenic activity of sex steroid hormones in the vagina. This study was undertaken to determine differential Determination of total (neural and endothelial) regulation of total NOS and arginase activities in NOS activity in rabbit vaginal tissue extracts the proximal and distal vagina by estrogen or replacement in the ovariectomized rabbit model. Total NOS activity (neural-NOS (n-NOS) and endothelial-NOS (e-NOS) in the tissue extract 14 was determined by conversion of L-[ C]arginine 14 38–40 Experimental to [ C]citrulline, as described previously. Briefly, aliquots of the tissue extract were incubated with tetrahydrobiopterin (3 mM), calmodulin (30 U/ 14 Animals ml), L-arginine (50 mM), L-[ C]arginine (2 mCi/ml), NADPH (2 mM) and CaCl2 (1 mM) at 371C for 45 min or 21C for 45 min. Non-specific enzymatic activity All protocols were approved by the Institutional was determined in parallel samples incubated with Animal Care and Use Committee at Boston Uni- 300 mMofL-NAME. Citrulline was separated from versity School of Medicine. Female New Zealand arginine using 1 ml columns of AG50W-X8 resin White rabbits (3.5–4.0 kg) were divided into several (Bio-Rad Laboratories, Hercules, CA, USA) and groups (5 animals per group) and kept either intact quantified by scintillation spectroscopy. Citrulline (control; C) or ovariectomized. At 2 weeks post- production was normalized to total soluble protein ovariectomy, animals were treated for a 2-week in the tissue. The activity measured in the presence period with vehicle (polyethylene glycol; V), testos- of the inhibitor L-NAME was subtracted from the terone (T), DHEA, DHT, D5-3b,17b androstenediol total activity to account for only the specific NOS (Adiol), estradiol (E), a combination of estradiol and activity. It should be noted that the total activity testosterone (E+T) or estradiol plus measured in this assay reflects both n-NOS and e- (E+P). Hormonal treatment of ovariectomized ani- NOS activities. Based on preliminary experiments, mals was carried out by implanting osmotic infusion we found that removal of endogenous arginine by pumps (model 2002; Alzet, Palo Alto, CA, USA) exchange chromatography did not influence the subcutaneously between the scapulas in a sterile measured activity. Similar observations were manner. The concentration of the steroid was reported in rabbit vagina tissue extracts.27 Further, calculated so that the pump would deliver estradiol we found that inducible NOS activity (-

International Journal of Impotence Research Nitric oxide synthase and arginase in the vagina AM Traish et al 399 independent) in the vagina of ovariectomized lulose membranes, samples were incubated with animals comprised less than 10% of the total mouse anti-rat n-NOS monoclonal antibody. The activity, in agreement with previous reports.41 To membranes were washed and incubated with horse- this end, we did not pursue measurements of iNOS radish peroxidase-linked goat anti-mouse IgG (sec- activity in this study. ondary antibody, Pierce Chemical Co., Rockford, IL, USA). Membranes were developed with an en- hanced chemiluminescence (ECL) kit and exposed to autoradiography film to obtain visible bands at Arginase activity in rabbit vaginal tissue 160 kDa. Rat pituitary lysate was used as positive control (10 mg/lane; rat neural n-NOS). The band intensities were quantified by scanning. It should be Crude tissue extracts were derived from homoge- noted that this assay provides estimate of n-NOS nates of rabbit vaginal tissue. Arginase enzyme only. activity in cytosolic extracts of vaginal tissue was assessed by the method of Ru¨ egg and Russell, as previously described.35 Briefly, 10 ml of tissue extract (triplicate aliquots) was incubated in buffer Data analysis (75 mM glycine, pH 9.0, 0.25 mM MnCl2) containing 14 300 000 dpm of [ C-guanidino]-L-arginine (51.5 mCi/mmol; NEN Life Science Products, Boston, Data were subjected to ANOVA. If the ANOVA MA, USA), 4 mM of unlabelled L-arginine in a final P-value was less than 0.05, multiple paired compar- volume of 100 ml. Samples were incubated for isons were performed using the Tukey–Kramer 43 60 min at 371C and reactions were terminated by test. Data were considered to be significantly the addition of 400 ml of 0.25 M acetic acid (pH 4.5), different when the two-tailed P-value was less than 7 M urea, 10 mM L-arginine. After the addition of 0.05. 500 ml of water, samples were passed through a 0.5 ml column of Dowex 50W-X8 resin (Bio-Rad Laboratories, Hercules, CA, USA). Tubes were Results rinsed twice with 500 ml of water and both rinses were poured onto the columns. Columns were washed with an additional 1 ml of water and all Effects of androgens on total NOS (e-NOS and effluent was collected in 20-ml vials. After the n-NOS) activities in proximal and distal addition of 16 ml of Liquiscint (National Diagnos- rabbit vagina tics, Atlanta, GA, USA), radioactivity was quantified by liquid scintillation spectroscopy. Urea produc- tion (pmol/min) was normalized to total soluble In tissue from control animals, total NOS activity protein in the tissue. was higher in the proximal (528778 pmol/mg protein) versus the distal vagina (391744 pmol/mg protein). As shown in Figure 1, ovariectomy enhanced total NOS activity in the proximal vagina, Determination of neural n-NOS protein expression but with little changes in the distal vagina. Treat- by Western blot analyses ment of ovariectomized animals with testosterone, DHT or Adiol further increased total NOS activity in Protein expression of n-NOS in vaginal tissue was the proximal vagina with DHEA having no signifi- determined by Western blot analyses, using isoform- cant effect. In the distal vagina, no significant specific monoclonal antibodies (Transduction changes in total NOS activity were observed by Research, Paducah, KY, USA), as described by ovariectomy or treatment with T or DHEA. However, Traish et al.39 Rabbit proximal or distal vaginal treatment with DHT or Adiol produced significant tissue powder was homogenized in 20 mM HEPES increase in total NOS activity. buffer (pH 7.2) containing 0.32 M sucrose, 0.5 mM EDTA and 1 mM DTT, and a cocktail of enzyme inhibitors including phenylmethyl sulfonyl fluor- Effects of estrogen on total NOS activity in proximal ide, aprotinin, leupeptin, pepstatin and bacitra- and distal rabbit vagina cine.42 The homogenates were centrifuged at 800 g for 30 min at 41C to obtain crude cytosolic extracts. Aliquots of each extract were assayed for protein As shown in Figure 2, treatment of ovariectomized concentration by the method of Lowry. Identical animals with estradiol with or without T or P amounts of protein (100 mg total protein/lane) from decreased NOS activity in the proximal vagina each extract were electrophoresed on 7.5% SDS relative to vehicle-treated, ovariectomized animals polyacrylamide gels. Following transfer to nitrocel- and normalized values relative to control. No signi-

International Journal of Impotence Research Nitric oxide synthase and arginase in the vagina AM Traish et al 400 Proximal Vagina 2000 + * + * * 1500 * 1000

NOS Activity 500 (pmol citrulline/mg prot) 0 C V T DHEA DHT Adiol

Ovariectomized

Distal Vagina 1000 + * 800 * 600

400 NOS Activity 200 (pmol citrulline/mg prot) 0 C V T DHEA DHT Adiol

Ovariectomized Figure 2 Effects of E, on NOS activity in rabbit proximal and Figure 1 Effects of androgens on NOS activity in rabbit proximal distal vagina. Proximal and distal vaginal tissues were obtained and distal vagina. Proximal and distal vaginal tissues were from intact animals (C) or ovariectomized animals treated with V, obtained from intact animals (control, C) or ovariectomized E, E+TorE+P. The tissues were processed for NOS activity as animals treated with V, T, DHEA, DHT or D5-androstenediol described in Figure 1. Data are the mean7s.e.m. of three (Adiol). The tissues were pulverized, pooled and homogenized. independent assays. *Po0.05 versus control; +Po0.05 versus The tissue extracts were assayed for NOS activity by conversion of vehicle. 14 14 L-[ C]arginine to [ C]citrulline and NO. Data are the mean7- s.e.m. of three independent assays. *Po0.05 versus control; +Po0.05 versus vehicle. (Figure 3a). No differential changes were observed in the distal vagina with androgen or estrogen ficant changes were observed in the distal vagina treatment (Figure 3c). Analyses of optical density with estradiol treatment. This observation suggests from several experiments showed consistent trends that estrogens differentially regulate e-NOS and n- of reduced expression with estrogens and increased NOS activities in the proximal and distal vagina. expression with androgens; however, no statistical This may have an important implication in the significance was reached. physiological function of these two compartments.

Effects of androgens on arginase activity Effects of androgens and estrogen on n-NOS protein in rabbit vagina expression in proximal and distal rabbit vagina Arginase activity was more predominant in the n-NOS protein expression in the proximal vagina distal (20678 nmol/mg protein) than in the proxi- was increased by ovariectomy and androgen treat- mal (6475 nmol/mg protein) vagina. Ovariectomy ment, but was reduced by estrogen treatment (Figure reduced arginase activity in proximal and distal 3a and b). Treatment of ovariectomized animals with vagina (Figure 4). Treatment of ovariectomized androgens (DHT or Adiol) resulted in increased NOS animals with DHEA, DHT or Adiol further reduced protein expression in the proximal vagina arginase activity in the distal and proximal vagina.

International Journal of Impotence Research Nitric oxide synthase and arginase in the vagina AM Traish et al 401

Figure 3 Effects of androgens and estrogens on NOS expression in rabbit proximal and distal vagina. Protein concentrations in the extracts of the proximal and distal vagina were determined and equal amounts of proteins from proximal and distal vagina extracts were subjected to SDS/PAGE. The proteins were then transferred onto nitrocellulose membrane and immunoblotted with isoform-specific antibody against n-NOS. The protein bands at approximately 160 kDa represent the n-NOS protein band. Panel a: Representative Western blot of n-NOS in proximal vaginal tissue from intact or ovariectomized animals, as described in Figures 1 and 2. Panels b and c: Densitometric scanning of n- NOS protein bands. Data were normalized to control samples and expressed as the percentage change in tissues from ovariecto- mized animals treated with V, E, T or E+T. Figure 4 Effects of androgens on arginase activity in rabbit proximal and distal vagina. Proximal and distal vaginal tissues were obtained from intact or ovariectomized animals (see Figures 1 and 2). The tissues were pulverized, pooled and homogenized. Interestingly, treatment with T produced an increase The tissue extracts were assayed for arginase activity by 14 14 in arginase activity in the proximal vagina while conversion of L-[ C]arginine to [ C]urea and ornithine. Data are the mean7s.e.m. of three independent assays. *Po0.05 decreasing arginase activity in the distal vagina. + versus control; Po 0.05 versus vehicle.

Effects of estrogen on arginase activity and the role of NO/cGMP in modulating clitoral and in rabbit vagina vaginal function has been the subject of several studies.23,26–30,41,44–46 Recent in vitro organ bath studies44 have shown that EFS elicited NANC Treatment of ovariectomized animals with estradiol relaxation responses in the rabbit clitoral corpus alone or in combination with T or P resulted in cavernosum smooth muscle and these responses are significantly increased arginase activity in the distal inhibited with L-NAME and ODQ, suggesting a role vagina (Figure 5), whereas in the proximal vagina, for NO/cGMP pathway in clitoral physiology. estradiol replacement with or without T did not Studies with rabbit vaginal strips in organ bath change arginase activity in ovariectomized animals. studies47 have suggested that the NANC neurotrans- E in combination with P resulted in reduced mitter does not involve NO. On the other hand, arginase activity compared to vehicle-treated 23 Giraldi et al presented data from organ bath ovariectomized animals. studies in rat vagina suggesting a key role for the NO/cGMP pathway in vaginal smooth muscle relaxation. However, neither the studies in the 44 23,40 Discussion clitoris nor that of the vagina demonstrated changes in blood flow in response to NO in vivo. Min et al24 showed that in the animal model, Nitric oxide (NO) has been recognized as a mediator administration of sildenafil enhanced vaginal and of nonadrenergic noncholinergic neurotransmission clitoral blood flow in response to pelvic nerve in many tissues, including the urogenital organs, stimulation. This observation lends further support

International Journal of Impotence Research Nitric oxide synthase and arginase in the vagina AM Traish et al 402 by sex steroid hormones in the various anatomical regions of the rabbit vagina. Treatment of ovariecto- mized animals with T resulted in increased expres- sion and activity of total NOS in the proximal vagina but not in the distal vagina. In contrast, treatment with DHT resulted in increased expression and activity of total NOS in both the proximal and distal vagina. Interestingly, treatment of ovariectomized animals with Adiol increased total NOS activity in proximal and distal vagina, similar to that obtained with DHT. These observations suggest that Adiol may be converted into DHT and modulates NOS activity in the vagina via an androgenic mechanism. Treatment of ovariectomized animals with E or E combined with T or P normalized NOS activity in the proximal vagina, to that obtained in control animals, while little or no effect was observed in the distal vagina. The downregulation of total NOS activity by estrogens in the proximal vagina is consistent with those reported previously25–28 in vagina of the rat and rabbit models. These observa- tions further suggest that in the proximal vagina, total NOS activity is modulated by androgens and estrogens in a reciprocal manner. n-NOS protein expression in the proximal and distal vagina paralleled enzymatic activity. Ovar- iectomy and androgen treatment increased n-NOS protein expression in the proximal vagina but not in the distal vagina. Estrogen treatment of ovariecto- mized animals decreased protein expression in the Figure 5 Effects of estadiol on arginase activity in rabbit proximal vagina. It remains to be determined if e- proximal and distal vagina. Proximal and distal vaginal tissues NOS isoform is equally regulated by estrogen and were obtained from intact or ovariectomized animals (see Figures androgens in proximal and distal vagina. 1 and 2). The tissues were processed for arginase activity as Vaginal arginase activity was reduced by ovari- described in Figure 4. Data are the mean7s.e.m. of three + ectomy and upregulated by estrogen replacement in independent assays. *Po0.05 versus control; Po0.05 versus vehicle. the distal vagina, suggesting that arginase is under regulation by estrogen hormones. Treatment of ovariectomized animals with DHEA, Adiol or DHT to the in vitro studies with tissues strips in organ resulted in decreased arginase activity in the baths. Recently, Kim et al25 demonstrated, in the proximal and distal vagina. Interestingly, treatment animal model, that administration of NOS and with testosterone increased arginase activity in the guanylyl cyclase inhibitors diminished vaginal proximal vagina but not in the distal vagina. At blood flow in response to pelvic nerve-stimulation. present, we do not know why T produced different These observations implicate NO/cGMP as one of effects from those obtained with Adiol or DHT. the key mediators of vaginal smooth muscle relaxa- Treatment of ovariectomized animals with E or E in tion and pelvic nerve-stimulated increase in blood combination with T or P increased arginase activity flow. in the distal vagina. These observations suggest that, In this study, we demonstrated that NOS and in vaginal tissue, arginase activity is modulated by arginase are differentially distributed among the two androgens and estrogens in a tissue- and region- distinct anatomic regions of the rabbit vagina. Total specific manner. NOS (n-NOS and e-NOS) activities were higher in It has been suggested that D5-Adiol mediates its proximal than in distal vagina. Arginase activity, activity by binding to the estrogen and androgen however, was greater in distal than in proximal receptors. If D5-Adiol binds to estrogen receptors vagina. The physiological significance for this and mediates estrogen-dependent activity, it would unique anatomical distribution of these enzymes, be expected to produce similar effects to those in this animal model, is unknown at present. obtained by E treatment, namely reduction of NOS Ovariectomy enhanced total NOS activity in the activity and increased arginase activity. To the proximal vagina with concomitant decrease in contrary, the data presented here show that D5- arginase activity in the proximal and distal vagina, Adiol produced effects similar to those obtained suggesting differential regulation of these enzymes with 5a-DHT. These observations indicate that D5-

International Journal of Impotence Research Nitric oxide synthase and arginase in the vagina AM Traish et al 403 Adiol acted specifically as an androgen in the rabbit vaginal vasocongestion. However, changes in e-NOS vagina. activity in the distal vagina may be confounded by Previous work from this laboratory and others the relative abundance of NOS isoforms from indicated that the NO/cGMP pathway may be various cellular elements (eg epithelial, smooth involved in increasing vaginal blood flow and muscle, neural and endothelial). This research vaginal wall compliance.23224 Recent studies have question needs to be clarified by future studies on shown that the NO/cGMP pathway is critical in the cellular and regional distribution of NOS iso- modulating pelvic nerve-stimulated vaginal blood forms in the proximal and distal regions of the flow in the rabbit model.25 Thus, understanding the vagina using immunohistochemical techniques or regulation of NOS activity and other factors that may selective inhibitors of varying NOS isoforms. affect this pathway is of physiological importance in In summary, we have characterized the distribu- peripheral sexual arousal. tion of NOS and arginase activities in the proximal Yoon et al30 have demonstrated increased clitoral and distal rabbit vagina and evaluated the effects of and vaginal e-NOS and n-NOS protein expression sex steroid hormone deprivation and replacement and activity in ovariectomized rabbits, and expres- on these enzymes. Androgens enhance total NOS sion and activity of NOS was reduced by estrogen activity in the proximal and distal vagina and administration. Since pelvic nerve-stimulated blood reduce arginase activity. Estrogens reduce total flow to the clitoris and vagina requires estrogen NOS activity in the proximal vagina and increase priming,45 it remains unclear how changes in NOS arginase activity in the distal vagina. These observa- expression and activity in response to estrogens tions suggest that anatomical distribution of these correlate with blood flow in these genital organs. key metabolic mediators and the circulating levels of The observations that estrogens reduced NOS androgens and estrogens may modulate the changes activity in the proximal vagina may appear para- in vaginal blood flow and compliance. doxical in light of the important role of estrogens in regulating vaginal blood flow. This would suggest that either NO is not a key mediator of blood flow in the vagina or that the effect of estrogens on NOS is Acknowledgements counterbalanced by other neurotransmitters in vivo. Alternatively, the anatomical regional distribution This work was supported by grants from the of NOS and arginase may explain, in part, the fact National Institute of Diabetes and Digestive and that estrogens reduce total NOS activity in the Kidney Diseases (R01-DK56846-01 and K01- proximal vagina but not in the distal vagina. Thus, DK02696-02) and The American Foundation for enhanced vaginal blood flow by estrogen may be Urologic Disease and the Institute for Sexual mediated by activation of e-NOS in the distal vagina, Medicine, Boston University School of Medicine. and this differential regulation is masked by changes in NOS isoforms from neural, epithelial smooth muscle and endothelial origins. Neither the studies by Al-Hijji and co-workers26–29 nor those by Yoon References et al30 examined NOS isoforms in the two subre- gions of the rabbit vagina. Thus, differential regula- 1 Levin RJ. Sex and the human female reproductive tractFwhat tion of e-NOS by sex steroid hormones in the proxi- really happens during and after coitus. Int J Impot Res 1998; mal and distal vagina remains to be established. 10(Suppl 1): S14 – S21. The rabbit vagina is comprised of two distinct 2 Park K et al. Vasculogenic female sexual dysfunction: the anatomical regions, an upper (proximal) region hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int J Impot Res 1997; 9: 27 – 37. which represents 2/3 of the length of the vagina 3 Henson DE, Rubin HB, Henson C. Analysis of the consistency and which is lined by a single layer of columnar of objective measures of sexual arousal in women. 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Hormones and sexuality in post- regulating total NOS activity in the proximal vagina, menopausal women: a psychophysiological study. J Psycho- may also upregulate expression and activity of blood som Obstet Gynaecol 1997;18: 126 – 133. 8 Davis SR. The therapeutic use of androgens in women. vessels e-NOS in the distal vagina. This would J Steroid Biochem Mol Biol 1999; 69: 177 – 184. result in increased vasodilation of blood vessels in 9 Davis SR, Tran J. Testosterone influences libido and well being the distal vagina and enhanced blood flow and in women. Trends Endocrinol Metab 2001; 12: 33 – 37.

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