Role of oestrogens in male erectile function 1

Chapter INTRODUCTION 1. INTRODUCTION Erectile dysfunction (ED), defined by the National Institutes of Health (NIH, 1993) consensus development panel as the inability to achieve or maintain an erection sufficient for satisfactory sexual performance, has gained much clinical attention with the emergence and unprecedented patient compliance to the oral phosphodiesterase (PDE) inhibitor, sildenafil citrate. Arising from intensive worldwide research efforts into the pathophysiology of ED over the last three decades, this pharmaceutical breakthrough has incidentally lifted the taboo of seeking medical attention for this important quality of life concern. However, ED is a symptom and not the disease per se and therefore the eventuality of successful pharmacotherapy with end-organ effectiveness should not deter us from recognising and delineating various precipitating causes and pathophysiology of presenting clinical situations for better patient evaluation, management and permanent resolve. From being relegated in the fifties as psychogenic in over 90% of ED cases (Wershub, 1959; Smith, 1981), the last two decades have ushered in a new era in the scientific outlook of this multifactorial disorder. Erectile physiology is a complex interaction of multiple organ systems coordinating a number of neurologic, vascular, endocrine and cellular inputs with processes in the penile corpora cavernosa (CC) and ED may therefore arise from various risk factors acting on any of these numerous systems / pathways (Mobley and Baum, 1998). Another important premise at this stage is that ED is progressively more prevalent in older men, because ageing is also associated with many of the risk factors and co-morbid conditions for ED including central and peripheral nervous system disorders, cardiovascular and peripheral vascular dysfunctions and concomitant medications (Korenman, 1998). As it is expected that the present number of men aged over 65 years will more than double by 2025 (Ponholzer et al., 2002), this negative correlation of aging with ED is a global concern. Furthermore, the aging process in man is also accompanied by a number of endocrine changes in addition to systemic illnesses. Several studies such as the Massachusetts Male Aging Study (Gray et al., 1991; Feldman et al., 2002) have clearly demonstrated age-related changes in serum levels of total testosterone, free testosterone and sex hormone binding globulin (SHBG). Consequently, several of these symptoms including ED may have been associated with such age-related hormone changes. Steroid hormone secretions in general, conform to health status in men and therefore understanding the dynamics of endocrine changes in man is also important because of their role in sexual and reproductive function. Normal male reproductive function depends on the secretion of luteinizing and follicle stimulating hormones (LH and FSH) by the pituitary gland under the influence of hypothalamic gonadotropin releasing hormone (GnRH). Luteinizing hormone-induced testicular / Leydig cells secretion of testosterone is associated with a diurnal fluctuations and negative feedback control. The present understanding is men produce a moderate amount of oestrogen along steroidogenesis of testosterone (T) (Berg, 1998) and 75 – 90% of this “female” hormone in the male is from a simple enzymatic conversion of testosterone called aromatization (Oettel, 2002a). While androgen and oestrogen constitute opposite sides of the same coin and their quantitative balance is what determines the physiological effect on target tissues (Sharpe, 1997), changes due to aging at the hypothalamo-pituitary-testicular axis may lead to decline in circulating T levels (Cohen, 1998). As a result of this decline, the “ying yang” physiological balance between androgen and oestrogen in normal man may be disturbed. Further compounding factors here are the significantly higher oestradiol (E2) levels in the aging male (Oettel, 2002a), changing E2/T ratio (Rubens et al., 1974) and the strong gonadotropin-suppressant effect of E2 compared to testosterone. Hence these factors will result in a state of secondary hypogonadism with decrements in LH and FSH release (Cohen, 1998). There sets in a vicious cycle with changes in E2/T ratio which may be further aggrevated by the continued peripheral conversion of the available testosterone to oestradiol through aromatization. Thus, the negative / detrimental effects of T deficiency will continue with advancing age leading to progressive and undeterred functional deterioration of testosterone compared to the relative and absolute overproduction of oestrogen. Considering this dual imbalance, it may be hypothesized that androgen deficiency is not the sole cause for the identified decrease in nocturnal penile tumescence (NPT) and spontaneous morning erections and loss of libido (Tserotas and Merino, 1998) and that E2 may play a decisive role. However, despite the concomitant elevation of oestrogen during this stage in the aging male, available scientific information on the physiological role of E2 on erectile function is too scant to correlate it with the aetiology of ED in these cases. The existing knowledge is that therapeutically, oestrogen was as effective as antiandrogens or GnRH analogues in countering the hypersexuality of paraphilias (Levitsky and Owens, 1999) and oestrogen supplementation prior to gender reassignment reportedly feminized male transsexuals (Goh, 1999). Indeed, months of oestrogen therapy is the mainstay for the development of breast and other female traits in these patients. In the process of such preoperative treatment, prospective male to female transsexuals have observed loss of NPT and morning erections and a gradual reduction in erectile and ejaculatory capacity (Adaikan, 1998). Hence, it appears that E2 plays a distinct negative physiological role in male erectile function. Together with the identification of oestrogen receptors alpha and beta (ERα and ERβ) at some sites of the male reproductive tract (Merchenthaler and Shughrue, 2000; Diagram 1), several reports from animal studies have indicated the positive role of oestrogen receptor activation in sperm production and fertility (Hess et al., 1997; Sharpe, 1997 and Berg, 1998). Diagram 1: Oestrogen Receptor Distribution (from Merchenthaler and Shughrue, 2000) Confirmatory changes are seen in the testes of the experimental ERα knock-out mice (ERαKO) model; an ineffectual fluid reabsorptive function of oestrogen is implicated in testicular atrophy from intraluminal fluid back pressure (Hess et al., 1997). Furthermore, this ER gene mutation model also shows that some components of male typical aggressive and sexual behaviours may be oestrogen-dependent (Ogawa et al., 1997; Rissman et al., 1999). In pair mating tests of these ERKO male mice with primed females, there were a statistically significant decrease in intromission and ejaculatory failure; in open-field behavioural studies, this animal model was docile. Taken together, these findings indicate a mediatory role for ER-alpha in the expression of some parameters of sexual and violent behaviours in male rodents. Underlying these reports is the hypothesis for this thesis in establishing the new identity for oestrogenic activity and its relationship to sexual dysfunction in males. It could be that some of the physiological effects once considered androgenic might instead arise after aromatization of androgen leading to abnormal or imbalanced oestrogen level. This will make the pathophysiological effects of excessive endogenous oestrogen clinically more significant in the male. Besides its occurrence in physiological aging, other precipitating conditions of such hyperestrogenism include aromatase hyperactivity (Elias et al., 1990), non-insulin dependent diabetes mellitus (Oettel, 2002a), rapid weight gain, obesity, hypercholesterolemia, cholelithiasis and chronic liver diseases (Thomas, 1993), idiopathic haemochromatosis (Stremmel et al., 1988), Klinefelter’s disease (Heinig et al., 2002), as well as tumours of male breast (Thomas, 1993; Heinig et al., 2002), Leydig cells (Mineur et al., 1987) and adrenal cortex (Veldhuis et al., 1985). Similar hypothesis of great health concern which is scientifically yet to be qualified/quantified is the oestrogenic activity of compounds of environmental and plant origin, structurally related to the endogenous oestrogen (Adlercreutz and Mazur, 1997). These oestrogen mimetics are hazardous to human reproductive health as they are also anti-androgenic (Danzo, 1998); indirect exposure of the foetus during sexual development affects differentiation and growth of the male reproductive tract (Sohoni and Sumpter, 1998) and is implicated in low sperm counts, cryptorchidism and increased risk for testicular cancer (Cheek and McLachlan, 1998). Recently, a population study suggested that these environmental agents could also be antierectile (Oliva et al., 2002). Thus, an excessive intake (phytoestrogens) / exposure to these chemical substances (xenoestrogens) on a daily basis are likely to affect not only the fertility but also the sexual profile of the male population. Oestrogenic activity of these exogenous substances is also seen by their binding affinity for endogenous oestrogen receptors and extracellular proteins such as SHBG, α fetoprotein and albumin fractions (Stephens, 1997); this indicates that such actions arising from modulations on the endogenous hormone levels may be more complex. Thus in light of these reports, the hypothetic basis of this study is the possibility of oestrogens compromising endogenous androgen milieu and erectile function. A systematic delineation of the effects of natural oestrogen mimetics on erectile parameters is aimed to provide a rational understanding to bridge the scientific void in this area. This study is therefore envisaged to explore the paracrine endpoints of oestrogens in male sexual function, in particular their effects on the normal physiological principles of penile erection and to propose a basis for such possible dysfunctional changes of hormone modulations in specific clinical presentations. 1.1. Erectile Physiology This component reviews the conceptual developments in the physiological control of penile erection, which include peripheral neuroanatomy, signal transduction, central pathways and coordinating vascular and humoral inputs based on directives from basic animal models and clinical epidemiologic designs. These parameters will be used as a rational framework for the investigations, understanding and discussions of the pathophysiological changes in erectile function secondary to the proposed experimental hyperostrogenism. 1.2. Historical Milestones and Current Thoughts 1.2.1. Autonomic Control of Penile Erection The innervation of the penis has somatic, sympathetic and parasympathetic components (Langley and Anderson, 1895). The somatic sensory nerve is carried in the pudendal nerve and divides into the inferior hemorrhoidal nerve, perineal nerve and the dorsal nerve of the penis. The sympathetic supply to the genitalia is derived from the twelfth thoracic and upper lumbar segments of the spinal cord. The parasympathetic innervation is from the sacral outflow at the second, third and fourth segments of the sacral cord and constitutes the preganglionic fibres to form the pelvic nerve or nervi erigente (nerve of erection). Autonomic innervation of the CC is comprised of the parenchymal and perivascular nerves (Christ et al., 1997). Studies at the cellular level demonstrate adrenergic nerve fibres in the penile tissues obtained from several animal species; these include fibres identified in the cavernosa of mice (Bock and Gorgas, 1977), rabbits (Klinge and Penttila, 1969; Fujimoto and Takeshige, 1975) and dogs (Bell, 1972). In human penile tissues, catecholamine fluorescent fibres and terminals were demonstrated in the cavernosa as well as the spongiosum (Benson et al., 1980). These fibres wind through the trabeculae, approach the walls of the cavernous spaces and extend into the spongiosum. In addition, the blood vessels of the cavernosum and spongiosum demonstrate adrenergic varicosities. Pharmacological demonstration of α- (contractile) and β- (relaxant) adrenoceptors in the human penis (Adaikan, 1979; Adaikan and Karim, 1981) concurred with these histological findings. Similarly, acetylcholinesterase positive nerve fibres have been identified in the penile tissues from rats (Dail and Hamill, 1989), rabbits (Klinge and Penttila, 1969) and primates (Steers et al., 1984). Ultrastructural examination of human CC reveals that these cholinergic terminals are located in close proximity to cavernous blood vessels and smooth muscle (Benson et al., 1980) and coexist with adrenergic and nonadrenergic, noncholinergic (NANC / nitrergic) fibres (Sathananthan et al., 1991). Furthermore, pathways for vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) have been implicated in penile erection. Nerve fibres immunoreactive to VIP have been identified in the CC and around the helicine arteries of human penis (Polak et al., 1981; Steers et al., 1984) and in the penis of various mammalian species (Alm et al., 1977; Larsson, 1977). Furthermore, VIP has been shown to relax cavernosal strips from dog (Carati et al., 1985) and man (Adaikan et al., 1984), in a concentration-dependent manner. This VIP-induced relaxation was inhibited by the nitric oxide (NO) synthesis blocker N-ω-nitro-L-arginine (Kim et al., 1995). In view of the colocalization of acetylcholine (ACh), VIP and neuronal nitric oxide synthase (nNOS) in parasympathetic neurons (Hedlund et al., 1999), it is believed that they may act synergistically through inhibition of α1 adrenergic activity by ACh and release of NO by VIP (Lue, 2002). CGRP has been immunohistochemically identified in cavernous smooth muscle, nerve endings and within the arterial walls of the human CC (Su et al., 1986; Stief et al., 1990). Both VIPergic and CGRP positive nerves may thus, play a supportive role in modulating penile erection. Other candidates include a relaxant factor acting through potassium (K+) channels (Okamura et al., 1998), non-NO dependent pathway (Reilly et al., 1997a) or involving cyclic adenosine monophosphate (cAMP) (Angulo et al., 2000). 1.2.2. Penile Erectile Process Corpus cavernosum smooth muscle (CCSM) contributes predominantly to the control of tumescence and erection (Adaikan et al., 1999). Its contraction mediates detumescence / flaccidity and relaxation determines tumescence or erection. The excitatory (adrenergic) neurotransmission elicits contraction and the inhibitory (NANC- nitrergic) component mediates relaxation (Adaikan and Karim, 1978; Adaikan, 1979; Adaikan et al., 1991a). Dual (contractile and relaxant) effectiveness have been demonstrated in the human CC for adrenoceptors (Adaikan and Karim, 1981), cholinoceptors (Adaikan et al., 1983), and histaminergic receptors (Adaikan and Karim, 1976) in addition to various prostaglandins (Adaikan, 1979; Hedlund and Andersson, 1985) and their receptors (Angulo et al., 2002; Moreland et al., 2003). 1.2.2.1. Erectile Neurotransmitters Three types of experiments have indicated the role of neurotransmitters in erectile physiology. 1. The different neuromediators that are synthesised and released by neurons innervating the penis of various mammalian species have been identified by immunohistochemistry. 2. In vitro experiments have demonstrated the contractile (anti-erectile) and relaxant (proerectile) neurotransmitters in the corpora cavernosa and corpus spongiosum to a variety of stimuli including through appropriate nerve stimulation. 3. Intracavernous injections of putative neurotransmitter and modulator substances into the flaccid and erect penis have been used to demonstrate their effectiveness in animals and man. The existence of non-adrenergic non-cholinergic / NANC erectile neurotransmission in human penis has been documented (Adaikan, 1979; Adaikan and Karim, 1978) which was eventually identified as nitric oxide (Rajfer et al., 1992). Scientific evidence suggests that NO from NANC nerve endings is indeed the important modulator of penile erection (Adaikan et al., 1991a) and it can also be physiologically derived from the endothelial cells (Furchgott and Zawadzki, 1980; Bredt and Snyder, 1992). Produced enzymatically 10 2001). At the receptoral level, these compounds may act as agonists (activate transcription) or antagonists (by mechanical presence and preventing activation of DNA response elements). They may also act as antioestrogens by competing for the oestrogen biosynthesizing (aromatase) and metabolizing (17 beta-hydroxy steroid oxidoreductase type1) enzymes (Santti et al., 1998). Additionally, lignan and isoflavone phytoestrogens may also be antiandrogenic due to their effects in inhibiting conversion of T to DHT (Stephens, 1997) and increased synthesis of SHBG with further modulations in hormone levels (Aldercreutz and Mazur, 1997). Due to their less affinity for extracellular binding proteins such as SHBG and α fetoprotein than E2, phytoestrogens may be available in higher free concentrations (Cheek and McLachlan, 1998) for peripheral tissue effects. Diagram 5: Structural Comparison of Isoflavones with Oestradiol (from Belcher and Zsarnovsky, 2001) Many processed foods and soybean proteins commonly consumed contain significant amounts of the isoflavones daidzein and genistein, either as the aglycone (unconjugated form) or as different types of glycoside conjugate (Setchell, 1998; Nicholls et al., 2002). 38 These compounds are further activated by the microbial enzyme systems in the gastrointestinal tract to heterocyclic phenols with hormonal properties (Setchell, 2001), the concentration of which vary widely between individuals given the same quantity (Webb and Glasier, 2001). Thus, both the precursors and metabolites are potent oestrogenic substances; they are handled by the body (transported to the liver and eliminated via kidneys) through similar endogenous pathways (Dornstauder et al., 2001). Early interest in the possible oestrogen-like effects of soy was prompted by the discovery of extremely high levels of isoflavones in blood and urine after soy food consumption. While the lesser prevalence of perimenopausal symptoms in Asian women especially in Japan, compared to their occurrence in other parts of the world (Ginsburg and Prelevic, 2000) may explain the therapeutic benefits of these SERMs, the possibility that there may detrimental hormonal effects on sexual health in the males cannot be ignored. There has been little progress towards a realistic assessment of whether environmental oestrogens pose serious health threat to humans. Besides the lack of known physiological endpoints of oestrogen action in males, the reason for the limited progress is the wide number of oestrogenic chemicals to be evaluated. For the researchers in male sexual health, the knowledge that there is inadvertent exposure to a number of oestrogenic substances from the environment is a matter of great scientific concern and evaluation. 1.4. Objective of the Present Study In the light of this review, the aim of the present study was to delineate the effects of oestrogen on various components of penile erectile physiology. In experimental pharmacology, induction of higher systemic oestrogen levels is important for understanding the pathophysiological changes of this provocation. This approach is similar to conventional methods such as induction of tone to study the relaxant effect on 39 smooth muscle or use of hypertensive rats to study the effects of antihypertensives. It is not possible in vitro alone to test the complicated interplay of different systems involved in the process of a final end organ response. For this reason, models to identify the effect of oestrogens on the whole animal as well as in vitro preparations will be used. This study therefore proposes to: • explore the possible aetiological relationship of elevated oestrogen to the cavernosal dysfunction, in suitable animal model and tissues. • investigate in detail, the changes in sexual behaviour, in vivo ICP changes during erection, the responses of CC strips from oestradiol treated animals to various endothelium-dependent and independent agents and mediators of nitric oxide induced relaxation and the second messenger estimations in cultured smooth muscle cells. • delineate ultrastructural details in the corpora cavernosa and receptor distribution to identify the cellular basis for the pathophysiology of ED from hormonal derangements. • achieve an insight into the pathophysiology of ED as a consequence to phytoestrogen intake using the above parameters. • extrapolate these changes with the hormone profile of a group of ED patients. To achieve these objectives, the following experimental models / procedures are used in this investigation: 1. Sexual behaviour studies (whole animal model) 2. ICP response during erectile stimuli (in vivo experiments) 3. Hormonal modulations secondary to treatment (hormone assays) 4. Neurotransmitter mechanisms of erectile function (in vitro tissue experiments) 40 5. cAMP and cGMP levels (cell culture studies) 6. Structural changes in the cavernosum (light microscopy) 7. Oestrogen receptor distribution and expression pattern in the cavernosum (immunohistochemical studies) 8. hormonal profile of patients with known history of erectile dysfunction 1.5. Implications An adequate knowledge of the actual pathophysiological changes in sexual function with aging is of concern to the patient and of value to the clinician in determining an appropriate therapy. Most of the currently available data on E2 is in the form of clinical impressions or single case reports. Basic research into the physiologic mechanisms of erectile process coupled with elucidation of alterations with the endocrine challenges of aging will undoubtedly optimise the therapeutic options in patients with erectile dysfunction. With these thoughts, the study is undertaken to evaluate ED with particular emphasis on its occurrence in oestradiol derangement and secondary to phytoestrogen intake. In addition to the scientific understanding of the mechanism of the role of oestrogens in male erectile function, the models established may be useful in investigations of andropause and clinical hyperoestrogenism. 41 Chapter OESTROGEN RECEPTOR IDENTITY IN THE RABBIT CORPUS CAVERNOSUM 42 2. OESTROGEN RECEPTOR IDENTITY IN THE RABBIT CORPUS CAVERNOSUM 2.1. Objectives The role of oestrogens in the regulation of certain cellular mechanisms in the male is documented now and similar to the female physiology, these actions are also mediated through interaction with specific intracellular receptor proteins (Faustini-Fustini et al., 1999). Two oestrogen receptor (ER) isoforms α and β have been isolated, cloned and characterized so far (Green et al., 1986; Kuiper et al., 1996). Using molecular and biologic probes, these receptors have been characterised as ligand-dependent transcriptional activators which regulate gene expression through complex mechanisms mediated by ligand binding, transformation, dimerization and interaction with specific cis DNA hormone response elements, co-activators and co-repressors (Parker et al., 1993; Rissman et al., 1999). Initial identity of these receptors was made possible through immunohistochemical principles and several biochemical approaches. The two forms of ER once bound to the steroid hormone may modulate the expression of different genes and protein synthesis (Pavao and Traish, 2001). Although recently ERα and ERβ were shown to have an overlapping distribution with androgen receptors (AR) in multiple tissues (Saunders et al., 1997; Faustini-Fustini et al., 1999), the AR-rich cavernosum of human foetus lacked ER at 18-22 weeks of gestation (Kalloo et al., 1993). However, while sexual maturation and aging of the cavernosum were associated with a decrease in AR content (Takane et al., 1991) and cavernous testosterone concentration was considered to be an indicator of AR density in the CC (Becker et al., 2001), it is not known whether these processes influenced expression of ER in the penile tissue. With the identification of ER in several 43 other regions of the male reproductive tract (Fisher et al., 1997; Merchenthaler and Shughrue, 2000), it seems reasonable to assume a possible co-existence of these receptors with those for androgen in the penile cavernosum. With this specific hypothesis, oestrogen receptor immunoreactivity was tested in the untreated rabbit CC as a preliminary attempt to locate these receptors in this hitherto unexplored region of the male reproductive tract. 2.2. Materials and Methods 2.2.1. Tissue Source Six adult male New Zealand White (NZW) rabbits weighing between – 3.5kg were used for this investigation. The animals were sacrificed with an inhalational overdose of carbon dioxide in a sealed gas chamber connected to carbon dioxide tank. The penile tissue was dissected from the surrounding tissues and trimmed into small pieces. 2.2.1.1. Tissue Collection and Storage The tissue samples following dissection were transferred to small polystyrene vials and immediately snap-frozen by placing into liquid nitrogen (-180°C) for 15minutes. The vials were then stored in a deep freezer at -80°C until cryosectioning. 2.2.2. Protocol of Immunohistochemistry 2.2.2.1. Cryosectioning and Fixatives The tissue was placed on a brass mount and embedded within a layer of freezing medium (Einbettmedium) to create a more uniform block. This apparent embedding does not involve tissue penetration but aids in holding the specimen firmly while sectioning and preventing the sudden impact of the knife’s cutting edge to the tissue. The specimen was then cryosectioned using Leica CM3050 cryostat to thin slices (7µ). The sections were 44 mounted on Polysine® (Sigma) coated slides and immediately fixed in acetonechloroform mixture for 10 minutes. After this, the slides were lightly dried and the sections circled with DAKO pen to create a shallow well for holding antibody solutions and preventing their dispersion to the periphery. The sections were then treated with 10% H2O2 in methanol to reduce endogenous peroxidase activity, which may lead to considerable background staining and washed in Tris buffered saline (TBS: 50mM TrisHCl and 150mM NaCl, pH: 7.5). To prevent nonspecific reactions, the sections were incubated in normal goat serum (from the same animal species that provided the secondary / link antibody, goat antimouse DAKO: 1:30 dilution in TBS, pH 7.5) for 30 minutes at room temperature, which was later drained and slightly dried. This was followed by immunostaining by the avidin-biotin peroxidase method (Sar and Welsch, 1999). 2.2.2.2. Primary Antibodies Sections were then blocked with the respective primary antibody as overnight incubation at 4°C in a well-sealed humid chamber. The ER antibodies were mouse monoclonal antibodies (GeneTex Inc, San Antonio, Texas) raised to recombinant 6-His fusion protein containing the region encoding amino acids 1-190 of human ERα (ER-alpha-IF3) or amino acids 1-153 of human ERβ (ER-β-14C8) expressed in E.Coli. The optimal working dilution of the respective antibody (40µg/ml for ERα and 10µg/ml for ERβ) was previously determined by serial incubation of sections with different antibody concentrations. Control samples were incubated with the same volume of TBS. 2.2.2.3. Secondary and Tertiary Conjugates Sections were then washed with TBS and incubated with the secondary antibody, goat antimouse IgG (Sigma) at a concentration of 1:150 for 45 minutes at room temperature. 45 The secondary / link antibody was directed against the immunoglobulins of the species used to produce the primary antibody. This was followed by washing with TBS and incubation with streptavidin-HRP (DAKO) (the third enzyme-conjugated antibody specific for the goat immunoglobulin in the secondary antibody) at a dilution of 1:300 for 30 minutes. Both the secondary and tertiary antibodies were conjugated to the same enzyme. The addition of a third layer of antibody serves to further amplify the signals since more antibodies were capable of binding to the previously bound secondary reagent. 2.2.2.4. Developing Colour Reactions After a minute wash with TBS, the sections were developed with a freshly prepared colour producing substrate system, 3,3’-diaminobenzidine (Sigma). The intensity of immunostaining was monitored under the microscope and the reaction was stopped before the background staining became prominent by dipping the slides in tap water. Sections were lightly counterstained with the compatible histological stain Mayer’s haematoxylin, dehydrated in graded ethanol and cleared in xylene. The slides were mounted with DePex® mounting medium and examined for colour signals using Aristoplan Microscope (Leitz Wedzler) and Imagepro analyzer (Zeiss, Aristoplan). 2.3. Results 2.3.1. Identity of ERα in the Rabbit Cavernosum Using the monoclonal antiserum raised against the specific peptide, ERα was immunolocalised to cell nuclei in multiple fields of the cavernosum in male rabbits compared to their absence in the control sample, which was not exposed to any specific primary antibody. The domain of ERα used for raising the antibody had no significant 46 sequence homology to that of ERβ, therefore the signals obtained were specific for the alpha receptors. There was a mild non-specific background staining in some areas of the control section which was clearly different from the pattern of typical nuclear staining for the primary antibody (Figures 3-5). Although the signals for ERα were weaker and of varying intensity in some fields, the receptors were predominantly located in the branch of the vasculature and a number of smooth muscle cells in its vicinity (Figure 4). The nuclei in the branch of the blood vessel (Figure 4) stained more intensely in comparison to those in the blood vessel seen in control sample (Figure 3). Staining was absent in most parts of the connective tissue and the cavernosal sinuses. 2.3.2. Identity of ERβ in the Rabbit Cavernosum Extensive signals for ERβ were identified in the nuclei of connective tissue and some smooth muscle cells and within the endothelial lining of the cavernosal sinusoids in all fields; the signals were accentuated bluish haematoxylin discolouration of the nuclear material in these regions. Immunopositive signals for these ERβ were also easier to obtain, appeared at a higher dilution of the antibody (1:100) compared to ERα (1:25) and although exact quantification of the individual receptoral density was not carried out in this investigation, from the careful scrutiny of the microscopic fields, it appeared that the ERβ was more preponderant than ERα in the rabbit CC (Figure 5). 47 Figure 3: Histochemistry of Control Rabbit Cavernosum Under typical colour development with 3,3’-diaminobenzidine and haematoxylin, control sample of rabbit corpus cavernosum (not exposed to the primary antibody) shows the normal distribution smooth muscle (blue arrow), blood vessel (red arrow), connective tissue (green arrow) and sinusoidal space (black arrow) x400. 48 Figure 4: Immunostaining of Rabbit Cavernosum for Nuclear Oestrogen α Receptor Accentuated colour intensity of nuclei staining positive for oestrogen alpha receptors in the blood vessel and smooth muscle cells (indicated by red arrows) of the rabbit corpus cavernosum tissue sample exposed to mouse monoclonal α receptor antibody (x400). 49 Figure 5: Immunostaining of Rabbit Cavernosum for Nuclear Oestrogen β Receptor Extensive colour signals of nuclei staining positive for oestrogen beta receptors in the smooth muscle, endothelial and connective tissue cells (indicated by blue arrows) of the rabbit corpus cavernosal sample exposed to mouse monoclonal β receptor antibody and subsequent conjugation reactions (x400). 2.4. Discussion and Conclusion This investigation gives the first known report on the existence of oestrogen receptors alpha and beta in the rabbit penile cavernosum. Oestrogen receptor α and ERβ have been shown to be homologous to the extent of 95% and 59% respectively for their DNA- and ligand-binding domains whereas the amino terminus for the two receptors is distinct (Sar and Welsch, 1998; Pavao and Traish, 2001). Since the antibodies used in this 50 investigation were raised against the amino acid sequence for the respective receptor protein, specificity and accuracy are expected (technical data from GeneTex, Inc., USA). One of the intriguing aspects of this identity concerns the source of oestradiol that may target these receptors in the penile cavernosum. Given the absence of documented evidence so far on the aromatase activity in the CC, erectile tissue is probably not one of the peripheral sites of E2 formation in the male. As mentioned, some previous studies have indicated the expression of oestrogen binding sites / ER in the male reproductive tract (see Fisher et al., 1997; Merchenthaler and Shughrue, 2000) and their coexistence with AR (Saunders et al., 1997; Faustini-Fustini et al., 1999); together with these reports, high aromatase activity and E2 concentrations have also been demonstrated in rete testis, efferent ductules and even the sperms (Hess et al., 1997; 1995). Testosterone physiology including receptor-mediated T effects facilitate the normal development and function of the male reproductive tract and early oestrogenic exposure was shown to interfere with the gene expression patterns in seminal vesicle (Pentecost et al. 1988) and prostate (Salo et al., 1997), alter the tissue response to testosterone through changes in the expression of AR and up-regulate ER (Prins and Birch, 1995; 1997). Similar biochemical processes could have resulted in the expression of ER in the penile cavernosum at some later stage since they were not identified during foetal life (see Kalloo et al., 1993); recently ER together with androgen receptors has been identified in the clitoral cavernosum, the female counterpart in rabbits (Sadeghi-Nejad et al., 1998). The functional role of this receptoral co-existence also needs to be delineated for instance, in the rabbit prostatic tissue, ER expression increased significantly after castration (Bodker et al., 1994). 51 Although both subtypes of oestrogen receptors are key mediators of oestrogenic effects, ERβ has been shown to have a lower binding affinity for E2 than ERα in the rat ovary and uterus (Hiroi et al., 1999) and a preponderant distribution in the male reproductive tract (Merchenthaler and Shughrue, 2000) including the penile cavernosum as seen in this investigation. With this knowledge, it may be hypothesized that selective ER ligands including the natural SERMs which preferentially bind to / activate one ER isoform over the other may help in the delineation of the differential roles of these two receptors in the cavernosum. However, most of the available agents to date are tissue specific and not receptor-specific (Paige et al., 1999) with some exceptional effect of ICI 182,780 (Faslodex) mediating increased proteolytic degradation of ERα while apparently protecting ERβ (Parker et al., 1993; Loose-Mitchell and Stancel, 2001). This is somewhat similar to the genetic knock-out of the alpha receptor in the experimental animal model. Given the importance of these findings, the emerging premise from this investigation is the need for delineation of the exact functional roles of the two receptors as key effectors of oestrogenic activity in erectile pathophysiology. Several known SERMs bind with high affinity to both the ER isoforms but after binding with these receptors, they may act as agonists of E2 in some tissues and antagonists in some others (Mitlak and Cohen, 1999). The antagonistic effect in these cases is mediated by competition with endogenous oestrogens (due to structural similarity) for binding to ER (Sadovsky and Adler, 1998). Unlike oestrogen-bound ER, SERM-bound ER may not stimulate the oestrogen response element or gene transcription adequately (Goldstein et al., 2000). Phytoestrogen isoflavones including daidzein, normally present in soy foods are being recognized as natural SERMs due to studies demonstrating their conformational binding to ER (Setchell, 2001). Through further ER-dependent reporter gene assay and 52 ER competition binding assay, their effects are considered to be oestrogenic and / antioestrogenic (Collins-Burow et al., 2000). Taken together, the identity of ER in the cavernosum in this investigation also implies that standard oestrogen action or even modifications in the effect profile of endogenous hormone may be mediated by regular intake / exposure to these phytoestrogens in the male population. Given the role of these receptors in mediating oestrogenic effects in the female tissues, they may not be inert in the male erectile process. The pharmacological detrimental effects of oestradiol/oestrogenic activity on erectile function, probably mediated through these receptors within the cavernosum, are demonstrated in the ensuing chapters. 53 [...]... belief in correlation of the physiological effect of the aging process to T decline (Morley, 2000) Although the inverse linear decline in androgen levels with increase in age was reported for the first time in the fifties (Hollander and Hollander, 19 58), uncertainty lingered on whether the age related decline in serum T levels in men was universal Initial indications in the 19 60s and 19 70s of decreased... consist of an N terminal involved in the transactivation function, an active DNA-binding domain responsible for specific DNA binding and dimerization and a C-terminal domain involved in ligand binding, nuclear localization and ligand-dependent transactivation function (Parker et al., 19 93; Rissman et al., 19 99) Recent scientific evidence indicates that ER mutations occur normally in tissues and cell lines,... and Arata, 19 98) 1. 2.7.3.2 Testosterone in Male Sexual Function The role of circulating androgens with regard to erectile physiology and sexual behaviour is incompletely understood (Schiavi et al., 19 91) since in men with normal gonadal function there is no correlation between testosterone levels and measures of sexual interest, activity or erectile function (Andersson and Wagner, 19 95) In lower animals,... deficiency of the aging male, PADAM” (Sternbach, 19 98) Since varying levels of sexual interest as well as activity persist in the aged men, the adequacy of erectile function and sexual intercourse are primary concerns to many This has led to attempts to counter the sexual changes of aging in males with simple hormone replacement therapy similar to those tried in the females (Tserotas and Merino, 19 98) due... result in increase of cytosolic calcium This step initiates the binding of calcium to calmodulin and the resultant calcium calmodulin complex phosphorylates light chain of myosin through myosin light chain kinase This promotes an interaction between actin and myosin and the subsequent phosphorylation and contraction of the smooth muscle (Chacko and Longhurst, 19 94) The next important step to initiate... open directly into the cavernous spaces (Lue, 2002) The cavernous spaces drain into a system of venules that coalesce on the outer surface of cavernosa just beneath the tunica albuginea These venules form a number of veins traversing the tunica, called the emissary veins which drain into the circumflex veins These in turn drain into the deep dorsal vein of the penis, in the dorsal midline of the penile... et al., 19 95) Therefore, possible oestrogen dysfunction should be routinely considered in the evaluation of male infertility (Berg, 19 98) The role of oestrogen in male sexual behaviour is still unknown Since the initial clinical report of impotence with hyperoestrogenism (Edwards, 19 76) and the immediate refutes (O’Regan, 19 76; McSherry, 19 76), very few studies have looked at the effect of oestrogens. .. implications of oestrogens in man that includes its role on erectile capacity Presently our knowledge is mainly confined to the understanding that oestrogens can act on many different tissues including the male reproductive tract Exact delineation studies in men will prove more difficult to design and interpret because of the absence of an equivalent dramatic hormonal menopause in them 1. 3.3 Oestrogens. .. modulate intracellular calcium stores (Lincoln and Cornwell, 19 91) to initiate and sustain cavernosal relaxation cAMP exerts its effects by stimulating cAMP dependent protein kinase A and cGMP causes the relaxation through protein kinase G; these events result in dephosphorylation of myosin 17 light chains mentioned above Studies indicate a complementary interplay between the two signaling mechanisms (Lincoln... prolactin inhibits the secretion of GnRH and in the periphery, it is shown to interfere with the activity of 5α reductase, an enzyme required for the formation of dihydrotestosterone (DHT) in target tissues (Hsueh, 19 88) 1. 2.7.2 Effect of Oxytocin Oxytocin plays a role in the hypothalamic regulation of erection and is released into circulation during sexual activity in humans (Carmichael et al., 19 87) . al., 19 91a) in nature. Clinically, intracorporal injection of phentolamine is a successful method of diagnosing erectile dysfunction. Other compounds such as trazodone, ketanserin and yohimbine. cytoplasm to initiate the release of calcium from internal storage vesicles. 16 17 Intracellular calcium ions play a major role in erectile physiology with increase in calcium maintaining flaccidity. result in increase of cytosolic calcium. This step initiates the binding of calcium to calmodulin and the resultant calcium calmodulin complex phosphorylates light chain of myosin through myosin