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Keywords:

  • oestrogen receptor α;
  • neuronal nitric oxide synthase;
  • clitoris;
  • mice

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

OBJECTIVE

To study the presence of oestrogen receptors (ER) and neuronal nitric oxide synthase (nNOS) in the mouse clitoris.

MATERIALS AND METHODS

A series of sections of the pelvic area, including the preputial glands and clitoris, of 10 mice were assessed by immunocytochemical studies specific for ER-α and -β, and nNOS; selected sections were also stained with Masson’s trichrome.

RESULTS

ERα was detected in the epithelium of the gland of the clitoris, and in the glandular tissue, preputial and apocrine gland. ERα was detected in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris. Cytoplasm ERα was detected in a few cells in an area ventral to the clitoral gland. There was also nuclear staining in the connective tissue cells surrounding the clitoris. Very light ERβ immunostaining was detected in the clitoris and in the tissue related to it. There were some cells with nuclear staining in the vessels of the cavernous tissue of the clitoris. nNOS immunostaining was detected in the clitoris, the preputial gland and the connective tissue.

CONCLUSION

ERα and β isoforms, and nNOS, are present in the clitoris and preputial glands of female mice in different cellular locations and with differing levels of receptivity. Functional studies would further elucidate the role of receptor functions and their relationship to the neuronal expression of NO.


Abbreviations
ER

oestrogen receptor

(i)(e)(n)NO(S)

(inducible) (endothelial) (neuronal) nitric oxide (synthase).

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Oestrogens are important in the growth, differentiation and function of female reproductive tissues [1]. Oestrogens appear also to be relevant to sensory function in the genital region, based on electrophysiological studies in animals indicating that oestradiol administration results in an expanded touch-receptor zone of the pudendal nerve [2].

Nitric oxide (NO) is the physiological mediator of penile erection, and its synthesizing enzyme, NO synthase (NOS), is localized in the pelvic nerve neurones innervating the corpora cavernosa and in neural plexuses of the adventitial layer of the penile arteries [3]. NOS inhibitors abolish electrophysiologically produced penile erections in laboratory animals and in man [4].

Oestrogen receptor-α (ERα) is the predominant form of ER expressed in uterine and vaginal tissue of the murine female reproductive tract. Expression of ERα and β was detected in vaginal tissue and in the ovary [5–7]. ERs were detected in basal and suprabasal cells of vaginal epithelium and epidermis of labia minora [8], and ERα was expressed in vulvar skin in dogs [9].

ERα and β have been described in the testis, prostate, efferent ducts and penis mostly in animals [10–12]. Recently it was reported that ERα and particularly β are regularly expressed in human testis, corpus cavernosum and male urethra [6,13]. Moreover, the presence of functional ER in differentiating male external genitalia was reported, and indicates a possible novel inhibitory role of oestrogens in the regulation of the development of these sex structures [14]. Oestrogens influence the action of local mediators in genital tissue; this idea is supported by experimental findings in the rat vagina showing the dependence of neuronal (n)NOS expression on the presence of oestrogen [15]. Decreased circulating levels of oestrogens alter vaginal and clitoral blood flow in experimental animals [16]. These findings are consistent with clinical evidence in women indicating that sufficient oestrogen levels preserve the vascular function of female genital tract structures [17].

Three types of NOS, inducible (i), endothelial (e) and nNOS, have been identified and characterized; iNOS is not expressed constitutively, eNOS is mostly membrane bound, whereas the nNOS has been identified in the cytosol [18]. eNOS is present in the deep arteries, veins and capillaries of the human vagina [19]. NOS isoforms are largely distributed in the cavernous tissue of the clitoris and human vagina, particularly in the nerve bundles, and vascular and sinusoidal endothelium [19–21], and in the vagina and uterus of experimental animals [22].

Aspects of ER status have been investigated in relation to the reproductive structures and vaginal tissue, and basic information about NO has focused on the internal female reproductive tract and penis, but has not included female external genital structures or the clitoris. There have been very few reports on the cellular location of ERα in the female rat reproductive organs [23,24] or in humans [6].

The aim of the present study was to assess the presence of ERα and β, and nNOS, and their relationships in the mouse clitoris.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

For the immunocytochemical experiments we used six adult female C57Bl/6 mice (aged 8–12 weeks, 35–45 g) obtained from Jackson Laboratories (Bar Harbor, Maine, USA). All procedures followed the National Institutes of Health Guide for the Care of Use of Laboratory Animals and a protocol approved by The Rockefeller University Animal Care and Use Committee. The mice were deeply anaesthetized with pentobarbital and perfused transcardially with 0.1 m of phosphate buffer and 0.1% heparin, followed by 4% paraformaldehyde. The external genitalia were then removed and post-fixed in the same fixative for 24 h, stored in 0.1 m phosphate buffer and 10% sucrose for a further 24 h, frozen and cut into 40 µm thick transverse sections on a cryostat. The sections were washed twice for 5 min in PBS (pH 7.4) then incubated in blocking solution (10% methanol, 3% H2O2 in PBS) for 10 min, rinsed twice in PBS, followed by the gelatine blocking step (0.75% gelatine in PBS) for 10 min, and rinsed twice in PBS. Matching series of sections from each mouse were incubated for 48 h at 4 °C, with the following antibodies in a humidified chamber: rabbit anti-ERα dilution 1:400 (Santa Cruz Laboratories, Santa Cruz, CA, USA), rabbit anti-ERβ 1 µg/mL (Zymed Laboratories Inc., San Francisco, CA, USA), rabbit anti-nNOS 2 µg/mL (Zymed). After incubation in primary antibody, the sections were then processed according to a standard procedure for the rabbit IgG Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Sections were then placed with agitation in two 5-min washes of PBS (pH 7.4) followed by a 5-min wash in 0.05 m TRIS buffer (Sigma Chemicals Corp., St. Louis, MO, USA) at pH 7.6. The sections were then immersed in 0.05% 3,3′-diaminobenzidine tetrahydrochloride (Sigma) with 0.03% H2O2 in 0.05 m TRIS buffer pH 7.6 and reacted for 10 min, rinsed with 0.05 m TRIS buffer pH 7.6, followed by distilled water. The reaction product appeared as a dark-brown stain. In the negative control the primary antibody was absorbed by pre-incubation with the respective synthetic peptide. One section in every 10 was lightly counterstained with haematoxylin and eosin. All sections were dehydrated through graded alcohols, cleared in xylene and coverslips applied with Permount.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

There was diffused and deep immunostaining for ERα in the epithelium of the gland of the clitoris, and in the glandular tissue, preputial and apocrine gland (Fig. 1). ERα was detected in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris (Fig. 2a,b). Cytoplasmic ERα was detected in a few cells in an area ventral to the clitoral gland (Fig. 3a). There was also nuclear staining in the connective tissue cells surrounding the clitoris (Fig. 3b).

image

Figure 1. Diffused and deep immunostaining for ERα in the epithelium of the gland of the clitoris, and glandular tissue, preputial and apocrine gland. There was also nuclear staining in the connective tissue cells surrounding the clitoris (×4).

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image

Figure 2. ERα staining in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris (a, ×20; b, ×40).

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Figure 3. Cytoplasm ERα was detected in a few cells in an area ventral to the clitoral gland (a, ×20; b, ×40).

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Very light ERβ immunostaining was detected in the clitoris and in the tissue related to it. However, there were some cells with nuclear staining in the vessels of the cavernous tissue of the clitoris (Fig. 4).

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Figure 4. There were some cells with nuclear staining in the vessels of the cavernous tissue of the clitoris (×20).

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nNOS immunostaining was detected in the clitoris, the preputial gland and connective tissue (Fig. 5a); although the pattern of staining was similar to that for ERα, the immunostaining detected for nNOS was lighter (Fig. 5b). For each figure, negative control experiments in which the complete immunocytochemical procedure was carried out, but leaving out the primary antibody, failed to show staining, thus confirming the specificity of the positive results (data not shown). Also, the epithelium of the vaginal wall was negative for the immunostain for ERα and β. Membrane-based nNOS was found in the vaginal wall, and not along the upper vaginal wall, but only in one part, closest to the vaginal opening (Fig. 6a–c). Sagittal sections show the difference in immunostaining in vaginal apocrine glands for ERα, and β and nNOS (Fig. 7a–c).

image

Figure 5. a, nNOS immunostaining was detected among the clitoris, preputial gland and connective tissue (×4); b, although the pattern of staining was similar to that with ERα, the immunostaining detected for nNOS was lighter (×20).

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image

Figure 6. Murine vaginal wall was negative for the immunostain for ERα (a) and ERβ (b) (both ×10); c, nNOS was detected in the membrane of the vaginal wall, but not along the upper vaginal wall, only in one part closest to the vaginal opening (×10).

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image

Figure 7. Immunostaining of apocrine gland of the vagina for ERα (a), ERβ (b) and nNOS (all ×10).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

Nuclear staining of ERα was detected in stromal cells near the epithelium in the clitoris; the ER were highly expressed in the epithelium and the glandular tissue (preputial and clitoral gland). In canine vaginal and vulvar tissue, there was nuclear staining for ERα in surface epithelium, stromal and smooth muscle cells. In vulvar skin ERα was also detected in the nuclei of apocrine sweat glands [9]. There was a higher staining intensity in stromal cells than in those cells stained in the epithelium. In general, there was a higher staining intensity for ERα in stromal cells than in epithelial cells in canine uterus, vaginal and vulvar tissue [9]. The nuclear immunostaining for ERα in the stroma of the clitoris suggests a higher receptivity to this hormone. All receptors identified in the clitoris tended to be more intensely expressed in stromal than epithelial cells, suggesting that there is a stromal–epithelial interaction induced by the different sex steroids.

ERβ immunostaining was only detected in a few cells in the vascular lumen of the cavernous tissue of the clitoris. By contrast with ERα, there was no staining in the glandular tissue, epithelium or stroma of the clitoris. These results suggest that ERβ is not essential for the normal functions that take place in the clitoris of the mouse. It is then likely that ERα is the ER subtype involved in the mediation of the major effects of oestrogen on the clitoris. Indeed, in mice deficient in ERα[25] there is atrophy of the oviduct and uterus. The study of Wang et al.[26] showed that ERα is the predominant ER in rat uterus, oviduct and vagina/cervix.

Notably, ERα can mediate the short-term effects of oestrogen on eNOS enzymatic activity and might assist vasodilatation by synthesis or activation of NO [27]. Furthermore, oestrogens cause rapid endothelial NO production because of the activation of plasma membrane-associated ER coupled to eNOS through Gαi[28].

nNOS was immunodetected with a similar pattern of distribution to that of ERα. NO might play a role in controlling blood flow and capillary permeability, the mechanisms of sexual lubrication due to cGMP action, induced by NO. The homeostasis of this system needs cGMP breakdown. It is possible that the physiological response to sexual arousal in the female follows the same biochemical pathway as in the male. cGMP/NO appears to be a key pathway mediating clitoral smooth muscle relaxation [29]. The final effect of NO synthesis, cGMP production, and smooth muscle relaxation regulated by NO-type 5 phosphodiesterase, is in fact the vascular engorgement of the clitoris and the anterior wall of the vagina [30].

ERα might also mediate the induction of nNOS by oestrogens, at least in rat hypothalamic ventromedial nucleus, which is rich in ERα. This is suggested by our finding that chronic treatment with oestradiol increased the expression of nNOS in the ventromedial nucleus of ovariectomized rats [31]. Thus, it is possible that oestrogens can act through ERα in the clitoris to enhance the nNOS/NO system to increase vascular engorgement of the clitoris.

NO production in response to the non-genomic activation of eNOS is critically involved in the cardiovascular protective actions of oestrogens. Many of these cardiovascular protective effects are due to non-genomic actions of the hormone on the vascular wall and a major factor is the activation of NO [32] production by the eNOS [33]. Further work showed that both ERα and β mediate non-genomic eNOS activation [27,34].

There are subpopulations of ERs (α and β) that are located in the plasma membrane of numerous cell types, including endothelial cells, where they mediate membrane-initiated signalling [35]. On the plasma membrane of endothelial cells, both ERα and β are coupled to eNOS in caveolae, which are a subset of lipid rafts known to compartmentalize signalling molecules [36].

In conclusion, knowledge about the relationship of oestrogen receptivity in the genital sensory field and clitoral vasculogenic processes would be advanced by a better understanding of the presence and anatomical location of nNOS and ER isoforms. Functional studies of varying oestrous states ERα- and β-knockout mice and the relationship to genital sensitivity and nNOS expression in genital tissue would further elucidate the role of ER function and their role in sexual behaviour.

CONFLICT OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES

None declared. Source of funding: Hess Roth Foundation, Rockefeller University.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONFLICT OF INTEREST
  8. REFERENCES