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1 Kyoto University Primate Research Institute, Inuyama, Aichi, Japan2 Department of Psychology, University of Richmond, Richmond, VA 23173, USA
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-dihydrotestosterone, had additive, independent effects on male potentia over a range of hormone concentrations, whereas androgens were confirmed to be the primary determinants of sexual motivation. We suggest that modulation of the serotonergic system by ‘female hormones’ may be fundamental to the regulation of male mating success in higher primates. This might also explain, at least in part, why significant correlations between steroid hormones and male copulatory behaviour have traditionally proven so elusive in this order, thereby warranting a re-evaluation of the current notion that male sexual behaviour has been emancipated from activational hormonal control in higher primates.
(Received 11 September 2005;
accepted after revision 13 December 2005; first published online 19 December 2005)
Corresponding author G. M. Barrett: Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 501 Smyth Road, Ottawa, Ontario, K1H 1C4, Canada. Email: zavalabarrett{at}yahoo.com.ar
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(Smith et al. 1994) have yielded conflicting results, although the small number of subjects and the likelihood of other coexisting mutations (Carani et al. 1997) preclude extrapolation as to the role of oestrogens in the sexual behaviour of human males in general (Sharpe, 1998). The study of human sexuality is rife with difficulties, and potentially confounding non-hormonal factors range from reporting biases to partner availability, stress, disease status and cultural influences, among others (Robbins, 1996). Non-human primates may represent an ideal model for the study of male sexual behaviour, owing to their highly developed social relationships, and physiological as well as phylogenetic propinquity to humans. Although relatively few studies have examined the role of oestrogens in the sexual behaviour of male non-human primates, pharmacological inhibition of aromatase activity significantly decreases sexual motivation and ejaculatory activity in testosterone-treated, castrate male cynomologus monkeys (Macaca fascicularis; Zumpe et al. 1993).
Oestrogens also modulate various neural systems, such as the cholinergic (Kompoliti et al. 2004), GABAergic (Wagner et al. 2001) and monoaminergic systems (McEwen & Alves, 1999), many of which have established or suspected roles in male reproductive functioning. Notably, the serotonergic system is implicated in myriad autonomic functions ranging from pituitary hormone secretion to sexual behaviour, satiety, cognition and affect (Jacobs & Azmitia, 1992), most or all of which are sensitive to oestradiol as well (McEwen, 1999). Although it is not yet known to what extent the serotonin neural system mediates the actions of oestradiol, oestrogens alter the expression of pivotal genes at various levels in a manner that appears to facilitate serotonin synthesis and neurotransmission in female macaques (Bethea et al. 2002). In male rats, both testosterone and oestradiol increase the density of the serotonin transporter (SERT) and 5-hydroxytryptamine2A receptor (5-HT2AR) mRNA and binding sites in brain (Sumner & Fink, 1998; McQueen et al. 1999). The fact that oestrogen mimics the action of testosterone but 5-dihydrotestosterone (DHT), an active testosterone metabolite and potent androgen that cannot be aromatized, has no effect suggests that the action of testosterone is due to its enzymatic conversion to oestradiol (Fink et al. 1999). Recently, Cornil et al. (2005) have shown that a single injection of oestradiol activates copulatory behaviour in castrated male quail, concomitant with a marked increase in brain serotonin activity. While substantial evidence now links functional interactions between oestrogen and serotonin in females (see Amin et al. 2005 for a comprehensive review), we are unaware of any previous study investigating the relationship between oestrogens and serotonergic function in male primates.
Serotonin (5-hydroxytryptamine, 5-HT) was previously believed to have a predominantly inhibitory role in the neuronal background and thus in sexual behaviour, although it is now clear that different brain regions respond differently to serotonergic influence. While the involvement of the serotonin neural system in the control of sexual behaviour has been well established in the male rat, comparatively few studies have been conducted in other species. The majority of investigations in primates thus far have focused on the effects of pharmacological alterations of serotonin neurotransmission, with both facilitative and inhibitory responses being observed, depending on the receptor subtype activated. Unfortunately, the lack of selective pharmacological agents has precluded direct examination of the 5-HT2 receptor subfamily, which is thought to be involved to the greatest extent in the regulation of behaviour (Sachenko & Khorevin, 2001). In one of the few studies in which serotonergic drugs were not employed, a significant correlation was observed between concentrations of the dominant serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in cerebral spinal fluid (CSF) and sexual behaviour of free-ranging male rhesus macaques (Mehlman et al. 1997).
Previously believed to contribute little to the control of male sexual behaviour, progesterone is also emerging as a ‘female hormone’ with a functional role in male reproduction. While it has been known for decades that pharmacological doses of synthetic progestins, such as cyproterone acetate and medroxyprogesterone acetate, are antiandrogenic and inhibit male sexual behaviour (Witt et al. 1995), more recent studies designed to produce physiological titres have shown a stimulatory effect of progesterone in the male (Witt et al. 1997). Using mice with a targeted disruption of the progesterone receptor (PR), Phelps et al. (1998) have shown that males heterozygous for the null receptor have impaired responses to testosterone, although they could not rule out the possibility that the influence of the PR is mediated by interactions with the ER after the aromatization of testosterone to oestradiol. Furthermore, progesterone administration appears to inhibit the uptake of oestradiol in the male macaque brain (Zumpe et al. 1997, 2001), thus potentially having a modulatory influence on the serotonergic system as well.
Here we endeavour to draw together the disparate knowledge concerning oestrogens and serotonin in the regulation of male primate sexual behaviour by asking whether behavioural variation is consequent on oestrogen modulation of the serotonergic system. Rather than pharmacologically manipulating the serotonergic system directly, we instead variously altered the endocrine milieu and examined the effects on whole blood serotonin, whole blood tryptophan (TRP) and behaviour over a range of sex steroid concentrations. We chose this approach in the hope of circumventing the various complexities associated with interpreting the effects of serotonergic drugs on sexual behaviour. We used as our subjects intact male Japanese macaques (M. fuscata), since levels of serotonin and its precursor tryptophan in blood are significantly correlated with CSF levels in this species (Yan et al. 1993).
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Nine intact, sexually mature male Japanese macaques (M. fuscata) were the subjects of this research, six of which served as treatment males and three as controls. The number of subjects represents a conscious effort to use the minimum number of animals required to reasonably address the problem in question while still allowing for statistical interpretation. A randomized, blinded, sham-treated and self-controlled, multitreatment cross-over design was employed. The study consisted of a baseline-sampling period followed by five experimental phases, each separated by a 10 day washout and/or establishment (WO/E) period for the various pharmacological agents in use. In addition to the nine subject males, three adult females of the same species were used as stimuli to elicit male-typical behaviours. The study was conducted from October 2002 to March 2003, corresponding to the natural breeding period we have previously observed in wild Japanese macaques (Barrett et al. 2002).
An important consideration in multitreatment cross-over studies is carry-over, or the so-called order effects, whereby the effects of one intervention persist and influence the subjects' response to an ensuing intervention. Although assay results can reasonably indicate whether or not the WO/E period was of sufficient duration to reset the animals' physiology in terms of the variables in question, our interpretation of observed effects is contingent upon behaviour being similarly reset and stabilized. Rather than operating under the assumption that this criterion had been fulfilled, we randomized experimental animals to one of two groups, designated Cohort 1 and Cohort 2, and administered treatments to these groups in reciprocal fashion. We performed repeated measures ANOVA for each of the variables under study to examine potential order of treatment effects. There were no significant effects of ‘cohort’ on any of the variables, so data from Cohort 1 and Cohort 2 experimental males were combined.
Subjects
The nine male subjects (mean weight 10.8 ± 1.0 kg mean ±S.E.M.) were maintained in individual squeeze-back cages (width 90 cm x height 82 cm x depth 76 cm) in a large temperature-controlled room with the three stimulus females (mean weight 6.9 ± 0.6 kg mean ±S.E.M.). Artificial lighting was provided for 12 h between 06.30 and 18.30 h, and a large window permitted entrance of ambient light. Diet consisted of twice-daily provision of a commercial monkey chow, supplemented with fresh fruits and vegetables. Water was available ad libitum at all times, and food was restricted only prior to the use of anaesthetic agents. The maintenance and experimental procedures were approved by our institutional animal welfare committee, and were in accordance with The Guide for the Care & Use of Laboratory Primates (2002) of the Kyoto University Primate Research Institute, which adheres to NIH standards.
Hormonal and drug treatments
Following an acclimation period, 2 weeks of sampling with no interventions were conducted in order to establish baselines. Five experimental phases, each lasting 2 weeks and bracketed by a WO/E period, ensued. Throughout the study, females received weekly injections of oestradiol cypionate (700 µg of steroid per subject in 0.3 ml sterilized sesame oil vehicle, I.M.), in order to maintain a constant level of attractiveness and receptivity as well as to override the menstrual cycle and prevent pregnancy (Bercovitch et al. 1987).
Phase 1: aromatase deficiency.
Males were given a twice-daily dose of the non-steroidal aromatase inhibitor anastrozole (1,3-benzenediacetonitrile,,,','-tetra-
methyl-5-(1H-1,2,4-triazol-1-yl-methyl). Anastrozole was purchased commercially in 1 mg tablets, pulverized, and administered orally before the morning and afternoon feeds in 0.5 mg doses incorporated into choice food. Subjects were strictly monitored for compliance and all readily ingested the drug. A similar dose given to male pigtailed monkeys (M. nemestrina) in preclinical pharmacological testing was found to effectively reduce circulating oestradiol concentrations (Dukes et al. 1996). Control males received only the food vehicle.
Phase 2: androgen deficiency. Subjects received the novel and potent gonadotropin-releasing hormone (GnRH) antagonist Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph-(Ac)-D4Aph(Ac)-Leu-Ilys-Pro-DAla-NH2]. Acyline (courtesy of Dr R. Blye,) the national institute of child health and human development USA (NICHD) was made available as a lyophilized powder, which we formulated in bacteriostatic water with 5% mannitol for tonicity at a concentration of 1 mg ml–1. The drug was administered on alternate days (50 µg kg–1, S.C.) using a very fine (27 gauge) syringe, and all injections were immediately followed by a reward of choice food. Placebo injections consisted of an equivalent volume of 5% mannitol in bacteriostatic water and were administered to control animals following an identical regimen.
Phase 3: oestrogen excess and androgen deficiency. Lengths of Silastic® tubing packed with crystalline 17ß-oestradiol (E2) 2.0 mm i.d. x 3.0 mm o.d., effective length 55 mm, two per male, were surgically implanted subcutaneously in the periscapular region of experimental males, while control males received empty implants of equal dimensions. Acyline and placebo injections were administered as in phase 2. Surgical procedures were carried out under full anaesthesia (a cocktail of 10 mg kg–1 ketamine hydrochloride (Sankyo, Tokyo, Japan) and 1 mg kg–1 midazolam (Yamanouchi, Tokyo, Japan), I.M.) and with due attention to sterile precautions.
Phase 4: oestrogen excess. In this phase, E2 implants remained in place, while the GnRH antagonist Acyline was allowed to clear from the subjects' systems.
Phase 5: oestrogen excess and aromatase deficiency. Anastrozole dosing was reinstated as per phase 1, with E2 implants still in place.
Behavioural testing
Prior to the initiation of the study, animals were given 6 weeks to acclimate to the experimental protocol, during which time compatible pairs for behavioural testing were established, compatibility being defined as the occurrence of one or more ejaculatory sequences during a 3 min test period. In order to minimize any potentially confounding influence of partner preference, a longitudinal rather than a cross-sectional design was employed, with each male being paired with the same female for the entire study period. Each male was tested with a stimulus female for a 30 min period twice weekly, for a total of four observational sessions per male per phase. The three females were housed in separate pair cages, and the subject males were transferred to the pair cages immediately prior to the test. All interactions were videotaped with the investigator out of the room, and subsequently analysed to determine the rates of the various behaviours of interest by a trained observer who was blind to both the experimental protocol and allocation of subjects. We report here on two measures of sexual behaviour, one concerning potentia (frequency of ejaculation) and the other libido or sexual motivation (latency to first mount). We also consider self-grooming, grooming performed, and Hinde's Index (Hinde & Atkinson, 1970), which is a function to quantify the relative contributions by males and females to the maintenance of proximity. Hinde's Index ranges from +1, indicating that the male is wholly responsible for proximity maintenance, to –1 where the female is wholly responsible. A value of 0 denotes either mutual attraction or mutual indifference. Hinde's Index has previously been shown to be a powerful predictor of both mating and reproductive success in this species (Soltis et al. 1997a,b), hence its inclusion in the present analysis.
Blood samples and assays
Weekly blood samples of 3 ml were collected in heparinized syringes from the cubital vein of unanaesthetized subjects briefly restrained by the squeeze-back mechanism. All samples were collected between 13.00 and 14.00 h on non-behavioural testing days. A 1 ml aliquot of whole blood was immediately transferred to a 1.5 ml polypropylene microcentrifuge tube containing 10 mg of ascorbic acid (to prevent oxidative degradation of monoamines during the freeze–thaw cycle), vortexed briefly, assigned a random number and stored at –30°C for use in monoamine assays as described below. The remaining 2 ml were centrifuged (3600 g, 15 min, 4°C) and the resulting plasma decanted, capped, assigned a random number and stored at –30°C. Plasma concentrations of testosterone (T), its active metabolites 17ß-oestradiol (E2) and 5-dihydrotestosterone (DHT), as well as sex hormone-binding globulin (SHBG), were determined using commercially available kits. 17ß-Oestradiol was quantified by enzyme immunoassay (EIA) (Cayman Chemical, Ann Arbor, MI, USA). The E2 antibody showed 100% cross-reactivity with oestradiol, 17% with oestradiol 3-glucuronide, 4% with oestrone and less than 1% with all other steroids tested. The detection limit was 8 pg ml–1. Testosterone was also quantified by EIA (Cayman Chemical). The assay had a detection limit of 6 pg ml–1 and showed 100% cross-reactivity with T, 27.4% with 5-DHT, 18.9% with 5ß-DHT, 3.7% with androstenedione, 2.2% with 11-keto testosterone and less than 1% with all other steroids tested. DHT levels were assessed by ELISA (IBL Immuno-Biological Laboratories, Hamburg, Germany). The detection limit of this assay was 6 pg ml–1, and cross-reactivities were 100% with 5-DHT, 8.7% with T, 2.0% with 5ß-DHT and less than 1% with all other steroids tested. SHBG was assayed by I125 IRMA (Orion Diagnostica, Espoo, Finland). The lower limit of detection was 1.3 nmol l–1 and there was no reported cross-reactivity with other serum proteins. Lastly, progesterone (P) was measured by EIA using the R4861 antibody, generously provided by Professors S. Shideler and B. Lasley, University of California at Davis. This assay has been described elsewhere (Daels et al. 1998). For all assays, inter- and intra-assay coefficients of variation were less than 10%. In cases where levels exceeded the linear range of the standard curve, samples were diluted and reassayed. Levels falling below the linear range were assigned the lower limit of detection of the assay (90% B/B0). Free testosterone concentrations were calculated from total T and SHBG levels and assuming a constant albumin concentration (Södergard et al. 1982). Calculations were performed by a computer program for simultaneous equations (developed by Scott Winters, Columbia University, NY, USA). The validity of calculated free T values has been previously discussed (Vermeulen et al. 1999).
Determination of monoaminergic levels
Whole blood 5-HT and its precursor tryptophan (TRP) were measured by high-performance liquid chromatography (HPLC) with fluorometric detection, according to a modification of an existing methodology (Anderson et al. 1981). Distilled, deionized water, further purified via a Millipore Ultra-Pure Water System (Tokyo, Japan) was used in the preparation of all aqueous solutions. 5-Hydroxytryptamine, TRP and 5-hydroxytryptophan (5-HTP) used as standards were purchased from Sigma (St Louis, MO, USA). All other reagents were of the highest quality commercially available and were used without further purification.
Samples were prepared by transferring 250 µl of whole blood into a 1.5 ml centrifuge tube, adding 25 µl of 10 µg ml–1 5-HTP as internal standard, 40.5 µl of 1.5 mol l–1 ascorbic acid, and vortexing for 2–3 s. Then, working quickly, samples were deproteinized by the addition of 50 µl of 3.4 mol l–1 perchloric acid, capped and vortexed vigorously for 15 s. Samples were placed on ice for 15 min before being centrifuged at 13 000g for 15 min at 4°C. The supernatant was transferred to a clean 1.5 ml polypropylene tube, centrifuged for an additional minute at 13 000g, and 20 µl of the resulting supernatant was manually injected into the injection loop port of the chromatograph.
The HPLC system consisted of a Hitachi L-7100 pump and a Shimadzu RF-10AXL fluorescence detector, with the analog output of the detector interfaced via a Hitachi L-7420 data recorder to a Hitachi D-7500 chromatointegrator. Separations were achieved on a reverse phase analytical column (Hitachi WH-C18, 4 mm i.d. x 150 mm) preceded by a guard column (WH-C18, 4 mm i.d. x 5 mm). Analytes were isocratically eluted with a solvent system of 950 ml of sodium acetate (pH 4.0, 10 mmol l–1) and 50 ml of methanol. The mobile phase was delivered at a flow rate of 1.0 ml min–1, yielding a pressure of 125–130 kg cm–2. Excitation and emission wavelengths were set at 285 and 345 nm, respectively. The identities of the 5-HT and TRP peaks were confirmed by superimposing the chromatograms of samples spiked with known amounts of standards on those of unspiked samples, as well as by the comparison of retention times in standards and samples.
A stock solution of standards containing 2 mg ml–1 5-HT and 10 mg ml–1 TRP was prepared and stored at –30°C in 1 ml aliquots. Working standard solutions were prepared immediately prior to each experiment, and a four-point calibration curve (range: 250–2000 ng ml–1 for 5-HT, 1250–10 000 ng ml–1 for TRP) was run on each day that analyses were conducted. The relationship between the area under the peak and concentrations of 5-HT and TRP in working standards was linear (coefficients of regression (r2) = 0.999 and 1.000 for 5-HT and TRP, respectively), and concentrations of all samples analysed fell within the range of the calibration curve. Intraday precision (relative standard deviation), assessed by analysing 10 replicates of a pooled whole blood sample, was 7.50% c.v. for 5-HT and 3.27% c.v. for TRP. Interday precision was 3.79% c.v. (5-HT) and 4.71% c.v. (TRP) for six replicate analyses of the same pool spanning 30 days. To determine analytical recovery, pooled whole blood samples (n= 6) were spiked with known amounts of standards over a range of concentrations and the response compared to the native sample. Mean recovery of added standards was found to be 73 ± 9% S.D. and 89 ± 8% S.D. for 5-HT (31.25–500 ng added) and TRP (156.25–2500 ng added), respectively.
Statistical analyses
Individual, weekly values of the physiological and behavioural variables were entered in a correlation matrix between the two sets of variables. We used a canonical correlation analysis to investigate the relationship between the behavioural (X set) and the physiological (Y set) of variables. Logistic regression analysis was performed on 105 weekly data points (9 individuals x 6 phases x 2 samples per phase = 108, 3 samples unusable = 105 data points) to establish which variables had significant independent and additive effects on ejaculation. The final logistic model was examined following parsimonious streamlining of the original model to remove non-significant terms. The Wald 
2 test was used to establish whether each explanatory variable had a significant independent and additive effect on the dependent variable. Owing to the continuous nature of the dependent variable, linear regression analysis was used to assess the independent effects of neuroendocrine and behavioural variables on mount latency. We hypothesized that treatment effects, temporal variation in this seasonal breeder, and the combination of the two factors were such that our data points would satisfy the intrinsic assumption of independence and therefore be suitable for use in multivariate analyses. Indeed, calculation of the Durbin-Watson coefficient based on the residuals of all regression models revealed no significant autocorrelation within the sample. Lastly, having determined which of our numerous variables contributed significantly to the behaviours of interest, we examined treatment effects on these parameters using repeated measures ANCOVAs with ‘group’ (experimental or control) as an independent categorical factor. All analyses were two-tailed.
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2(25)= 96.2, P < 0.00001). The variance extracted by the physiological set of canonical roots was 100% with 19.7% redundancy, and that extracted by the behavioural set was 100% with 14.2% redundancy. Given that physiology and behaviour were so interconnected in our study that a combination of linear equations was able to explain most of the variance in the dataset, we combined the two sets in a regression model in order to determine which variables were significantly involved in affecting male potentia.
Data from all individuals were dichotomized based on the presence or absence of ejaculation during the behavioural session, and the relative contribution of experimental and control males to the two groups did not differ significantly. Logistic regression analysis was used to determine which factors had an additive and independent effect on ejaculation, considered as a binary (Yes/No) variable. E2, TRP and H Index were found to correspond to these criteria, and the model composed by these variables produced the best fit for positive occurrences of ejaculation (2(4)= 111.8, P < 0.001; –2log likelihood = 73.8, Table 1). A correlation matrix (Table 2) revealed a number of distinct associations that differentiate sessions where ejaculation occurred (n= 60) from those where it did not (n= 45). Most notable was the fact that TRP was significantly correlated with the active metabolites of T (DHT and E2) in ejaculating males only. In terms of mean absolute values, several parameters differed significantly between the two groups, although the two variables directly manipulated (T and E2) did not (Table 3). We finally considered the latency to first mount within the ejaculating group, calculating a linear regression model to determine what factors contributed to sexual motivation in those individuals who copulated successfully. DHT, Free T and GRM SELF were found to have additive, independent effects on latency. The resulting regression model was robust (F8,60= 4.03, P < 0.001, Table 1), and was able to explain a significant portion of the variance (r2= 0.35). As the observed relationship between androgens and sexual motivation merely reconfirms what has previously been demonstrated in numerous studies on a multitude of species, we will not elaborate further on this association here.
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The most common approach currently used to assess serotonergic function in human and non-human primates is the measurement of the dominant serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in CSF following lumbar puncture. However, as the collection of CSF involves either anaesthesia or catheterization when repeated, sequential measures of serotonergic activity are desired, as in the experimental study of behaviour, efforts have been made to establish peripheral markers of brain serotonergic status. Multiple lines of evidence suggest that the measurement of either serotonin or its amino acid precursor tryptophan in the periphery are biologically meaningful and may constitute accessible models of central serotonergic function. Although serotonin itself does not cross the blood–brain barrier, blood and brain serotonin share similarities in metabolic production, storage, degradation and responses to pharmacological agents (Stahl & Meltzer, 1978; Moffitt et al. 1998), and alterations in blood serotonin are positively correlated with brain serotonin levels in rats (Malyszko et al. 1993). Numerous studies in psychobiology have focused on levels of serotonin in the periphery, and significant associations have been reported with suicidal behaviour, epidemiological levels of violence, various psychiatric disorders and depression, among others (Banki, 1978; Mann et al. 1992; Quintana, 1992; Moffitt et al. 1998; Pfeffer et al. 1998).
Measurements of tryptophan in blood are theoretically of greater appeal as a surrogate assay for central serotonergic activity, since physiological changes in plasma tryptophan concentration influence brain serotonin levels (Fernstrom & Wurtman, 1971). The activity in brain of the enzyme tryptophan hydroxylase (TPH) is thought to be the rate-limiting step in the biosynthesis of serotonin from tryptophan, although the concentrations of tryptophan in neurones are insufficient to saturate the hydroxylase under normal physiological conditions (Jequier et al. 1969), thus suggesting that serotonin synthesis is limited by tryptophan availability (Fernstrom & Wurtman, 1971). Indeed, it is well established that serotonin production in the brain is directly related to blood tryptophan levels in humans and other mammals (McKinney et al. 2005), despite the fact that distinct central and peripheral isoforms of TPH have now been recognized (Walther & Bader, 2003). Oral tryptophan administration leads to elevated CSF 5-HIAA in humans and animals (Bender, 1983), and dietary tryptophan depletion leads to a rapid reduction in cerebral serotonin synthesis (reviewed by Russo et al. 2003). Tryptophan levels in blood are associated with hostility and levels of aggressive behaviour in healthy men (Wingrove et al. 1999), and tryptophan depletion has been reported to lower mood in the normal male (Young et al. 1985). Alterations in tryptophan availability are concomitant with a host of mood and other disorders, and it has recently been suggested that tryptophan may serve as a link between psychopathology and somatic states (Russo et al. 2003). Lastly, in a study of normal, drug naïve male vervet monkeys, blood tryptophan concentrations were correlated with a number of behaviours thought to be serotonergically influenced, and it was suggested that blood tryptophan better reflects central serotonergic activity than does blood serotonin (Raleigh et al. 1981).
We measured progesterone levels retrospectively because we hypothesized that, via several mechanisms, this steroid may in part account for the disparate physiological and behavioural profiles that characterize the two groups. Firstly, progesterone has the highest affinity for the enzyme steroid 5-reductase of all known steroids, and is preferentially converted before testosterone (Lephart, 1993). While DHT formation is usually a passive function of testosterone levels (Wilson, 1975), competitive inhibitors, including progesterone, may modulate 5-reductase activity (Cassidenti et al. 1991; Krieg et al. 1985), and Cassidenti et al. (1991) have shown that in vitro, progesterone inhibits the conversion of testosterone to DHT by 97.0 ± 5.3%. Thus, the elevated progesterone levels in non-ejaculating males may account for the fact that this group had significantly lower levels of DHT despite similar availability of substrate. Secondly, the administration of pharmacological doses of progesterone to castrated, testosterone-treated male cynomolgus monkeys reduced by 80% the uptake of [3H]oestradiol in the preoptic area (Zumpe et al. 1997, 2001), the brain structure thought to most directly influence sexual performance in males (Everitt, 1990). As oestradiol appears to have a stimulatory effect on serotonin synthesis, it is conceivable that the elevated progesterone levels in non-ejaculating males resulted in less uptake of oestrogen in the brain, thus inhibiting serotonergic activity. This appears consistent with the significantly lower levels of tryptophan observed in this group compared to males who copulated successfully.
There is some evidence in the clinical literature for an association between serotonergic function and male potentia. A large percentage of men suffer from sexual dysfunction, which is often comorbid with disorders of mood and is affected by psychotropic medications, including selective serotonin reuptake inhibitors (SSRIs; Rosen et al. 1999). SSRIs are commonly associated with delayed ejaculation and absent or delayed orgasm, while a specific association with libido (i.e. sexual motivation) has not been consistently demonstrated. Furthermore, other studies have shown SSRIs to have a positive impact on men suffering from premature ejaculation (Althof et al. 1994). Taken together, this suggests an involvement of the serotonergic system in male potentia as opposed to motivation, a finding confirmed in the present study. It would indeed be interesting to examine the relationship between active testosterone metabolites and serotonergic status in that subset of patients who suffer sexual dysfunction versus those who do not. We would predict a decoupling of the two in the former that, if amenable to adjunct intervention, would have significant ramifications in terms of SSRI treatment compliance and quality of life of those patients so affected. Further work is needed to determine which agents would best address this putative decoupling, although in light of our observation of the powerful inhibitory effect of progesterone on ejaculation frequency, an antiprogestin may well be a worthwhile starting point.
It is widely held that although hormones regulate the copulatory behaviour of most mammalian species, primates are more complex and copulatory ability has been emancipated from biological controls in this order (i.e. Wallen, 2001; but see Wilson, 1999). According to this view, psychological and social forces in primates have subjugated the hormones that mediate copulatory behaviour in other species. Without detracting from the central importance of these factors in the expression of primate copulatory behaviour, we suggest that the general perception that they are the sole determinants of male potentia is perhaps too parochial. Our study, examining a previously unexplored mechanism of hormone action in the male, suggests that steroid hormones may in fact play a significant contributory role in male potentia. This opens the door to a reinterpretation of a host of studies focusing on the factors regulating male mating and reproductive success in primates. If male reproductive success is viewed as contingent upon a suitable physiology to accommodate potentia, and if this physiology is susceptible to social, environmental and psychological perturbation, classic theories in sexual selection, such as the theory that male reproductive success is limited only by access to sexually receptive females, may require a footnote. At the very least, and in consideration of our limited sample size, these results provide a way forward to investigate the neuroendocrine regulation of male potentia in higher primates. Much more work is needed to determine what aspects of serotonergic function are modulated by oestradiol in the male and how this translates into differential mating success.
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