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Effect of Testosterone on Intracavernous Pressure Elicited With Electrical Stimulation of the Medial Preoptic Area and Cavernous Nerve in Male Rats

From the Department of Urology, Sapporo Medical University, Hokkaido, Japan.

Correspondence to: Dr Shin-Ichi Hisasue, Department of Urology, Sapporo Medical University School of Medicine, S1-W16 Chuo-ku, Sapporo, Hokkaido, Japan 060-8543 (e-mail: hisasue{at}sapmed.ac.jp).

Received for publication May 21, 2006; accepted for publication August 21, 2006.

	Abstract

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We studied the effects of castration and testosterone (T) replacement on intracavernous pressure (ICP) elicited with electrical stimulation of the medial preoptic area (MPOA) and cavernous nerve (CN) in male rats. We measured the ICP during electrical stimulation of the MPOA and CN in castrated male rats with and without testosterone replacement. The experimental group consisted of 20-week-old male rats at 2 weeks (n = 8), 4 weeks (n = 8) and 8 weeks (n = 8) after castration, and at 8 weeks after castration with T replacement (n = 4). Intact 20-week-old rats (n = 8) served as controls. The erectile response was expressed as the ICP/blood pressure (BP) ratio. The ICP/BP ratios during CN stimulation of the animals at 2, 4, and 8 weeks after castration were significantly lower than those of the intact animals. However, the erectile responses were not eliminated. In contrast to these peripherally evoked responses, erectile responses elicited by electrical stimulation of the MPOA were eliminated following castration. After testosterone replacement, both erectile responses were restored. Testosterone plays important roles in both the central and peripheral neural pathways for the maintenance and restoration of erectile capacity. The central control of erection shows more extensive changes following testosterone replacement than the peripheral control.

     Key words: MPOA, penile erection

Numerous studies have demonstrated that testosterone and its metabolites enhance sexual desire and sexual activities (Meisel and Sachs, 1994). On the other hand, the effects of the hormone on erectile function remain to be investigated. Previous studies have demonstrated that testosterone ensures penile erection through maintenance of nitric oxide synthase (NOS) activities in the peripheral nervous system (Burnett, 2003; Saenz de Tejada, 2004), and that deprivation of the androgen may reduce the number of corporal smooth muscle cells through apoptosis (Traish et al, 1999). These findings confirm the peripheral effects of androgens on erectile physiology. However, the central effects of testosterone that are involved in initiating and maintaining penile erection remain unclear (Saenz de Tejada, 2004). Therefore, further research is required to determine the direct actions of testosterone on centrally induced penile erection.

To evaluate the central effects of testosterone on erectile capacity, we have to use animal models with centrally evoked penile erection (ie, the penile reflex with administration of apomorphine), non-contact erections or erections elicited by peripheral and central electrical nerve stimulations (Meisel and Sachs, 1994; Sachs, 2000). We have developed an in vivo model that allows measurement of the intracavernous pressure (ICP) elicited by electrical stimulation of the medial preoptic area (MPOA), which is designated as central stimulation, and of the cavernous nerve (CN), which is designated as peripheral stimulation, in the same animal (Sato and Christ, 2000). This rat model displays penile erection through activation of the central and peripheral neural pathways. Comprehensive analysis of ICP responses during electrical stimulation of the MPOA and CN in the same animal may have important implications for the central regulation of testosterone for penile erection. In this regard, the goal of the current investigation was to quantify the effects of castration and testosterone replacement on the ICP responses to electrical stimulation of the MPOA and CN in male rats.

	Materials and Methods

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Experimental Design

Sprague-Dawley rats (300–350 g), 20 weeks of age, were used in the present study. The rats were housed in a 24-hour airconditioned room on a 12-hour light/dark cycle. The rats were divided into the following five groups: 1) controls (n = 8), 2) 2 weeks after castration (n = 8), 3) 4 weeks after castration (n = 8), 4) 8 weeks after castration (n = 8), and 5) 8 weeks after castration followed by testosterone replacement (n = 4). In the fifth group, castration was performed at the age of 16 weeks, followed by intraperitoneal administration of 50 mg testosterone propionate 2 weeks later. The blood testosterone levels were 3060 ± 165.0 ng/dL at 3 days and 838.3 ± 73.4 ng/dL at 2 weeks. Electrical stimulation of the MPOA and CN was performed 2 weeks after the administration of testosterone.

After a 0.5-mL blood sample was collected from the vein of each rat, the blood concentrations of total testosterone were determined by solid-phase radioimmunoassay (DPC Total Testosterone kit; Nippon DPC Inc, Tokyo, Japan) at 3 days and 2 weeks after administration to 4 rats.

All of the experiments were performed according to the recommendations of the Guidelines for Animal Experiments of Sapporo Medical University.

Measurement of ICP

ICP elicited by electrical stimulation was measured according to our previously described method (Sato and Christ, 2000). Briefly, rats were placed in the supine position, with their four limbs strapped. Anesthesia was induced by intraperitoneal administration of pentobarbital (35 mg), followed by the administration of 5–10 mg/kg pentobarbital every 45–60 minutes, to maintain sedation. The sympathetic ganglia and CN on both sides of the rats were identified. The unilateral crus of the corpus cavernosum was then punctured by a cannula to which a 22G needle was attached. After confirming penile erection by the infusion of physiologic saline, a pressure transducer was connected for ICP monitoring. Central arterial pressure (BP) was monitored by inserting a 22G cannula into the carotid artery on the left side of the incised neck.

For electrical stimulation of the peripheral nerves, stainless steel bipolar wire electrodes (SNE-100; Rhodes Medical Instruments, Woodlands Hills, Calif) and a pulse generator were used. Electrical stimulation (10 mA) was given for 10 seconds on the separated CN on both sides, which were picked up with hooks. For electrical stimulation of the MPOA, the upper half of the strapped rat was turned 180° so that the head could be held in a horizontal orientation in a stereotaxic head-holder (Kopf 900; David Kopf Instrument, Tujima, Calif) (Sato and Christ, 2000). An electrode needle was then inserted into the MPOA and electrical stimulation (100 µA, 30 Hz, 2-ms duration) was applied. The MPOA was located in accordance with the atlas of Paxinos and Watson at 0.4–0.6 mm lateral and 0.2–0.8 mm caudal, and at a depth of 8.8–9.0 mm from the bregma. We have confirmed previously that these procedures are sufficiently precise to evaluate the results for the MPOA (Sato et al, 1999; Sato and Christ, 2000; Adachi et al, 2003).

Since the levels of electrically stimulated ICP are strongly affected by differences in blood pressure among rats, which might result in bias, we used ICP/BP ratios in evaluating erectile responses to stimulation. ICP and BP were measured with the Mac Lab 8/e (ADI Instrument, Milford, Mass) and Mac ETH-400 (ADI Instrument) ADI instruments, and the ICP/BP ratios were calculated.

Statistical Analysis

For the statistical analysis, the StatView software was used. Significant differences were tested by the Mann-Whitney U test. All differences were considered significant at P less than .05.

	Results

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Serum Testosterone Levels in Castrated Rats with Testosterone Replacement

Radioimmunoassay of 4 rats revealed serum testosterone levels of 3060 ± 165.0 ng/dL at 3 days after testosterone replacement and 838.3 ± 73.4 ng/dL at 2 weeks after testosterone replacement.

Characteristics of Changes in the ICP/BP Ratios Induced by MPOA and CN Electrical Stimulations Following Castration

Significant increases in the magnitudes of the ICP/BP ratios were observed with electrical stimulation of the CN of rats 2, 4, and 8 weeks after castration, as compared to the ratios before stimulation (Table 1). In contrast to these peripherally stimulated responses, the ICP responses elicited in rats by electrical stimulation of the MPOA were eliminated by castration.

Effects of Castration and Testosterone on ICP/BP Ratios Induced by Peripheral and Central Electrical Stimulation

In the control group, sufficient penile erection, as determined by the ICP/BP ratio, was achieved by CN electrical stimulation (Figure 1A). In rats at 2, 4, and 8 weeks after castration, the ratios were significantly lower than in the control group (P < .01 by the MannWhitney U test) when the stimulation was given. In rats with testosterone replacement, the ICP/BP ratio during CN stimulation was significantly higher than in castrated animals, and returned to a magnitude comparable to that of the control.

Electrical stimulation of the MPOA produced erectile responses that could be evaluated by the ICP/BP ratio in the control group (Figure 1B). Castration markedly reduced the ratios. In castrated rats, significantly lower MPOA-stimulated ratios were found by electrical stimulation of the MPOA in rats at the given intervals (P < .01 by the Mann-Whitney U test). Similar to the ICP response to CN stimulation, the responses to MPOA stimulation following testosterone replacement recovered to a level that was significantly higher than those in rats with castration but without testosterone replacement.

	Discussion

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Testosterone or its metabolites may play a critical role in the central modulation of male sexual function. Reports on the central control of male sexual behavior have confirmed the direct effect of testosterone on mounting behavior (Meisel and Sachs, 1994; Sato et al, 1998). Recently, testosterone replacement was reported to play a key role in the central control of penile erection (Baba et al, 2000a,b). Our results show that testosterone is essential to ensure penile erection through activation of the central nervous system in male rats. These novel findings emphasize the importance of testosterone replacement for erectile dysfunction in patients with hypogonadism (Heaton, 2000; Buvat and Montorsi, 2001; Rampin and Giuliano, 2001).

View larger version (20K): [in this window] [in a new window]   The effects of castration and testosterone replacement on the magnitudes of the intracavernous pressure/blood pressure ratios by electrical stimulation of (A) the cavernous nerve and (B) the medial preoptic area in rats. Statistical differences were determined by the Mann-Whitney U test. ICP: intracavernous pressure; BP: blood pressure; CN: cavernous nerve; MPOA: medial preoptic area; T-replace: testosterone replacement.

Although previous studies using various animal models have demonstrated centrally mediated proerectile effects of testosterone (Makenna, 2004), the significance of our results is not reduced. While our MPOA animal model involves erectile responses elicited by smooth muscle relaxation but not by contraction of the penile striated muscle (Sato and Christ, 2000), other central models show erectile responses induced by smooth muscle relaxation and contraction of the penile striated muscle. Thus, the current results demonstrate the elimination and restoration of penile erection induced exclusively by smooth muscle relaxation due to castration and testosterone replacement, respectively. The importance of this characteristic is supported by the fact that human penile erection is initiated and maintained dominantly by the relaxation of penile smooth muscles (Saenz de Tejada, 2004).

One of the mechanisms for the central action of testosterone in modulating penile erection is the enhancement of NOS activities in the central nervous system. Previously, with testosterone replacement, it was confirmed that histological restoration of neuronal NOS in the peripheral nervous system after castration contributed to the recovered erectile response (Zvara et al, 1995; Baba et al, 2000a,b). Moreover, the collagen tissue/smooth muscle ratio, which also affects normal erectile function (Takahashi et al, 1991), was significantly higher in castrated animals. However, previous investigations have suggested that central nitric oxide (NO) as well as that in the end organ (ie, penile tissues) may play an important role in penile erection (Melis and Argiolas, 1997; Sato et al, 2001).

Gonadal steroids enhance NOS activity in the MPOA (Pu et al, 1996). The results of our previous studies are compatible with this hypothesis, as testosterone replacement significantly enhanced NOS activity in the MPOA and paraventricular nucleus, which is the critical nucleus in the hypothalamus (Sato and Tsukamoto, 2000). A recent study has demonstrated a clear relationship between MPOA nNOS immunoreactivity and copulatory measures in testosterone-induced restoration of castrated animals (Sato et al, 2005). Central NO is directly associated with the centrally evoked ICP response. An intrathecal NO donor and NOS inhibitor increased and decreased the MPOA-stimulated ICP response, respectively, in the same rat model used in our previous investigation (Sato et al, 2001). Although testosterone replacement may systemically affect not only the central NO but also other neural systems and organs that modulate sexual function, enhancement of the central NO response following testosterone replacement may contribute to the restoration of erectile responses evoked both centrally and peripherally.

Another interesting finding in the current study is that at 8 weeks after castration, a significant increase in the ICP response was elicited by electrical stimulation of the CN, in contrast to the elimination of the MPOA-stimulating ICP response. This implies that CN-stimulating ICP responses consist of testosterone-dependent and testosterone-independent components. Reilly et al (1997) have reported that relaxation of the cavernous smooth muscle is under partial androgenic control. Our results are consistent with this hypothesis. In contrast to the peripheral erectile response, the central response was eliminated, as found in the current study. The MPOA is one of the CNS areas that is richest in androgen and estrogen receptors (Pu et al, 1996; Sato and Tsukamoto, 2000; Sato et al, 2005). As mentioned earlier, gonadal steroids regulate nuclear function through activation of NOS and other neuromodulators, such as dopamine (Sato et al, 1998; Putnam et al, 2003). Castration may induce a decrease in the number of NOS-containing neurons, whereas gonadal steroid replacement restores these populations. In addition to these findings at the cellular level, physiological observations support the strong androgenic dependency of the centrally evoked erectile response. Nocturnal penile erection, which is considered to be controlled by central modulation, depends strongly on androgenic conditions (Foresta et al, 2004). On the other hand, this might explain the reflexogenic erection in patients who have undergone bilateral orchiectomy but who can still have coitus. Although the androgenic threshold for maintaining penile erection remains to be discovered, information derived from the findings of the present study enhances our understanding of the physiology of erectile function and its dysfunction following partial androgen deficiency in the aging male.

Many studies have shown that the MPOA is one of the critical brain areas involved in the modulation of male sexual function. The MPOA sends neural fibers to the nuclei in the spinal cord, which connects with the CN, a critical autonomic pathway for penile erection. Thus, electrical stimulation of the MPOA may activate a putative physiological pathway that induces erectile response (Meisel and Sachs, 1994; Heaton, 2000; Sato and Christ, 2000; Rampin and Giuliano, 2001). However, a question that arises from our current results is where the most likely site of action of testosterone is located, since the central-to-peripheral neural pathway is quite long. The candidate sites presumably exist in the MPOA, spinal cord, and preganglionic or postganglionic neurons. Giuliano et al (1993) have indicated that postganglionic parasympathetic neurons are the targets for gonadal steroids. However, if testosterone or its metabolites act on a part or the entirety of the proerectile neural pathway, the cumulative effect may have a greater impact on erectile function than the postganglionic effect alone. In the near future, we should investigate the exact site of action of testosterone with histological confirmation. Another limitation of this study is the small number of animals used. In future studies, we need to use more animals in the testosterone replacement group, to ensure appropriate statistical comparisons among the groups. Moreover, we should confirm the possibility of the actions of the metabolites estradiol and dihydrotestosterone.

The current results suggest that the actions of testosterone and its metabolites on both the central and peripheral neural pathways are crucial for maintaining and restoring erectile capacity. The mechanism of enhanced erection with testosterone depends not only on the peripheral neural pathway but also on the central neural pathway. In the near future, we hope to conduct a clinical trial of testosterone replacement for the treatment of erectile dysfunction with the use of peripherally and centrally mediated medications, such as phosphodiesterase inhibitors and apomorphine.

	Acknowledgments

 
We express our special gratitude to Dr Shiro Hinotsu and Dr Takahiro Suetomi of Tsukuba University for advice on the statistical analysis, Ms Toshie Kurohata for technical assistance in performing experiments, and Mr M. Kim Barrymore for help in preparing the manuscript.

	Footnotes

 
Supported by grants-in-aid for scientific research (12671547 and 16591605) from the Japanese Society for the Promotion of Science.

	References

Top Abstract Materials and Methods Results Discussion References

 
American Association of Clinical Endocrinologists. American Association of Clinical Endocrinologists Medical Guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male patients - 2002 update. Endocr Pract. 2002; 8: 440 –456.[Medline]

Adachi H, Sato Y, Kato R, Hisasue S, Suzuki K, Masumori N, Itoh N, Tsukamoto T. Direct evidence of facilitative actions of dopamine in the medial preoptic area on reflexive and noncontact erections in male rats. J Urol. 2003;169: 386 –389.[CrossRef][Medline]

Baba K, Yajima M, Carrier S, Akkus E, Reman J, Nunes L, Lue TF, Iwamoto T. Effect of testosterone on the number of NADPH diaphorase-stained nerve fibers in the rat corpus cavernosum and dorsal nerve. Urology. 2000a;56: 533 –538.[CrossRef][Medline]

Baba K, Yajima M, Carrier S, Morgan DM, Nunes L, Lue TF, Iwamoto T. Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis. BJU Int. 2000b;85: 953 –958.[CrossRef][Medline]

Burnett AL. Neurophysiology of erectile function: androgenic effects. J Androl. 2003; 24: S2 –5.[Free Full Text]

Buvat J, Montorsi F. Safety and tolerability of apomorphine SL in patients with erectile dysfunction. BJU Int. 2001; 88(suppl 3): 30 –35.[Medline]

Comhaire FH. Andropause: hormone replacement therapy in the ageing male. Eur Urol. 2000; 38: 655 –662.[Medline]

Foresta C, Caretta N, Rossato M, Garolla A, Ferlin A. Role of androgens in erectile function. J Urol. 2004; 171: 2358 –2362.[CrossRef][Medline]

Giuliano F, Rampin O, Schirar A, Jardin A, Rousseau JP. Autonomic control of penile erection: modulation by testosterone in the rat. J Neuroendocrinol. 1993; 5: 677 –683.[Medline]

Heaton JP. Central neuropharmacological agents and mechanisms in erectile dysfunction: the role of dopamine. Neurosci Biobehav Rev. 2000;24: 561 –569.[CrossRef][Medline]

Makenna K. Further commentary: Experimental model for the study of male sexual function. In: Lue TF, Basson R, Rosen R, Giuliano F, Khoury S, Montorsi F, eds. Sexual Medicine: Sexual dysfunctions in men and women. The Second International Consultation on Sexual Medicine. Plymouth: Plymbridge Distributors Ltd.; 2004; 241 –286.

Meisel RL, Sachs BD. The physiology of male sexual behavior. In: Knobil E, Neill JGS, Greenwald GS, Markert CL, Pfaff DW, eds. The Physiology of Reproduction, Vol. 2. New York: Raven Press; 1994; 3 –107.

Melis MR, Argiolas A. Role of central nitric oxide in the control of penile erection and yawning. Prog Neuropsychopharmacol Biol Psychiatry. 1997;21: 899 –922.[CrossRef][Medline]

Mills TM, Reilly CM, Lewis RW. Androgens and penile erection: a review. J Androl. 1996; 17: 633 –638.[Free Full Text]

Morales A, Heaton JP. Hypogonadism and erectile dysfunction: pathophysiological observations and therapeutic outcomes. BJU Int. 2003;92: 896 –899.[CrossRef][Medline]

Morales A, Lunenfeld B. Investigation, treatment and monitoring of late-onset hypogonadism in males. Official recommendations of ISSAM. International Society for the Study of the Aging Male. Aging Male. 2002;5: 74 –86.[Medline]

Morley JE, Perry HM 3rd. Androgen deficiency in aging men. Med Clin North Am . 1999;83: 1279 –1289.[CrossRef][Medline]

Pu S, Xu B, Kalra SP, Kalra PS. Evidence that gonadal steroids modulate nitric oxide efflux in the medial preoptic area: effects of N-methyl-D-aspartate and correlation with luteinizing hormone secretion. Endocrinology. 1996; 137: 1949 –1955.[Abstract]

Putnam SK, Sato S, Hull EM. Effects of testosterone metabolites on copulation and medial preoptic dopamine release in castrated male rats. Horm Behav. 2003; 44: 419 –426.[CrossRef][Medline]

Rampin O, Giuliano F. Brain control of penile erection. World J Urol. 2001; 19: 1 –8.[CrossRef][Medline]

Reilly CM, Zamorano P, Stopper VS, Mills TM. Androgenic regulation of NO availability in rat penile erection. J Androl. 1997; 18: 110 –115.[Abstract/Free Full Text]

Sachs BD. Contextual approaches to the physiology and classification of erectile function, erectile dysfunction, and sexual arousal. Neurosci Biobehav Rev. 2000; 24: 541 –560.[CrossRef][Medline]

Saenz de Tejada I. Further commentary: Physiology of erectile function and pathophysiology of erectile dysfunction. In: Lue TF, Basson R, Rosen R, Giuliano F, Khoury S, Montorsi F, eds. The Second International Consultation on Sexual Medicine: Sexual Medicine-Sexual dysfunctions in men and women. Plymouth: Plymbridge Distributors Ltd.; 2004; 289–343.

Sato S, Braham CS, Putnam SK, Hull EM. Neuronal nitric oxide synthase and gonadal steroid interaction in the MPOA of male rats: co-localization and testosterone-induced restoration of copulation and nNOS-immunoreactivity. Brain Res. 2005; 1043: 205 –213.[CrossRef][Medline]

Sato Y, Christ GJ. Differential ICP responses elicited by electrical stimulation of medial preoptic area. Am J Physiol Heart Circ Physiol. 2000;278: H964 –970.[Abstract/Free Full Text]

Sato Y, Christ GJ, Horita H, Adachi H, Suzuki N, Tsukamoto T. The effects of alterations in nitric oxide levels in the paraventricular nucleus on copulatory behavior and reflexive erections in male rats. J Urol. 1999;162: 2182 –2185.[CrossRef][Medline]

Sato Y, Shibuya A, Adachi H, Kato R, Horita H, Tsukamoto T. Restoration of sexual behavior and dopaminergic neurotransmission by long term exogenous testosterone replacement in aged male rats. J Urol. 1998;160: 1572 –1575.[CrossRef][Medline]

Sato Y, Tsukamoto T. Effects of nitric oxide stimulation on the brain. Drugs Today (Barc). 2000; 36: 83 –92.

Sato Y, Zhao W, Christ GJ. Central modulation of the NO/cGMP pathway affects the MPOA-induced intracavernous pressure response. Am J Physiol Regul Integr Comp Physiol. 2001; 281: R269 –278.[Abstract/Free Full Text]

Takahashi Y, Hirata Y, Yokoyama S, Ishii N, Nunes L, Lue TF, Tanagho EA. Loss of penile erectile response to intracavernous injection of acetylcholine in castrated dog. Tohoku J Exp Med. 1991; 163: 85 –91.[Medline]

Traish AM, Park K, Dhir V, Kim NN, Moreland RB, Goldstein I. Effects of castration and androgen replacement on erectile function in a rabbit model. Endocrinology. 1999; 140: 1861 –1868.[Abstract/Free Full Text]

Zvara P, Sioufi R, Schipper HM, Begin LR, Brock GB. Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. Int J Impot Res. 1995; 7: 209 –219.[Medline]

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This Article Abstract Full Text (PDF) All Versions of this Article: 28/2/218    most recent Author Manuscript (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, N. Articles by Tsukamoto, T. Search for Related Content PubMed PubMed Citation Articles by Suzuki, N. Articles by Tsukamoto, T.