The Official "Androgens and your Armpits" thread

[lanky]

Lets face it, your armpits can smell sometimes,,some peoples smell worse than others, It all starts during puberty when your armpits start to smell,,this is thought to occur due to androgens mediating the develpement, enlargement, andi ncrease in activity of the apocrine glands in your pits, and your genital region....

AAS just dont act on your muscles, prostate, and scalp alone...in the nexyt few days or so depending on my schedule i will post some interesting findings on how androgens induce armpit odor( And what you can do about it)

I will try and answer the following questions.

lanky, can you tell me why do my armpits smell?

Lanky,Can certain AAS make your pits smell good?

Lanky, Which ones will make them smell bad?

Lanky, does the smell of the testosterone metabolites my armpits secrete with or without the prescence of bacteria acting on them actually attract women?

Lanky, do you have any scientific evidence to back this up?

well the last question i think you know the answer to..lol

I would also like to know your experience on different AAS and the odor of your armpits while on them. and the thickness of secretions .be descriptive.

 

[UA_Iron]

I got the foulest Armpit BO while on Tren Acetate.

Androgens are awesome.

 

[lanky]

Male Axillary Extracts Contain Pheromones that Affect Pulsatile Secretion of Luteinizing Hormone and Mood in Women Recipients1

lankys quick notes---"Women were less tense (exact, two-tailed, significance = 0.012) and more relaxed (exact, two-tailed, significance = 0.022) during exposure to the male extract relative to the control stimulus"

"Our data are the first evidence that male axillary extract causes neuroendocrine and mood alterations in women. Male extract brings on a subsequent LH pulse sooner than it would occur under endogenous rhythms, suggesting that one or more extract components influence the GnRH pulse generator"

Human underarm secretions, when applied to women recipients, alter the length and timing of the menstrual cycle. These effects are thought to arise from exposure to primer pheromones that are produced in the underarm. Pheromones can affect endocrine (primer) or behavioral (releaser) responses, provide information (signaler), or perhaps even modify emotion or mood (modulator). In this study, we extracted underarm secretions from pads worn by men and placed the extract under the nose of women volunteers while monitoring serum LH and emotion/mood. Pulses of LH are excellent indicators of the release of GnRH from the brain's hypothalamus. In women, the positive influence of GnRH on LH affects the length and timing of the menstrual cycle, which, in turn, affects fertility. Here we show that extracts of male axillary secretions have a direct effect upon LH-pulsing and mood of women. In our subjects, the putative male pheromone(s) advanced the onset of the next peak of LH after its application, reduced tension, and increased relaxation. These results demonstrate that male axillary secretions contain one or more constituents that act as primer and modulator pheromones.


gonadotropin-releasing hormone, luteinizing hormone, menstrual cycle, neuroendocrinology, pheromones


INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES


Odors play an important role in mammalian reproductive biology. In nonhuman mammals, chemical signals, known as pheromones, emitted from one animal can cause a variety of changes in behavior and/or physiology when received by a conspecific ([1, 2] for review). Historically, two broad categories of pheromones were defined: releaser pheromones, which generate immediate, primarily behavioral responses (such as sexual attraction and/or copulation); and primer pheromones, which generate slower physiological/endocrine/neuroendocrine responses, including hormonal changes that alter reproductive function [3]. A third category, signaler pheromones, was introduced to encompass chemical signals in which information is conveyed but no obvious primer or releaser effect could be established [4]. Jacob and McClintock [5] recently introduced the concept of modulator pheromones as an additional group of potential chemical signals. These are chemicals that have the potential to affect the state or mood of the recipient and/or regulate multisensory inputs during exposure. The authors [5] suggest that two steroids may fulfill the criteria as modulators when used at concentrations well above reported endogenous levels. Recently, Chen and Haviland-Jones [6] provided data suggesting that information about human emotional state is contained within axillary secretions and that other people could accurately infer the emotional state of the donor after sniffing the secretions.

In humans, several studies have indicated that interpersonal relations among women, as well as between men and women, may alter reproductive endocrinology, suggesting the presence of primer pheromone activity. These relations include menstrual synchrony among women, first documented in all-women groups [7] and later replicated in a variety of other situations ([1, 2] for review).

Russell et al. [8] were the first to present evidence that suggested menstrual synchrony could be mediated by axillary secretions. Further studies employing these secretions from women also suggested that extracts of pooled samples, collected from women across the menstrual cycle, could be used to bring a recipient group of women into synchrony with the donors [9]. Extracts of male axillary secretions also appear to affect women with a history of irregular cycle lengths. Lengths of the menstrual cycles of these women showed a significant shift (vs. controls) toward the normal cycle length of 29.5 ± 3 days [10] upon application of male axillary secretion to the upper lip of the recipient women.

Additional support for axillary components from women having primer-pheromone activity is contained within the studies by Stern and McClintock [11] and Shinohara et al. [12]. These studies have focused on female axillary secretions being applied to women recipients. The results of Stern and McClintock [11] suggest that exposing women with normal menstrual cycles to axillary extracts from women in their follicular phase (the days following menses but several days prior to ovulation) shortens the length of the recipient's menstrual cycle by 1.7 ± 0.9 days. Exposing these same women to axillary extracts collected near the time of ovulation of the donors lengthens the recipient's menstrual cycle by 1.4 ± 0.5 days. Interestingly, similar effects on the lengths of the estrous cycles of female rats were noted when the rats were exposed to the follicular and ovulatory odors of other rats [13, 14]. Studies that have sought to provide a measure of primer pheromone activity by looking for a change in the length of the menstrual cycle have received some criticism for statistical and/or methodological errors [15–18]. Other criticisms have been fueled by the intra- and intersubject variability found in normal, consecutive menstrual cycles [19, 20].

Shinohara et al. [12] recently demonstrated changes in frequency of LH pulses in women exposed to female axillary secretions. These investigators demonstrated an average 28% decrease in LH pulse frequency in women receiving follicular phase secretions and a 16% increase in LH pulse interval in women receiving ovulatory phase secretions. The authors suggest that the changes in LH pulsing caused by axillary components are modulating the time of ovulation and changing cycle length; these changes may provide an objective measure of human primer pheromone activity.

Exogenous stimuli, such as a conspecific's chemical signal, appear to alter reproductive function by influencing the hypothalamic GnRH pulse generator. Although GnRH levels are not easily measured in humans, alterations in GnRH output can be inferred by measuring LH pulsing in blood. Changes in pulse frequency, height, and interval occur across a normal menstrual cycle. As women move from the early follicular phase of the menstrual cycle toward ovulation, LH pulses increase in frequency; however, LH pulse amplitude decreases in the midfollicular phase and increases at the time of ovulation. LH pulse frequency and amplitude progressively decrease across the luteal phase as the next menses draws near [21].

We hypothesized that a human male primer pheromone could act to alter menstrual cycle length and timing by altering LH parameters in a fashion analogous to that previously reported for female goats [22] and ewes [23]. In these animals, exposure of estrogen-primed females to either the male goat or his hair decreased the LH pulse interval, bringing on the next pulse after exposure to male stimuli faster than exposure to control conditions [22, 23]. Data presented herein demonstrate that applications of extracts of male axillary secretions cause a significant decrease in the latency to the next LH pulse when compared with application of a control substance. In addition to this primer pheromone effect, women also reported experiencing less tension and being more relaxed during exposure to the male extract, suggesting the presence of a modulator pheromone. These effects may serve as objective measures of primer and modulator pheromone activity that could guide an analytical isolation of the active axillary constituents.


METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES


Eighteen heterosexual women were enrolled in the protocol, which was approved by the Institutional Review Board at the University of Pennsylvania.

Subjects were given a gynecologic exam and Pap smear; each had a full history taken. Each subject met the following criteria: 1) was between 21 and 45 yr of age (median and average ages: 27 yr and 28.2 ± 5.7 yr [SD], respectively); 2) had regular menstrual cycles of 25 to 35 days duration in the previous year (by self-report) and must not have skipped any cycle; 3) had stable exercise and sleep patterns; 4) had not taken any hormonal medication in the 3 mo preceding the study; 5) had a normal physical exam with no acne, hirsutism, or galactorrhea, and had body weight within 15% of normal according to the Metropolitan Life tables (Metropolitan Life Insurance Co., 1960); 6) had normal thyroid function tests; 7) had serum prolactin levels <15 ng/ml; and 8) was heterosexual. All subjects displayed normal olfactory ability and no general anosmia as evidenced by their ability to perceive dilute solutions of trimethylamine (0.25%) and 3-methyl-2-hexenoic acid (0.1%).

Once admitted to the study, subjects participated for three menstrual cycles discussed below as baseline, test, and final cycle.

Baseline Cycle

Beginning with the first or second day of menses, subjects recorded their basal body temperature on a standard calendar every morning. Morning urine samples were tested for LH daily, from Day 10 until the LH surge (using a commercially available kit; OvuQuick One Step, Henry Schein, Melville, NY), to determine the time of ovulation. Approximately 7 days following ovulation, serum progesterone levels were measured; a level >4 ng/ml was required for subjects to be included in the study.

Test Cycle

Basal body temperature, ovulation detection, and luteal phase progesterone measures were recorded as in the baseline cycle. However, within the first 7 days of the test cycle onset (the first day of menses is Day 1 of the menstrual cycle), subjects were admitted to the General Clinical Research Center (GCRC) at the Hospital of the University of Pennsylvania for a 12 h period: 0800–2000 h. During this time, subjects received 0.5 ml of control (ethanol) or male extract applied to the nasal region/upper lip every 2 h using a cotton pad. As described below, both extract and control were fragranced to be indistinguishable. Blood samples (5 ml) were drawn through a heparin lock every 10 min. The nurses who drew the blood and applied the stimuli as well as the subjects were blind to the nature of the applied materials.

Since we had no a priori knowledge regarding potential lasting effects of male extracts upon LH pulsing parameters, half of the women were assigned to receive the control treatment during the first 6 h (0800–1400 h) of their stay in the GCRC and the male extract during the final 6 h (1400–2000 h); the other half received the treatments in reverse order.

Final Cycle

As a check on any changes in menstrual cycle length, patients were followed for an additional cycle by basal body temperature and urine LH measurements just as they were during their baseline cycle. Luteal phase progesterone levels were measured 7–10 days after the urinary LH indicated ovulation had occurred.

Male Extract and Control Solutions

The extract was created by using axillary secretions from four to six male donors. Secretions were collected on cotton pads as previously described [10, 24]. Three extracts were employed in the course of this study. Male donors were all healthy and ranged from 22–45 yr of age. All refrained from using deodorants/antiperspirants and washed/bathed using only plain soap with little to no fragrance (Ivory Soap, Proctor and Gamble, Cincinnati, OH) for a total of 4 wk. One week into this protocol, axillae were swabbed and axillary odor was assessed. Axillary swabs were used to generate bacteria cultures and determine the type and number of endogenous cutaneous bacteria. Following this, subjects collected axillary secretions on cotton pads three times per week. All pads were frozen (-10°C) until extracted. Male donors had no contact with and were not familiar with the female recipients.

We had no knowledge about the structural nature of the active axillary constituents; consequently, we arbitrarily chose to standardize all extracts to 0.5 µg/ml of 3-methyl-2-hexenoic acid (3M2H), the major analytical odorant found in the male axillary extracts [24]. This was done in the following manner: half of each extract was concentrated to 10 ml, and a 5 µl portion was analyzed by gas chromatography/mass spectrometry, as per previous studies [24], to determine the concentration of 3M2H. After quantifying the concentration of 3M2H, portions of the concentrated extract were added to the more dilute extract such that the concentration of 3M2H in the extract to be applied to subjects was 0.5 µg/µl.

The extract was then scented with a deodorant fragrance supplied by the Mennen division of Colgate-Palmolive (Piscataway, NJ) to a level of 0.005%. A similar volume of ethanol was used to dilute an aliquot of fragrance to 0.005% for use as an odor-matched control.

Mood Ratings

At 1 and 4 h into each 6-h exposure session, subjects were asked to provide ratings on a 7-point, Likert-type, categorical scale on the following: energetic, sensuous, tense, tired, calm, sexy, anxious, fatigued, relaxed, and active (see [25] for a full discussion of this type of scaling method). Subjects were allowed to self-interpret these adjectives. Horizontal scales, each on a separate sheet of paper, were composed from 1, labeled "I am not at all...," to 7, which was labeled "I am extremely...." Adjectives were scored by circling a numeral one at a time. After completing each scale, the forms were collected by the nurse so that subjects could not refer to previous ratings.

LH Levels and Pulse Detection

Each blood sample from our subjects was analyzed for LH using a standard immunoradiometric assay [26] by the Chemistry Core Lab of the GCRC of the Hospital of the University of Pennsylvania. After collection, blood samples were allowed to stand on ice for 30 min and then centrifuged. Serum was separated and frozen at -20°C until analysis. The minimum detectable level of LH was 0.1 mIU/ml of blood. Analyses of serum samples from groups of three to six subjects were run at different times during the course of this study. A coefficient of variation for the assay was calculated for each "batch" of samples by analysis of a pooled serum sample doped with standards. The coefficient of variation ranged from 2.8% to 12.3% during the course of this study.

The location, amplitude, and frequency of the LH pulses in the data were determined using the pulse detection software Cluster Analysis of Urban et al. [27]. With this program, significant pulses were detected using 2 x 1 cluster sizes (2 test samples for a nadir and 1 for a peak) and t-values of 2 x 2 to minimize false positives and negatives in the pulse detection. In addition to these parameters, we employed 0.75, 1.0, and 1.5 mIU/ml as minimum peak sizes for an LH pulse. All analyses yielded similar results. The results using 1.5 mIU/ml as a minimum peak size for a pulse are presented here.

Data Analysis

The mean LH levels, LH pulse amplitude, LH pulse frequency, and latency to next LH pulse in the control and extract periods of the follicular phase were compared using repeated measures analysis of variance (ANOVA) for each variable independently. Sequence of applications formed the independent groups factor; all others were repeated measures. Latency to the next LH pulse after an application also was analyzed with the nonparametric Wilcoxon signed ranks test. Possible diurnal effects were evaluated with paired-samples t-tests while ignoring type and order of stimulus application.

Categorical mood ratings were analyzed with the nonparametric sign test or the Wilcoxon signed ranks test. Prior to analyses of mood ratings, the two ratings in each phase (male extract and control) were compared with the sign test to determine whether there were any significant differences between ratings within each experimental condition. There were none; therefore, the two ratings in each phase of the experiment were averaged for subsequent analysis.


RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES


Eighteen ovulating, heterosexual women completed the protocol. The ages of the women ranged from 25 to 45 yr.

Post-hoc interviews with all subjects demonstrated that none correctly guessed the true nature of the stimuli that were being delivered to their nasal region. All subjects perceived "alcohol" and/or "fragrance." None guessed that sweat/axillary-secretion extracts had been applied when it was revealed to them at the end of the interview. No changes in menstrual cycle length attributable to our 6-hr application of extract were noted.

Figure 1 presents examples of the pulse patterns seen in our female recipients. Relative to the average latency to the next pulse during control applications (59 ± 5 min), male axillary extracts shortened the time to the next LH pulse by an average of 20% (to 47 ± 5 min; see Fig. 2). Since our study was a repeated measures design, the error bars in Figure 2 can be misleading. Hence, average latency difference scores were calculated by subtracting the average latency to the next pulse during control applications from the average latency to the next pulse during application of male extract. These difference scores are seen in Figure 3.





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FIG. 1. LH pulse patterns in four representative subjects. The times of control (C) and extract (E) applications are indicated, as are the LH peak tops (*). As described above, all LH pulses (peak tops) have been objectively determined by the Cluster Analysis software [27]







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FIG. 2. Average latency to the next LH peak subsequent to the application of male axillary extracts (extract; 47 ± 5 min) applied three times, spaced by 2 h each, or subsequent to the application of the control solutions (control; 59 ± 5 min), also spaced by 2 h each. In an analysis of variance (ANOVA), the main effect of stimulus type on latency to the next pulse was significant (F1,16 = 28.34, P < 0.001)







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FIG. 3. Average latency difference (in minutes) generated by subtracting the average latency to the next LH peak subsequent to the application of the control stimulus from the average latency to the next LH peak subsequent to the application of male axillary extracts. Sixteen of the 18 women had an average latency to the next LH pulse that was shorter in the extract condition than in the control condition; one woman had latencies that were equivalent in both conditions (Wilcoxon signed ranks test = 3.54; two-tailed P < 0.001)



The reduction in time between exposure to the extract and the onset of the next LH pulse was most effective following the first two applications (see Fig. 4) and suggests that there may be a refractory period following repeated exposures to male stimuli. Relative to the control condition, male extract did not affect the average number of LH pulses, the average LH pulse amplitude, or the basal level of LH (see Table 1).





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FIG. 4. Minutes to the next LH peak after each application of male axillary extracts (male extract), spaced by 2 h each, or after application of control solutions (control), also spaced by 2 h each. In an analysis of variance (ANOVA), the main effect of application number on latency to the next pulse was significant (F2,32 = 4.59, P < 0.02). Order of presentation was not a significant factor (F1,16 = 0.61, P > 0.44) and did not enter into any significant interaction with other factors (P > 0.60 for all F-values)






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TABLE 1. Number of LH pulses and blood levels of LH during exposure to male axillary extract or control solutions and a statistical evaluation of any difference between them



We also noted significantly greater average LH pulse heights in the second 6-h interval (afternoon to early evening [1400–2000 h]; 4.51 mIU/ml) than in the first interval (morning; 3.85 mIU/ml; t(17) = 5.18, P < 0.001) and significantly higher basal LH levels in the afternoon (3.10 mIU/ml) than in the morning (2.80 mIU/ml; t(17) = 3.44, P < 0.005), both of which appear to agree with published findings that LH levels in the afternoon are elevated relative to the morning [28]. The average number of pulses in the morning (4.3) was not different from the number in the afternoon (4.2). We also examined the possibility that a diurnal influence might be responsible for the decreased latency to the next LH pulse that was observed subsequent to application of male extract. We ignored the type of stimulus application and compared the latencies with the next LH pulse after an application in the first 6 h (0800–1400 h) to the latencies noted during the next 6 h (1400–2000 h). The results of the ANOVA indicated that there was no significant difference between the morning (latency = 55 ± 5 min) and afternoon (latency = 51 ± 4 min) sessions (F1,17 = 1.75, P > 0.20).

Women were less tense (exact, two-tailed, significance = 0.012) and more relaxed (exact, two-tailed, significance = 0.022) during exposure to the male extract relative to the control stimulus (Fig. 5). No other effects on mood were noted. Mood ratings also were reanalyzed to determine whether there was a diurnal effect: there was none. The effects on moods appear to be specific to the male extract: a woman nurse was in close contact with the subject throughout the experiment, yet the mood effect was noted only during application of the male extract.





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FIG. 5. Mood ratings were analyzed with nonparametric statistics; hence we present the median ratings for tension and relaxation during the control and male extract phases. Responses could range from 1 ("I am not at all tense/relaxed") to 7 ("I am extremely tense/relaxed"). Scales of this type are routinely used to collect such data [43, 44]. Complete data for tension were available for 17 women; 16 had ratings of tension while exposed to the male extract that were equal to or lower than their ratings of tension while exposed to the control stimulus, which is unexpected in a random distribution (Wilcoxon signed ranks test = 2.61; two-tailed P = 0.009). These same women also accounted for the increased ratings of relaxation, although data were available for all 18 women. The other mood ratings were not significantly different between control and male extract conditions




DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES


Our data are the first evidence that male axillary extract causes neuroendocrine and mood alterations in women. Male extract brings on a subsequent LH pulse sooner than it would occur under endogenous rhythms, suggesting that one or more extract components influence the GnRH pulse generator. In nonhuman mammals, studies have demonstrated that conspecific chemosignals may cause a rapid increase in LH. Although the neuroendocrine response in women was not like that of female rodents [29], it did rival that seen in goats. In ovariectomized female goats, which had been primed with estradiol to suppress the GnRH pulse generator, exposure to the chemical signals present on the hair of male goats decreased the intervals between LH pulses by about 10%, relative to control exposures, during 4 h postexposure [30]. In contrast to these effects in female goats, under normal cycling conditions in women we observed a decrease in latency of 20%, an effect that was twofold greater than noted in animals that had been hormonally manipulated to maximize an effect.

This study presents a potential mechanism by which male axillary secretions may alter menstrual cycle length and timing. The change in LH pulsing is predominantly seen during the first two applications of extract and suggests that the male stimulus may have only a transient effect upon the GnRH pulse generator. In addition to the noted endocrine change, which may be accurately described as a primer pheromone effect, the mood data, demonstrating a lessening of tension and increased feeling of relaxation, suggest the presence of a modulator pheromone. Whether these two effects are mediated by the same or different compounds, or sets of compounds, is unknown. No other behavioral effects were noted as a result of extract application.

All subjects perceived that alcohol or fragrance was being applied to them during their stay in the GCRC; none guessed the true nature of the stimuli or the delivery sequence. This suggests that conscious awareness of body odor is not necessary to elicit the primer and modulator pheromonal responses we observed.

LH pulse maxima (heights) during extract application were significantly greater relative to control applications in the afternoon and early evening. Further, LH pulse maxima during afternoon control applications were greater than both morning control and morning extract maxima. These results suggest a diurnal shift in LH pulse maxima. LH has been reported to increase to maximal levels during the 1100–1700 h time-frame [28]; this time encompasses 3 h in our morning/afternoon session (0800–1400 h) and 3 h in our afternoon/early evening session (1400–2000 h). Alternatively, male extract may have a "lingering effect," which is reflected as higher levels of LH during afternoon applications of the control stimulus. Male extract may "uncouple" the controlling effect that endogenous opiates have upon the GnRH pulse generator. In this regard, its action appears similar to a small, acute dose of an opiate antagonist such as naloxone. In the follicular phase of the human menstrual cycle [31], as well as in ovoids [32], chronic administration of opiate antagonists raises tonic LH levels. As reported above, we may have observed this in subjects receiving male extract; however, we cannot say with certainty that a diurnal effect is not responsible for the elevated LH. Future studies should attempt to resolve this question by employing the same 12-h protocol reported herein and exposing subjects to the control stimulus every 2 h for the entire 12 h.

Other studies that have examined the effects of male and female axillary secretions upon the menstrual cycle have relied upon changes in cycle length as a measure of activity. The results reported here present a potential neuroendocrine measure of extract activity and a mechanism by which axillary secretion components may change cycle length. A periodic acceleration of LH pulses might be expected to occur during intimate heterosexual encounters as part of a stable heterosexual relationship. This pattern may ultimately result in a greater frequency of pulses and entrain the pulse generator as well as stimulate ovarian follicles with a subsequent increase in tonic estrogen levels; this increase has been demonstrated in domestic ruminants [33, 34] and is suggested in data from humans where plasma estrogen levels in women having regular, weekly heterosexual intercourse are higher than in women having infrequent or no heterosexual intercourse [35].

The recent study by Shinohara et al. [12] describes a reduction in LH pulse interval in women exposed to the follicular phase secretions of women donors and an increase in the LH pulse interval in women exposed to ovulatory phase secretions. These results suggest that female axillary secretions affect other women; however, the effects are different from those of male axillary secretions used in our study. Aside from the first pulse after application, we did not observe a significant increase or decrease in pulse intervals across the time we monitored LH. There are, however, significant methodological differences between the studies. The application schedule and protocol of Shinohara et al. [12] differed from ours. These investigators applied their stimuli every 30 min; we applied every 2 h. In addition, they did not use each subject as her own control as we did. In their study, women were randomly assigned to receive isopropanol (control), follicular phase secretions, or ovulatory phase secretions. The latter two were presented on axillary pads moistened with isopropanol swiped on the upper lip; we used an ethanol extract of axillary pads. Despite these differences, these results support and strengthen the impact of our data: 1) axillary secretions from women affect the LH pulses of other women, and 2) the changes seen in LH pulses are of the same magnitude as those seen by us. Shinohara et al. [12] reported that follicular phase secretions decrease LH pulse interval by 28%, and ovulatory phase secretions increase pulse intervals by 16%; we noted a decrease in the latency to the next LH peak by 20%. Shinohara et al. [12] did not examine this parameter. Based on earlier studies by the same authors [36, 37], they propose androstenol (5-androst-16-en-3-ol) as the axillary constituent that is responsible for the change in LH. One of these earlier studies reported that women who exhibited menstrual synchrony had lower olfactory thresholds to androstenol [36]. In another study [37], women exposed to 2.5 mM androstenol in isopropanol (applied above the upper lip hourly for 4 h) experienced an increase in their LH interpulse interval vs. controls. The level of androstenol applied by these investigators to their subjects (300 µl of the 2.5 mM solution; personal communication to G.P.) far exceeds endogenous levels, reported to be 4–10 ng in male axillae [38, 39]. Furthermore, at the level used, the odor of androstenol would have been obvious to the recipients; no control odor was used to exclude the possibility that the observed results were spurious as the result of a novel odor.

Studies by Jacob and McClintock [5] utilized putative "human pheromones" reported to "stimulate the (human) vomeronasal organ" (VNO) ([40, 41]; however, see [42] for a review of experimental evidence for and against the existence of a functional, human VNO). These compounds are steroids, but only one, 4,16-androstadienone, has been reported in the axillae in picomolar quantities [38], which are far lower than the levels used by Jacob and McClintock [5] (9 nmol/recipient; the other compound, estra-1,3,5(10)tetraen-3-yl acetate, is a synthetic compound never reported in the axillae). Jacob and McClintock [5] used such high levels (1000 times greater than endogenous concentration) because they wished to increase the compound's concentration near the pits of the "vomeronasal organ." Previous studies from our laboratory have not revealed the presence of this steroid in axillary extracts produced by the extraction techniques we employed in the current study [39]. Consequently, although Jacob and McClintock [5] reported modulator pheromone activity for androstadienone when used at nonphysiological concentrations, we do not speculate on it or any other compound's role in our study.

 

[lanky]

Just givving you a quick lesson on what the chemical structure is on this next metabolite of testosterone that the armpit secretion contains......trying to give you a picture in your head of what this is......this is a cool metabolite...it has no hydroxy or keto on carbon 17,,,just a double bond that connects carbon 16 to carbon 17..on carbon 3 it has a hydroxy group,,it is also a 5 alpha reduced androstane,,

Effects of 5-Androst-16-en-3-ol on the Pulsatile Secretion of Luteinizing Hormone in
Human Females
Kazuyuki Shinohara, Masayo Morofushi, Tosihya Funabashi, Dai Mitsushima and Fukuko Kimura
Department of Physiology, Yokohama City University School of Medicine, 3–9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

Correspondence to be sent to: Kazuyuki Shinohara, MD, PhD, Department of Physiology, Yokohama City University School of Medicine, 3–9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. e-mail: [email protected]

Abstract

We examined the effects of 5-androst-16-en-3-ol (3-androstenol) on pulsatile luteinizing hormone (LH) secretion in human females. The frequency of the LH pulse in the follicular phase was decreased by exposing the women to 3-androstenol.

Introduction

Menstrual synchrony, the convergence of the onset date of the menstrual flow, is observed in women living together (Weller and Weller, 1993). Menstrual synchrony has been shown to be mediated by axillary odour (Stern and McClintock, 1998). Axillary odours from women in the follicular phase (FP) of the ovulatory cycle shorten both the time to ovulation and the length of the menstrual cycle in the recipients whereas axillary odours in the ovulatory phase (OP) delay ovulation and lengthen the menstrual cycle. However, the pheromones in the axillary compounds, which regulate both the time to ovulation and the length of the menstrual cycle, have not hitherto been identified.

When we examined the relationship between menstrual synchrony and the ability to smell a putative pheromone, 5-androst-16-en-3-ol (3-androstenol), the women who showed menstrual synchrony had a high sensitivity to 3-androstenol (Morofushi et al., 2000). It is therefore possible to speculate that human females use 3-androstenol to synchronize their menstrual cycles. We recently reported that axillary compounds in the FP increased the frequency of pulsatile secretion of luteinizing hormone (LH) and axillary compounds in the OP decreased it, suggesting that axillary compounds change the length of the menstrual cycle by changing the frequency of the pulsatile LH secretion (Shinohara et al., 1999). In the present study, we examined the effect of 3-androstenol on the pulsatile secretion of LH to explore the possibility that 3-androstenol is one of the active substances in axillary compounds which regulate the length of the menstrual cycle.

Materials and methods

Eleven female college students, aged 19–25 years and with a history of regular ovulation, were involved in this study. From 10:00 to 18:00 on days 5–7 after the menstrual onset, blood samples were collected through an i.v. heparin-locked catheter at 10 min intervals. The subjects took 100 kcal isocaloric snacks and 100 ml water hourly for 8 h. They were not exposed to anything for the first 4 h. Afterwards, 2.5 mM 3-androstenol in 70% isopropyl alcohol (IA) was applied hourly to half of them by wiping above their upper lip with a pad containing the solution for the next 4 h. IA alone was applied to the others in a similar way to 3-androstenol. Blood plasma was separated by centrifugation at 2000 g for 10 min and stored at –30°C until the LH assay (SAPC-S LH RIA kit; Daiichi Radioisotope Laboratory, Tokyo, Japan). The mean of the minimally detectable amount of LH was ~0.3 mIU/ml. The intra-assay coefficients of variation (CV) which were calculated on the basis of duplicated measurement of pooled samples containing 7.78 and 42.1 mIU/ml were 4.17 and 0.66%, respectively. An LH pulse was defined as when both the ascending and descending CVs were >1.7 times the intra-assay CV. The protocol observed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Yokohama City University. The subject gave written informed consent after the procedures had been fully explained.

Results

As seen in a representative subject (Figure 1a), the intervals between consecutive peaks of pulsatile LH secretion were 70, 70 and 70 min before the application of 3-androstenol, but after the start of the application the intervals became longer: 90 and 80 min. On the other hand, IA alone had no effect on the interpulse intervals: 60, 60 and 50 min before IA application and 60, 60 and 60 min during the application (Figure 1b). The mean (± SE) interpulse interval between LH pulses (n = 6) was significantly (P < 0.05, paired t-test) increased from 53.9 ± 3.9 to 66.0 ± 1.5 min in response to 3-androstenol. No change in the mean interpulse interval between LH pulses was observed in response to IA (P > 0.05, paired t-test). The mean interpulse intervals between LH pulses (n = 5) before IA application and during the application were 54.0 ± 4.9 and 53.8 ± 4.7 min, respectively. The data were expressed as the frequency are shown in Table 1. 3-Androstenol decreased the frequency while IA alone had no effect.





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Figure 1 Representative examples of the effect of 3-androstenol (a) and isopropyl alcohol (b) on pulatile LH secretion.






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Table 1 Effects of 3-androstenol and isopropyl alcohol on the frequency (the number of pulses/4 h) of LH pulses



Discussion
The present results demonstrate that the frequency of pulsatile secretion of LH is decreased by exposing women to 3-androstenol. This finding indicates that 3-androstenol retards the growth and maturation of ovarian follicles and consequently delays the timing of ovulation. It is therefore possible that 3-androstenol is involved in menstrual synchrony by increasing the period of menstrual cycles.

3-Androstenol is known to be secreted from the axillae in humans (Gower and Ruparelia, 1993). The steroid has been demonstrated to act as a pheromone in pigs (Gower and Ruparelia, 1993). It is secreted in the saliva of the boar and acts as a pheromone effective in eliciting the characteristic immobilization response of the estrous sow to the advance of her mate (Gower and Ruparelia, 1993). (Z)-7-Dodecen-1-yl acetate is used as a pheromone by the females of more than 126 species of insects and the elephant (Rasmussen et al., 1996). 3-Androstenol has been reported as affecting women’s mood (Gower and Ruparelia, 1993) and sexual arousal (Gower and Ruparelia, 1993), so it also may not be a species-specific pheromone.

Furthermore, women whose menstrual cycles became synchronized with that of room-mates within 3 months had higher olfactory acuity for 3-androstenol than non-synchronized women, suggesting that the ability to perceive the odour emitted by 3-androstenol is related to the menstrual synchrony (Morofushi et al., 2000). We recently showed that the frequency of the LH pulse was increased by axillary compounds in the FP, which shorten the menstrual cycle, and decreased by axillary compounds in the OP, which lengthen it (Shinohara et al., 1999). Together with these findings, the present results suggest that 3-androstenol may be a pheromone included in axillary compounds secreted in the OP, which lengthen the menstrual cycle.

Just summing everything up....although women nag and complain that we are smelly pigs...deep down they really like it..lol

 

[lanky]

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane-X-receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3-hydroxysteroid dehydrogenase, 5-reductase and 3-hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17-hydroxylase/17,20-lyase (P450c17), which possesses dual 17-hydroxylase and 17,20-lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16-ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK-293) cells with various amounts of vectors expressing P450c17, NADPH-cytochrome P450 reductase, and cytochrome b5. Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3-ol, without the formation of the precursor 17-hydroxypregnenolone. Cytochrome b5 has a much stronger effect on the 16-ene-synthase activity than on the 17-hydroxylase/17,20-lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16-androstadien-3-ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.

Keywords: 16-ene-synthase; 17-hydroxylase/17; 20-lyase; cytochrome b5; 5,16-androstadien-3-ol; pregnenolone.

Abbreviations: P450c17, 17-hydroxylase/17,20-lyase; P450red, NADPH cytochrome P450 reductase; cyt b5, cytochrome b5; 3-HSD, 3-hydroxysteroid dehydrogenase/54 isomerase; 3-HSD, 3-hydroxysteroid dehydrogenase; preg, pregnenolone; DHEA, dehydroepiandrosterone; 17-OHpreg, 17-hydroxypregnenolone; androstadienol, 5,16-androstadien-3-ol; androstenol, 5-16-androsten-3-ol; CAR, constitutive androstane receptor; PXR, pregnane-X-receptor; RXR, retinoid-X-receptor; HEK-293, transformed human embryonic kidney 293 cells


Introduction
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Summary
Introduction
Experimental procedures
Results
Discussion
References


It has been established that human cytochrome P450c17 (product of the CYP17 gene) has two distinct activities responsible for the synthesis of glucocorticoid and sex steroid precursors from pregnenolone (preg). A 17-hydroxylase activity, which converts preg into 17-OHpreg, is necessary for cortisol synthesis, and a 17,20-lyase activity further transforms 17-OHpreg into dehydroepiandrosterone (DHEA), the precursor of sex steroids (Fig. 1). Several different studies have revealed that these two activities are differentially modulated by many factors, two of the most important being the abundance of the redox partner cytochrome P450reductase (P450red) [1,2] and the interaction with cytochrome b5 (cyt b5), an allosteric effector [3-6].





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Fig. 1. Central role of cytochrome P450c17 in the biosynthetic pathways leading to the formation of 16-ene steroids, sex steroids and glucocorticoids.




As for 16-androstenes, the precise mechanism by which they are biosynthesized has been until now subject for debate. Early studies reported testosterone to be a precursor for these steroids [7-12]. Later, however, testosterone and a large number of other compounds, including epitestosterone, DHEA, 16-hydroxypregnenolone and 16-hydroxyprogesterone, were excluded as precursors for 16-androstenes, whereas preg and progesterone were found to be putative precursors. Multiple pathways have been suggested for the transformation of C21-steroids into 16-unsaturated C19-steroids in porcine testicular homogenates. These included 20-reduction (preg pregnenediol 5,16-androstadien-3-ol) [12], 21-hydroxylation (preg 21-OHpreg 5,16-androstadien-3-ol) [10,13] and 16–17-dehydrogenation (preg 17-OHpreg 16-dehydro-preg 5,16-androstadien-3-ol) [7,14]. A concerted process (preg 5,16-androstadien-3-ol) has also been suggested. Finally, results published by Weusten et al. provided evidence that androstadienol was synthesized from preg in a single step by a 16-ene-synthase enzyme system in human testicular homogenates [15]. However, the molecular mechanism responsible for this biosynthesis remains to be elucidated.
It is well recognized that 16-androstenes are produced by Leydig cells of porcine testis [8] and that these steroids have pheromonal activity in pigs. In humans, the physiological role of 16-androstene steroids is still ill-defined. It has been proposed that these compounds may have significant effects on behavior, namely reducing nervousness, tension and other negative emotional states in women [16]. Another study demonstrated a positive relationship between menstrual synchrony and the ability to smell certain 16-androstene steroids [17]. Recent reports in the literature show that androstenol (5-16-androsten-3-ol) could modulate the activity of two orphan receptors, the recently renamed CAR (constitutive androstane receptor) [18], previously known as constitutively active receptor [19], and PXR (pregnane-X-receptor) [20]. It has been suggested that androstenol is an endogenous ligand for these receptors [20], which share a common hetero-dimerization partner, RXR (retinoid-X-receptor), and are subject to cross talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways [21,22].

The purpose of this research is to examine, at the molecular level, whether the human P450c17 overexpressed in HEK-293 cells possesses 16-ene-synthase activity and how it differs from the 17-hydroxylase and 17,20-lyase activities. We also compare the human P450c17 with its porcine counterpart.


Experimental procedures
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Summary
Introduction
Experimental procedures
Results
Discussion
References


Construction of P450red, cyt b5 and P450c17 expression vectors
The cDNA fragments containing the entire coding regions of human NADPH-cytochrome P450reductase (P450red, EC 1.6.2.4) [23,24] and cyt b5 were isolated as previously described [25]. The cDNAs were then subcloned into a pCMV expression vector. Porcine P450c17 cDNA was amplified by PCR using Taq DNA polymerase (Perkin-Elmer Cetus, Emerville, CA, USA) [26] and an oligo-primer pair (5'-GGGGTCGACATGTGGGTGCTCTTGGTTTTCTTCTTG-3' and 5'-GGGGTCGACTCAGGAGGTACTCCCCTCAGTGTGGGC-3') and poly(A)+ RNA isolated from pig testis. The cDNA was then subcloned into a pCMV expression vector. The cDNA coding for human P450c17 (EC 1.14.99.9) was kindly provided by Y. Tremblay (CHUL Research Center, Quebec, Canada).

Transient expression in transformed human embryonic kidney (HEK-293) cells
Vectors expressing P450c17 (pCMV-P450c17), P450red (pCMV-P450red) and cyt b5 (pCMV-cyt b5) were transfected into HEK-293 cells using the Ex-gene kit according to the manufacturer's instructions (MBI Fermentas, Amherst, NY, USA). Cells were initially plated at 5 x 105 cells per well in six-well falcon flasks and grown in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, USA) at 37 °C under a 95% air, 5% CO2 humidified atmosphere.

Assay of enzymatic activity
Determination of the activities was performed in intact cells transiently transfected with P450c17 and/or P450red and/ or cyt b5 as previously described [25]. Briefly, [3H]preg, [3H]17-OHpreg or [3H]DHEA was added to freshly changed culture medium in six-well culture plates. For enzymatic assays performed with intact cells in culture, we have previously established 16 h to be an appropriate incubation time period, as the activity vs. time graph still shows linearity. After 16 h of incubation, the steroids were extracted twice with 2 mL of ether. The organic phases were pooled and evaporated to dryness. The steroids were solubilized in 50 µL of dichloromethane, applied to a Silica Gel 60 TLC plate (Merck, Darmstadt, Germany) before separation by migration in the toluene/acetone (4 : 1, v/v) solvent system. Substrates and metabolites were identified by comparison with reference steroids, revealed and quantified using phosphoimaging, Storm 860 (Molecular Dynamics Inc., Sunnyvale, CA, USA). Nonlabeled reference steroids were revealed with a solution of molybdate/sulfuric acid (10 : 10, v/v).

Analysis by HPLC
3H-Labelled steroids were analyzed using Waters Nova-Pak reverse-phase C18 HPLC column (3.9 x 150 mm, 4 µm). The mobile phase was MeOH/H2O (80 : 20, v/v) with 2 mm ammonium acetate and 0.1% acetic acid, with a flow rate of 1 mL·min-1. Radioactivity was monitored in the eluent using Beckman 171 HPLC Radioactivity Monitoring System. Nonlabeled androstadienol and preg standards were monitored using UV at 216 nm.


Results
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Summary
Introduction
Experimental procedures
Results
Discussion
References


Identification of metabolites by HPLC analysis and corecrystallization
To verify the nature of the metabolites obtained from the transformation of preg by human and porcine P450c17, we identified by HPLC analysis, extracts of HEK-293 cells transfected with P450c17 and cyt b5. 3H-Labeled preg, 17-OHpreg, and DHEA, used as standards, showed elution peaks at 4.70, 2.30 and 2.50 min, respectively (Fig. 2). In both porcine and human assays using preg as a substrate, an additional peak of elution appeared at 15 min (panels C and D, respectively). This additional peak coincides with the elution time of nonlabeled commercial androstadienol monitored using UV at 216 nm. This data shows that one of the metabolites obtained in assays using human and porcine P450c17 is androstadienol. In addition to comigratory behavior on both HPLC and TLC analyses, the identity of the radiolabeled androstadienol product was confirmed by cocrystallization with commercial steroid (data not shown).





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Fig. 2. Identification by HPLC of pregnenolone metabolites from HEK-293 cells transfected with P450c17 and cyt b5. (A) [3H]standard preg (left panel), 17-OHpreg (middle panel) and DHEA (right panel), (B) nonlabeled preg (left panel) and androstadienol (right panel). Products extracted from cells transfected with 1 µg of pCMV-cyt b5 and 0.1 µg of (C) human or (D) porcine pCMV-P450c17. Separation and identification of metabolites were performed as described in Experimental procedures.




Assessment of the 16-ene-synthase, 17-hydroxylase and 17,20-lyase activities of human and porcine P450c17
In order to produce DHEA from preg, P450c17 first transforms preg into 17-OHpreg through its 17-hydroxylase activity and then transforms this intermediate into DHEA through its 17,20-lyase activity. In order to determine whether the transformation of preg into androstadienol requires prior synthesis of 17-OHpreg or DHEA, we performed enzymatic assays using human and porcine P450c17 in the presence of various substrates – preg, 17-OHpreg and DHEA – and analyzed androstadienol formation from each substrate. As observed in Fig. 3, the biosynthesis of androstadienol in humans (A) and pigs (B) does not require prior formation of 17-OH-preg and DHEA. The lack of androstadienol synthesis in the presence of 1 µm of ketoconazole (C), an inhibitor of cytochrome P450, further demonstrates the specific implication of P450c17 in the formation of this metabolite.




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Fig. 3. Thin layer chromatography showing the transformation of pregnenolone, 17-OHpregnenolone and DHEA by human and porcine P450c17. HEK-293 cells transfected with 1 µg pCMV-cyt b5 and 0.1 µg (A) human, or (B) porcine pCMV-P450c17 were treated with 5 nm of the indicated 3H-labeled substrates and analyzed for their ability to produce androstadienol after overnight incubation (16 h). (C) HEK-293 cells transfected with 1 µg pCMV-cyt b5 and 0.1 µg human P450c17 were incubated with 5 nm of [3H]preg in the absence/presence of 1 µm ketoconazole. Metabolites were analyzed after overnight incubation (16 h). (D) Nonlabeled standards revealed with molybdate/sulfuric acid (10 : 10, v/v).




Formation of androstadienol by human and porcine P450c17, with and without cyt b5
Using porcine and human P450c17 expressed in HEK-293 cells in culture, we compared the formation of androstadienol from preg in the pig and the human. As illustrated in Fig. 4, both human and porcine enzymes have the ability to produce androstadienol in presence of cyt b5. When exogenous cyt b5 is omitted from the transfection assays, both human and porcine P450c17 poorly catalyze the formation of androstadienol from preg (less than 2% of preg transformation). Porcine P450c17 shows a slightly stronger stimulation by cyt b5, its activity increasing to 15% of preg transformation while the activity of human P450c17 increases to 12%. These results show that human and porcine P450c17 have similar catalytic activities and that, in both species, P450c17 is involved in the biosynthesis of androstadienol.




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Fig. 4. Role of cyt b5 in the formation of androstadienol from pregnenolone by human and porcine P450c17. HEK-293 cells were transfected with 0.1 µg human or porcine pCMV-P450c17 in the presence or absence of 1 µg pCMV-cyt b5. Their ability to catalyze the transformation of 5 nm of [3H]preg into androstadienol after overnight incubation (16 h) was determined. Transfections and enzymatic assays were performed as described in Experimental procedures. The results are the mean ± SEM of three independent experiments.




Effect of cyt b5 on DHEA and androstadienol biosynthesis
In order to determine the effect of cyt b5 in P450c17 16-ene-synthase and 17-hydroxylase/17,20-lyase activities, we performed transfection assays with increasing amounts of DNA fragments encoding cyt b5 and monitored the formation of androstadienol and DHEA. As shown in Fig. 5 the stimulation of DHEA and androstadienol production from preg increases with increasing amounts of cyt b5 in presence of endogenous levels of P450red. In presence of these low levels of P450red, cyt b5 shows a slight stimulatory effect on DHEA formation. However, we observe a more profound effect on the synthesis of androstadienol. More precisely, an increase of androstadienol formation was observed at a cyt b5/P450c17 ratio of 5 : 1 (Fig. 5). The activity reached a maximum at a ratio of 12 : 1. Thus, the influence of human cyt b5 changes dramatically as the cyt b5/P450c17 ratio varies.




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Fig. 5. Influence of increasing concentrations of cyt b5 on the relative formation of androstadienol and DHEA by human P450c17. HEK-293 cells were transfected with 0.1 µg pCMV-P450c17 and the indicated amounts of pCMV-cyt b5. The transfected cells were analyzed for their ability to catalyze the transformation of 5 nm of [3H]preg to androstadienol. An increase in 16-ene-synthase activity is observed at a cytb5/P450c17 ratio of 5 : 1 (0.25 µg cytb5/0.1 µg P450c17) while the optimal stimulation is observed at a ratio of 12 : 1 (1 µg of cytb5/0.1 µg of P450c17). Transfections and enzymatic assays were performed as described in Experimental procedures. The results are the mean ± SEM of three independent experiments.




Effect of P450red on DHEA and androstadienol synthesis
To further investigate the modulation of human P450c17 16-ene-synthase activity, we proceeded to analyze the relative effect of P450red on 17-hydroxylase/17,20-lyase and 16-ene-synthase activities. To do so, we cotransfected P450c17 and cyt b5 in amounts determined to be optimal for androstadienol formation along with increasing amounts of P450red. It can clearly be seen in Fig. 6 that the addition of P450red, even in small amounts, has a profound effect on 17-hydroxylase/17,20-lyase activities. In presence of only endogenous P450red levels, DHEA formation from preg is below 10%. Increasing P450red up to 0.25 µg causes a drastic increase of DHEA formation, reaching levels up to 50% of preg transformation. On the other hand, increasing amounts of P450red do not significantly stimulate 16-ene-synthase activity. For this activity, endogenous levels of P450red seem to be sufficient for optimal cyt b5 stimulation as increasing amounts of P450red do not further stimulate androstadienol production. These results clearly show a differential modulation of 17-hydroxylase/17,20-lyase and 16-ene-synthase activities by P450red and cyt b5.




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Fig. 6. Influence of increasing amounts of P450red on the relative formation of DHEA and androstadienol by P450c17. HEK-293 cells were transfected with 0.1 µg pCMV-P450c17, 1 µg pCMV-cyt b5 and the indicated amounts of pCMV-P450red. The transfected cells were analyzed for their ability to catalyze the transformation of 5 nm of [3H]preg into DHEA () and androstadienol (). Transfections and enzymatic assays were performed as described in Experimental procedures. The results are the mean ± SEM of three independent experiments.





Discussion
Top
Summary
Introduction
Experimental procedures
Results
Discussion
References


It is already known that human cytochrome P450c17 possesses two distinct activities, a 17-hydroxylase and a 17,20-lyase activity, responsible for the biosynthesis of glucocorticoid and sex steroid precursors. In this report we show that human and porcine P450c17 also possess a 16-ene-synthase activity that catalyzes the transformation of preg into androstadienol, a precursor in the biosynthesis of androstenol (Fig. 1). This reaction differs from the production of DHEA through the 17-hydroxylase/17,20-lyase activity, in that it does not require the 17-OH-preg formation step and it is strongly stimulated by optimal amounts of cyt b5. Indeed, results obtained from assays using 17-hydroxypregnenolone as a substrate demonstrate that the synthesis of androstadienol diverges from the biosynthetic pathway of sex steroids at the level of preg transformation and that it does not involve the 17-hydroxylase activity. Furthermore, time course experiments (data not shown) using preg as a substrate do not show the production of any intermediates in the formation of androstadienol which suggests that it is synthesized from pregnenolone in a single step. Therefore, although the synthesis of glucocorticoid/sex steroid precursors and 16-ene-steroids result from the same enzyme, the activities responsible for their formation and the regulation of these activities are distinct. Gower et al. had previously demonstrated, using porcine testis microsomes, the formation of 16-androstenes from 17-hydroxypregnenolone [8]. However, this situation is not observed in the intact transfected cell system using porcine or human P450c17. In combination, these results suggest the presence, in pig testis, of another system that can use 17-hydroxypregnenolone to form a different product.
P450c17 is therefore a crucial enzyme, not only in the formation of sex steroid precursors, but also in the production of androstadienol which is considered to be an intermediate in the 16-androstene pathway leading to the biosynthesis of androstenol and of 5(16)androsten-3-one, a pheromonally active steroid in the pig [8,14]. A recent report by our group [27] shows that further transformation of androstadienol into androstenol involves the classic enzymes of the steroidogenic pathway, namely 3-HSD, 5-reductase and 3-HSD. Interestingly, because of the lack of either a 17-keto or a 17-hydroxy group, this pathway does not require the enzyme 17-HSD which is specific to sex steroid biosynthesis.

Although it is well known that androstenol is a pheromone in the pig, its role in the human is still ill-defined. Recent findings show that androstenol is able to modulate the expression of certain cytochrome P450s and alcohol dehydrogenases through interactions with the orphan receptors CAR and PXR [18,20]. These receptors belong to the P450-regulatory nuclear receptors, in the subfamily NR1 (nuclear binding site 1) [21]. Other members of this NR1 orphan nuclear receptor gene subfamily are PPAR (peroxisome proliferator-activated receptor), LXR (liver X receptor) and FXR (farnesol-X-receptor). They share a common hetero-dimerization partner, the RXR, and are subject to cross-talk interactions with other nuclear receptors and with a broad range of other intracellular signaling pathways, including those activated by certain cytokines and growth factors [21,22]. It has been shown that the steroids androstenol [16(5)-androsten-3-ol] and 5-pregnanedione (5-pregnane-3,20-dione) modulate the action of these receptors and thus are putative endogenous ligands for these receptors [20]. Upon binding to the ligands, PXR and CAR bind DNA as a heterodimer with the RXR and modulate the expression of cytochromes P450, especially CYP2B and CYP3A families. Because P450s play an essential role in the detoxification of drugs and of a large series of exogenous compounds from the environment, androstenol and 5-pregnanedione that modulate cytochrome P450 levels could have a profound effect on the detoxification process.

Because of the low affinity of these orphan nuclear receptors (in the range of 1–10 x 10-6 m) and their relatively broad spectrum of ligand specificity, many researchers that are familiar with classic steroid receptors, namely androgen, estrogen, progesterone, glucocorticoid and mineralocorticoid receptors, that bind to their corresponding specific ligand with a very high affinity (10-10-10-9 m), are skeptical about the idea of androstenol and 5-pregnanedione being ligands for these orphan receptors. However, it is noteworthy that, as classic active steroids are diluted in the blood, their concentration is very low and thus they require high affinity receptors to pick them up. On the other hand, the orphan receptor ligands are most probably produced locally in the various tissues. Because of the small volume of the cell, the production of a little amount of ligand will give a relatively high concentration (up to 1–10 x 10-6 m). We hypothesize that this low affinity combined with the local biosynthesis of ligands represents a mechanism allowing the selective regulation of the action of the receptor: ligands that enter the cell or tissue by chance will not have a high enough concentration to turn on the receptor. Only for ligands that are produced locally or accumulated in the tissue (probably through active transport or hydrophobicity) is the concentration high enough to modulate the receptor activity.

As suggested above, local biosynthesis in various tissues such as the liver, could constitute a way to selectively regulate the activity of nuclear orphan receptors such as CAR and PXR. Although there is no evidence of P450c17 expression in the human liver, many other enzymes such as 3-HSD, 5-reductase and 3-HSD, whose activities lead to androstenol synthesis from androstadienol, are present in the liver and many other peripheral tissues. Furthermore, androstadienol is found in circulation suggesting that it is synthesized at the sites of expression of P450c17 and eventually converted to androstenol by different enzymes in peripheral tissues such as the liver and adipose tissue. Our study aimed at elucidating the nature and the mechanism of the reactions involved in the local formation of androstenol is thus of major importance

 

[lanky]

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Biol. Lett.doi:10.1098/rsbl.2005.0332Published onlineWomen’s preference fordominant male odour:effects of menstrual cycleand relationship statusJan Havlicek1,*, S. Craig Roberts2and Jaroslav Flegr31Department of Anthropology, Faculty of Humanities, CharlesUniversity, Husnikova 2075, 155 00 Prague, Czech Republic2Evolutionary Psychology and Behavioural Ecology Research Group,School of Biological Sciences, University of Liverpool, LiverpoolL69 3BX, UK3Department of Parasitology, Faculty of Science, Charles University,Vinicna 7, 127 44 Prague, Czech Republic*Author for correspondence ( [email protected])Body odour may provide significant cues abouta potential sexual partner’s genetic quality,reproductive status and health. In animals, akey trait in a female’s choice of sexual partner ismale dominance but, to date, this has not beenexamined in humans. Here, we show thatwomen in the fertile phase of their cycle preferbody odour of males who score high on aquestionnaire-based dominance scale (inter-national personality items pool). In accordancewith the theory of mixed mating strategies, thispreference varies with relationship status, beingmuch stronger in fertile women in stablerelationships than in fertile single women.Keywords: attractiveness; scent; smell; good genes;mate choice; sexual selection1. INTRODUCTIONIn many systems, dominance-associated traits havebeen suggested as honest signals of male geneticquality. Several studies on rodent species havereported preferences for the odour of dominant males(e.g. Mossman & Drickamer 1996; Kruczek 1997;Gosling & Roberts 2001). Odour cues may also playa substantial role in human mate choice. For instance,women prefer the smell of men with low fluctuatingasymmetry (Thornhill & Gangestad 1999), which isconsidered to be a marker of genetic and develop-mental stability and is an important factor influencingvisual attractiveness (Gangestad & Simpson 2000). Inaddition, humans prefer the scent of opposite-sexindividuals with major histocompatibility complex(MHC) genes that are dissimilar (Wedekind et al.1995; Wedekind & Füri 1997) or intermediatelydissimilar (Jacob et al. 2002) to their own (see alsoThornhill et al. 2003). Such preferences might resultin more viable offspring (Penn 2002).It has also been observed that preference formen’s scent depends on the menstrual cycle phaseof women. In controlled experiments, only thewomen near peak fertility within their cycle pre-ferred scent of men with low fluctuating asymmetry(Gangestad &Thornhill 1998; Rikowski & Grammer1999; Thornhill & Gangestad 1999; Thornhill et al.2003). Similarly, research on facial attractivenessindicates that female preference for visual masculi-nity (a trait putatively correlated with dominance)varies across the cycle (Penton-Voak et al. 1999) andwith partnership status (Little et al. 2002). In thisstudy, we investigated whether women’s preferencefor odour of dominant males also varies cyclicallyand between single women and those in stablerelationships.2. METHODS(a) Odour stimuliForty-eight male students aged between 19 and 27 were asked tocomplete an 11-item questionnaire on dominance from the inter-national personality items pool (http://ipip.ori.org/ipip/; Goldberg1999) and to wear cotton pads in their armpits for 24 h. Pads(Premium cosmetic pads, Boots, www.boots.co.uk) were 100%cotton, elliptical in shape, approximately 9!7 cm at their longestaxis and held in place using MicroporeTMsurgical tape (Boots).The questionnaire was used in its original form and corresponds tothe scale ‘Narcissism’ in the widely used California psychologicalinventory (CPI). Subjects were instructed to avoid spicy and smellyfood, alcohol, smoking or using any scented cosmetics on both theevening before and during the day when they were wearingthe pads.(b) Subjects and experimental procedureFreshly collected pads were presented to 30 female students (meanage 20.6 years) in their follicular phase (days 9 to 15) and to 35female students (mean age 20.2 years) in other phases of the cycle.The range of days included as falling into the follicular phase (i.e.fertile period) was based on results showing that probability ofconception is highest within this ‘fertile window’ (Wilcox et al.2000). None of the women were using hormonal contraception.Each of them rated the odour of 10 pads for their intensity, sexinessand masculinity using a 7-point scale. The ratings from eachwoman were converted to z-scores to compute the correlationbetween male odour and male dominance as measured by thequestionnaire. The obtained correlation coefficients showed anormal distribution and, therefore, were compared with randomexpectation (rZ0) using one-sample t-tests. Although our design isbetween-subjects in nature, this should tend to make our resultsconservative compared with a within-subjects design.3. RESULTSWe found a positive correlation between male psycho-logical dominance assessed by the questionnaire andodour sexiness when rated by women in their fertilephase (t29Z3.1, pZ0.004, mean rZ0.20) but not inother phases of their cycle. Subsequently, we testedseparately the women who reported to be single andthose who were in a heterosexual romantic relation-ship. A strong association between male odoursexiness and psychological dominance was only foundfor non-single women in the fertile phase of theirmenstrual cycle (t12Z4.4, pZ0.0008, rZ0.29;figure 1). There was no significant correlationbetween male psychological dominance and perceivedmasculinity of their body odour when rated by singlewomen, regardless of phase of their cycle. In contrast,we found a negative correlation between maledominance and intensity of body odour for bothfemale subsamples (fertile phase of the cycle,t29Z2.3, pZ0.03, rZK0.13; rest of cycle, t34Z3.0,pZ0.005, rZK0.18; figure 2). As this effect wasobserved irrespective of menstrual cycle phase, theshifts in attractiveness of dominant males cannot beexplained by variation in odour sensitivity across thecycle (Doty et al. 1981).Received 14 March 2005Accepted 4 April 2005q 2005 The Royal Society
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4. DISCUSSIONOur results indicate that psychological dominance isassociated with odour attractiveness. The preferencefor the odour of dominant men varies with menstrualcycle phase and partnership status of women. Thepublished evidence that men who are visually per-ceived as dominant are also rated attractive is,however, ambiguous. A positive correlation betweenattractiveness and perceived dominance was found inone study (Neave et al. 2003), but others have foundnegative correlations (Perrett et al. 1998; Swaddle &Reierson 2002). None of the studies on facial attrac-tiveness investigated actual dominance in local hier-archies or psychological dominance (i.e. tendency todominate) of the target subjects. Therefore, it is notclear whether dominant-looking men have a genuinetendency to dominate or whether the attribution ofdominance based on facial appearance is misplaced.Evidence for the former suggestion comes fromMueller & Mazur’s (1997) study, which found thatdominant-looking men reach higher military rankcompared with those who look rather submissive.However, even in this case, it remains possible thatachieved rank is influenced by dominant appearancewithout necessarily implying a direct link withpsychological dominance.Although we find that psychological dominancepredicts odour attractiveness, we find no significantcorrelation between dominance and perceived odourmasculinity. It has been shown that women rate thesmell of androstenone more positively around thetime of ovulation (Hummel et al. 1991; Grammer1993). This substance is a significant constituent ofaxillary odour and is found in much higher concen-trations in men than in women (Gower et al. 1985).More objective measurement of odour masculinity(e.g. levels of 16 androstenes in the axilla) wasunfortunately not available in our study. Dominanceis stereotypically attributed to more masculine faces(Perrett et al. 1998). However, it is possible that therelationship between dominance and both perceivedand measured odour masculinity differs qualitativelyfrom the relationship between dominance and facialmasculinity.There is no common agreement on the interpret-ation of the association between dominance andattractiveness. Some researchers have suggested thatthe high mating value of dominant men is a result oftheir tendency to reaching higher socio-economicalstatus and, therefore, gaining the resources that theymay invest in their mate and offspring (Mueller &Mazur 1997). Alternatively, dominance has beensuggested to honestly reflect male genetic quality.Tendency to dominate is a risky strategy in competi-tive encounters and is associated with higher levels oftestosterone, which may reduce immunocompetencein various species (Folstad & Karter 1992); domi-nance could, therefore, reliably indicate male con-dition. There is also evidence that males of highgenetic quality have a tendency for lower parentalinvestment (Waynforth 1998). In response, a mixedmating strategy may have evolved in females: theyprefer genetically superior males as short-term orextra-pair sexual partners while, at the same time,they seek males who are more willing to invest in theiroffspring as long-term or social partners (Reynolds1996; Penton-Voak et al. 1999; Blomqvist et al. 2002;Foerster et al. 2003). This interpretation is consistentwith our findings that women in stable relationshipshave a strong tendency to prefer the smell ofdominant men when in the fertile phase of their cycle,while single women and all women in non-fertilephases lack this preference.Changes in preference for mate-related traitsduring the menstrual cycle have been demonstratedrepeatedly. Researchers have focused particularly onbody odour, symmetry and facial masculinity. Theresults of four different studies show that the bodyodour of symmetrical men is rated as more attractiveFigure 1. Mean (Gs.e.m.) correlation coefficient betweenthe male dominance score and their odour attractivenessrated by single (open bars) or partnered women (grey bars)in the fertile and non-fertile phases of their cycle.Figure 2. Correlation between standardized ratings of odourintensity and males’ dominance score. (a) Ratings bywomen in the fertile phase of their cycle, (b) Ratings bywomen in other phase of their cycle. The two correlationsare significant ( p!0.05).2 J. Havlicek and others Male dominance and odour attractivenessBiol. Lett.
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by women in the fertile phase but not other phases ofthe cycle (Gangestad & Thornhill 1998; Rikowski &Grammer 1999; Thornhill & Gangestad 1999;Thornhill et al. 2003). Several studies also report thatwomen in the fertile phase of their cycle preferrelatively more masculine faces (Penton-Voak et al.1999; Penton-Voak & Perrett 2000; Johnston et al.2001). The relative preference for more masculinefaces was found also when rated by single women orin a short-term partnership context (Little et al.2002). All of the above-mentioned studies are con-gruent with our findings and support the hypothesisabout female mixed mating strategies dependent ontheir cyclical and partnership states.The proximate mechanism responsible for thecorrelation between psychological dominance andodour sexiness is unknown. Nevertheless, previousstudies have shown that emotional state (e.g. fear orhappiness) may influence perception of body odourquality (Chen & Haviland-Jones 2000; Ackerl et al.2002). The higher self-confidence of dominant malesmay also have an impact on the perceived sexiness oftheir body odour.We thank all volunteers for their participation in the study,two anonymous referees for their comments on the manu-script and Jindra Jileckova for language corrections. Thisstudy was supported in part by the NATO Science Fellow-ship and the Owen F. Aldis Fund (J.H.) and grant no.0021620828 awarded to J.F. by the Czech Ministry ofEducation.REFERENCESAckerl, K., Atzmueller, M. & Grammer, K. 2002 The scentof fear. Neuro Endocrinol. Lett. 23, 79–84.Blomqvist, D., Andersson, M., Küpper, C., Cuthill, I. C.,Kis, J., Lanctot, R. B., Sandercock, B. K., Székely, T.,Wallander, K. & Kempenaers, B. 2002 Genetic similaritybetween mates and extra-pair parentage in three speciesof shorebirds. Nature 419, 613–615.Chen, D. & Haviland-Jones, J. 2000 Human olfactorycommunication of emotion. Percept. Mot. Skills 91,771–781.Doty, R. L., Snyder, P. J., Huggins, G. R. & Lowry, L. 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