Kwangsung Park, MD, PhD, Department of Urology, Chonnam National University Medical School, 8 Hak-dong, Dong-gu, Gwangju 501-757, Republic of Korea. Tel: +82-62-220-6701; Fax: +82-62-227-1643; E-mail: firstname.lastname@example.org
Introduction. Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. There has been little research on the role of AQPs in the female sexual arousal response.
Aim. The purposes of this study were to investigate the localization and functional roles of AQP1, AQP2, and AQP3 in rat vagina.
Methods. Female Sprague-Dawley rats (230–240 g, N = 20) were anesthetized. The vaginal branch of the pelvic nerve was stimulated for 60 seconds (10 V, 16 Hz, 0.8 ms), and the animals were sacrificed either immediately or 5 minutes later. The expression and cellular localization of AQP1, 2, and 3 were determined by Western blot and immunohistochemistry of the vagina. The intracellular membrane and plasma membrane fractions of the proteins in vaginal tissue were studied by immunoblot analysis with the differential centrifugation.
Main Outcome Measures. The expression and cellular localization of AQPs, and pelvic nerve stimulation induced translocation of AQPs in rat vaginal tissue.
Results. Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the vagina. AQP2 was expressed in the cytoplasm of the epithelium, and AQP3 was mainly associated with the plasma membrane of the vaginal epithelium. AQPs were found to be present primarily in the cytosolic fraction of untreated tissues. The translocation of AQP1 and 2 isoforms from the cytosolic compartment to the membrane compartment was observed immediately after nerve stimulation and had declined at 5 minutes after nerve stimulation, while the subcellular localization of AQP3 was not changed by nerve stimulation.
Conclusions. These results showed a distinct localization of AQPs in the rat vagina. Pelvic nerve stimulation modulated short-term translocation of AQP1 and 2. These results imply that AQPs may play an important role in vaginal lubrication. Park K, Han HJ, Kim SW, Jung SI, Kim S-O, Lee H-S, Lee MN, and Ahn K. Expression of aquaporin water channels in rat vagina: Potential role in vaginal lubrication. J Sex Med 2008;5:77–82.
Female sexual arousal is a neurovascular phenomenon which is characterized by clitoral engorgement and vaginal lubrication responses resulting from increased clitoral and vaginal blood flow [1–2]. Vaginal lubrication is one of the indicators of genital arousal and tissue integrity. In the basal state, the vaginal epithelium reabsorbs sodium from the capillary plasma transudate. During arousal in humans, a dramatic increase in capillary inflow in the submucosa overwhelms sodium reabsorption, leading to 3–5 mL of vaginal transudate, and enhancing lubrication essential for pleasurable coitus .
The structure of the vaginal wall consists of the inner mucosal epithelial stratum, a lamina propria containing thin-walled veins, the intermediate muscularis stratum, and the external adventitial layer . The vaginal epithelium consists of stratified squamous epithelium devoid of glands, but its surface is usually covered with a film of fluid . The lamina propria of the mucosa contains blood vessels contributing to the diffusion of the vaginal fluid across the epithelium . Shabsigh et al.  demonstrated that the sub-epithelial region of the vaginal wall contains a dense and rich network of capillaries that perfuse the epithelium. Levin  hypothesized that vaginal epithelial cells have transport mechanisms capable of modifying plasma to create vaginal fluid.
Recently, water channel proteins, aquaporins (AQPs), have been widely investigated in many fluid-transporting tissues, such as kidney tubules and glandular epithelia, as well as nonfluid-transporting tissues [8–9]. The AQPs are membrane proteins that transport water and small solutes such as glycerol. The AQPs are a family of hydrophobic, integral membrane proteins that are expressed widely, 13 members having been identified so far in mammals . AQPs are expressed in many fluid-containing tissues, and their roles are to facilitate transepithelial fluid transport, urinary concentration, fluid secretion from glands, etc [8–10]. AQP1, AQP2, AQP4, AQP5, and AQP8 are specifically water permeable, whereas AQP3, AQP7, AQP9, and AQP10 are permeable to water and small solutes, such as glycerol .
Recently, the localization of AQP1 was reported in the smooth muscle cell membrane of rat vagina . However, there has been little research on the role of AQPs in the female sexual arousal response. Therefore, the purposes of this study were to investigate the localization and functional roles of AQP1, 2, and 3 water channels in rat vagina.
Materials and Methods
Female Sprague-Dawley rats (230–240 g, N = 20) were maintained on a standard diet and then fasted overnight before the experiment. Animals were premedicated with xylazine (2.2 mg/kg, IM), and anesthetized with a zolazepam/tiletamine cocktail (4.4 mg/kg, IM). A midline abdominal incision was made, and the vaginal/clitoral branch of the pelvic nerve was exposed. A Harvard subminiature electrode was placed around the vaginal/clitoral branch of the pelvic nerve. Unilateral nerve stimulation was performed at 10 V, 16 Hz, 0.8 msec, and then animals were sacrificed either immediately afterward or 5 minutes later.
Histology and Immunohistochemistry
The lower one-third of the vaginal tissue was dissected from both lateral walls. The tissue was placed in 4% paraformaldehyde fixative for 16 hours and then processed for washing and dehydration. The tissue was routinely embedded in paraffin, and 5 µm sections were prepared. Tissues were stained with hematoxylin and eosin.
Immunohistochemistry was performed using an immunoperoxidase procedure (Vector ABC Kit; Vector Laboratories, Burlingame, CA, USA). The tissue sections were deparaffinized in xylene, dehydrated in a graded series of ethanol, rinsed twice in phosphate-buffered saline (PBS), and then treated with 3% H2O2 in 60% methanol for 30 minutes to quench endogenous peroxidase activity. After washing twice (5 minutes) in PBS, the sections were incubated for 12 to 14 hours with antibodies for AQP1, 2, or 3 (Chemicon International, Inc., Temecula, CA, USA) in PBS with 0.3% bovine serum albumin. For a negative control, the sections were incubated in PBS containing 5% normal goat serum only. The sections were then rinsed three times in PBS and incubated sequentially for 30 minutes each with the biotinylated secondary antibody and the ABC reagent. Then the sections were incubated for 5 minutes with the peroxidase substrate solution (diaminobenzidine) contained in the kit. Finally, the tissue sections were examined and photographed under a light microscope.
The tissue sections were deparaffinized in xylene, dehydrated in a graded series of ethanol, rinsed in PBS, and then treated with normal goat serum for 30 minutes to block nonspecific binding. After washing in PBS, the sections were incubated with antibodies for AQP2, or 3 (Chemicon International, Inc., Temecula, CA, USA) in PBS with 0.3% bovine serum albumin for 12 to 14 hours at 4°C. For a negative control, the sections were incubated in PBS containing 5% normal goat serum only. The sections were then rinsed in PBS and incubated for 30 minutes with the antirabbit IgG conjugated to fluorescein (Vector Laboratories, Burlingame, CA, USA). Finally, the tissue sections were examined and photographed under a fluorescence microscope.
Intracellular Membrane or Plasma Membrane Fraction Preparation
Membrane fractions enriched in plasma or intracellular microsomal membranes were prepared from vaginal tissue by differential centrifugation as previously described . Briefly, vaginal tissues were separated and homogenized in 0.3 M sucrose containing 0.1 mM phenylmethanesulfonyl fluoride and 0.1 mM leupeptin. The plasma membrane fraction was obtained by centrifugation at 200,000 × g for 60 minutes on a discontinuous 1.3 M sucrose gradient. After removing the plasma membrane band, the sucrose gradient was sonicated, diluted to 0.3 M, and centrifuged at 17,000 × g for 30 minutes. The resulting supernatant was centrifuged at 200,000 × g for 60 minutes to yield the intracellular membrane fraction. The protein level in each fraction was quantified using the Bradford procedure .
Western Blot Analysis
The cell homogenates (30 µg of protein) were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose article. The blots were then washed with Tris-Buffered Saline Tween-20 (TBST) (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20). The membrane was blocked with 5% skimmed milk for 1 hour and incubated with the appropriate primary antibody the dilution recommended by the supplier. The membrane was then washed, and the primary antibodies were detected with goat antirabbit-IgG conjugated to horseradish peroxidase. Antibody incubations were performed in a 4°C incubator. The bands were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, England).
The results are expressed as the mean ± the standard error. All experiments were analyzed by anova. In some experiments, a comparison of the means (treatment vs. control) was made using a Bonnferroni-Dunn test. A P value <0.05 was considered significant.
Localization of AQPs in Rat Vagina
Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the vagina. However, AQP1 was not expressed in the vaginal epithelial layer. Immunolabeling of AQP2 showed expression throughout the epithelial cells of the superficial layer in rat vagina. AQP3 immunolabeling was mainly associated with the plasma membrane of the epithelium of the middle and deep layers (Figure 1). For further histological confirmation of the differential localization of AQP2 and 3 in rat vaginal epithelium, immunofluorescent staining was performed. The expression of AQP2 and AQP3 demonstrated localizations very similar to those seen by immunohistochemistry (Figure 2).
Effect of Pelvic Nerve Stimulation on AQPs Translocation
In order to examine the effect of pelvic nerve stimulation on AQP1 and AQP2 translocation of vaginal tissue, the vaginal tissue was stimulated with electricity and separated for Western blotting analysis immediately after electrical stimuli or 5 minutes after electrical stimuli. The intracellular membrane fraction of AQP1 and AQP2 significantly decreased immediately after nerve stimulation and recovered to control level after 5 minutes, whereas the plasma membrane fraction of AQP1 and AQP2 increased significantly immediately after nerve stimulation (P < 0.05) and then seemed to decrease after 5 minutes (Figures 1B and 3A).
In the present study, we have shown the presence and immunolocalization of water channel proteins, AQP1, AQP2, and AQP3 in the rat vagina. Immunolabeling showed that AQP1 was expressed mainly in the subepithelial vessels in the vagina; in contrast, AQP2 and AQP3 were expressed in the vaginal epithelium. Gannon et al.  reported that AQP1 is expressed in the cell membrane of smooth muscle cells of the rat vagina and uterine tube. They suggested that the presence of AQP1 in vaginal smooth muscle might explain the rapid movement of water either into or out of the vaginal smooth muscle cells. This fluid contributes to the distention and elongation of the vagina during sexual stimulation. However, our results showed that AQP1 was mainly expressed in the subepithelial capillaries and venules. AQP1 has been identified in the cell membrane of vascular smooth muscle cells of large arteries . Shabsigh et al.  demonstrated that there is an active interaction between the microvasculature and the epithelial cells of the vaginal wall.
Aquaporin 2 is known to be expressed in the renal collecting duct, especially in the apical membrane and intracellular vesicles in collecting duct principal cells [8–15]. AQP3 transports glycerol as well as water; therefore, it is called an aquaglyceroporin . AQP3 is expressed in the renal collecting duct, epidermis, airway epithelium, conjunctiva, large airways, and urinary bladder . Ma et al.  reported that AQP3 is expressed strongly in the basal layer of keratinocytes in mammalian skin. Hairless mice lacking AQP3 showed reduced hydration of the stratum corneum, which is located at the most superficial layer of skin [16–18]. However, there has been no report on AQP2 and 3 in the vagina. In the present study, AQP2 was expressed throughout the epithelial cells of the superficial layer in rat vagina. And AQP3 immunolabeling was mainly associated with the plasma membrane of the epithelium of the middle and deep layers, with sparse labeling in the cytoplasm of the epithelium in the vagina. These findings suggested that AQP2 and AQP3 may play an important role in water transport in the vaginal epithelium.
Female genital sexual arousal is a physiologic process which results in pelvic vasocongestion and vaginal engorgement. Vaginal engorgement is believed to result in plasma transudation, allowing a flow through the epithelium onto the vaginal surface [1,19]. In an animal model study, pelvic nerve stimulation resulted in increased vaginal and clitoral blood flow, as well as increased vaginal wall pressure, vaginal length, and clitoral intracavernosal pressure . In the present study, pelvic nerve stimulation induced the translocation of AQP1 and AQP2 from the intracellular membrane to the plasma membrane. This phenomenon occurred immediately after pelvic nerve stimulation and recovered about 5 minutes after nerve stimulation. Our study revealed that AQP1 was expressed in the capillaries, venules, and smooth muscle of vagina. This finding implies that pelvic nerve stimulation may activate the AQP1 water channel, and allow plasma transudation from the capillaries to the subepithelial layer. In the epithelial layer, translocation of AQP2 may open water channels at the cell membrane, and facilitate transepithelial fluid transport to the vaginal lumen. In the current study, increased plasma membrane targeting of AQP3 was not observed by pelvic nerve stimulation, even though it was expressed strongly at the plasma membrane of the vaginal epithelium. These findings support the view that the subcellular localization of AQP1 and AQP2 are regulated differently than that of AQP3. The role of AQP3 should be clarified in future studies.
In this study, we evaluated the potential role of AQP1, 2, and 3 as water channel proteins of the vaginal epithelium; however, these proteins are only part of the AQP family of proteins. Further studies are needed to investigate the localization of all the AQP family members in the vagina, and their regulation by sex steroid hormones or other pharmacological agents [21–23].
In this study, we have demonstrated that AQP1, 2, and 3 are constitutively expressed in the rat vagina. AQP1 is expressed in the subepithelial vessels, whereas AQP2 and 3 are expressed in the epithelial layer. These results showed a distinct localization of these AQPs in the rat vagina. Furthermore, we showed that pelvic nerve stimulation modulated short-term translocation of AQP1 and 2 from the intracellular membrane to the plasma membrane in rat vagina. This result implies that AQPs may play an important role in vaginal lubrication.
This study was supported by a grant R01-2007-000-11925-0, Korea Science and Engineering Foundation. We thank Ms. Jennifer Macke for assistance in editing the text.