Πέμπτη 3 Σεπτεμβρίου 2015

Sexual behavior reduces hypothalamic androgen receptor immunoreactivity

doi:10.1016/S0306-4530(02)00036-7
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Abstract

Male sexual behavior is regulated by limbic areas like the medial preoptic nucleus(MPN), the bed nucleus of the stria terminalis (BST), the nucleus accumbens (nAcc) and the ventromedial hypothalamic nucleus (VMN). Neurons in these brain areas are rich inandrogen receptors (AR) and express FOS-immunoreactivity in response to mating. In many species sexual satiation, a state of sexual behavior inhibition, is attained after multiple ejaculations. The mechanisms underlying sexual satiation are largely unknown. In this study we show that sexual activity reduces androgen receptor immunoreactivity (AR-ir) in some of the brain areas associated with the control of male sexual behavior, but not in others. Thus, one ejaculation reduced the AR-ir in the MPN and nAcc, but not in the BST and VMN. Copulation to satiation, on the other hand, reduced AR-ir in the MPN, nAcc and VMN, and not in the BST. The AR-ir reduction observed in the MPN of sexually satiated rats was drastic when compared to that of animals ejaculating once. Serumandrogen levels did not vary after one ejaculation or copulation to exhaustion. These data reveal that sexual activity reduces AR in specific brain areas and suggest the possibility that such a reduction underlies the sexual inhibition that characterizes sexual satiety.

Keywords

Steroid hormones play an important role in the regulation of male sexual behavior. Thus, in general, castration decreases while exogenous androgen administration restores copulation (Meisel and Sachs, 1994). Interestingly, several factors such as previous experience, photoperiod, age, and others also influence copulatory behavior without necessarily modifying the endocrine milieu (Chubb and Desjardins, 1984, Clemens et al., 1988, Miernicki et al., 1990, Phoenix and Chambers, 1986 and Wallen, 2001). It is clear that steroids act on the brain to induce sexual behavior. Within the rat brain thehypothalamus has been proposed to play a dual role in the control of this behavior. On one side, several hypothalamic nuclei are directly involved in its neural control and, on the other, hypothalamic neurons that contain gonadotropin hormone realising factors (GnRH) participate in its neuroendocrine modulation. Regarding the latter function, lesions in certain hypothalamic areas produce gonadal atrophy and thereby suppress sexual behavior (Heimer and Larsson, 1966/1967) by three possible mechanisms: (a) direct lesions of the GnRH neurons continuum primarily located in the telencephalon-diencephalon limit; (b) lesions that damage the majority of the GnRH nerve fibers traversing to the median eminence; or (c) isolation of hypothalamic areas (like the mediobasal hypothalamus) that contribute to the functional integrity of the neuroendocrine network (Silverman, 1994). In contrast, extensive bilateral electrolytic lesions encompassing the medial preoptic region and the anterior part of the hypothalamus suppress male rat sexual behavior without causing gonadal atrophy. Attempts to arouse the lesioned rats by handling or by changing the stimulus female are ineffective in inducing sexual behavior; chronic testosterone treatment is similarly ineffective, further indicating that the effects of mPOA lesions are not an indirect result of an altered gonadal regulation (Paredes et al., 1993). The androgen sensitive neurons in the mPOA participate in the regulation of copulation, since implantation of testosterone into this brain area of castrated male rats restores sexual behavior, while selective blockade of androgen receptors (ARs) in this region inhibits mating (McGinnis et al., 1996). In the mPOA the sexual dimorphic nucleus or medial preoptic nucleus (MPN), particularly rich in ARs (Handa et al., 1996), specifically participates in the control of masculine sexual behavior (De Jonge et al., 1989). Besides, the mPOA, other limbicregions like the bed nucleus of the stria terminalis (BST) and the nucleus accumbens(nAcc), play an important role in the control of male sexual behavior. In general, it is considered that the BST participates in the transmission of the olfactory information necessary for copulation (Emery and Sachs, 1976). The nAcc has also been implicated in the control of copulatory behavior (Mitchell and Gratton, 1994) particularly sexual motivation (van Furth et al., 1995). Thus, elevations in dopamine release within this area coincide with preparatory sexual activity and copulation (Pfaus et al., 1990, Damsma et al., 1992 and Mas et al., 1990).
Mating in male rats, as many other behavioral processes, importantly increases FOS expression (the protein product of the c-fos proto-oncogene) in the MPN, the BST and to a much lesser extent in the VMN ( Coolen et al., 1996). Significantly increased neuronal FOS responses have also been reported to occur in the nAcc of males that displayed non-contact penile erections or had two ejaculations ( Kelliher et al., 1999).Immunohistochemical and in situ hybridization analyses have shown that the distribution of the AR protein and the AR mRNA within the brain follows an identical pattern and reaches peak density in the MPN and the principal nucleus of the BST ( Handa et al., 1996). Additionally, using a radiolabeled ligand it was established that the highest concentration of nuclear androgen receptors is found in the VMN ( Roselli et al., 1989). Interestingly, Beatrice Gréco and co-workers recently demonstrated that mating-induced FOS expression occurs almost exclusively in AR-containing neurons ( Gréco et al., 1996 and Gréco et al., 1998) suggesting a primary role of sex-steroid sensitive neurons in male rat copulation.
The male rat may ejaculate several times before reaching a state of sexual inactivity that lasts for various days. This state, named sexual satiation, is common to many species, but differs in its duration and in the amount of sexual activity necessary to be reached (Larsson, 1956 and Meisel and Sachs, 1994). Little is known on the control of this phenomenon. Recently, we have established a sexual satiation procedure in which male rats are allowed to copulate ad libitum during a 4 h period. Twenty-four hours later a major population shows a complete inhibition of sexual activity. In the control of sexual satiety, we have reported the involvement of several neurotransmitter systems (Rodríguez-Manzo and Fernández-Guasti, 1994 and Rodríguez-Manzo and Fernández-Guasti, 1995), possibly coupled to a dopamine-mediated motivational change (Rodríguez-Manzo, 1999). In support of this idea, an increase in dopamine release has been found in the nucleus accumbens during the anticipatory phases of sexual behavior (Fiorino et al., 1997).
In the present study we analyze whether sexual activity alters AR-ir in the MPN, BST, nAcc and VMN. These brain areas were selected because of their involvement in the neural and/or neuroendocrine control of sexual behavior. A putative participation of neuroendocrine factors in the process of sexual satiation is almost unexplored. Since the AR-ir has been found to depend on the circulating levels of androgens (Handa et al., 1996, Kruijver et al., 2001 and Wood and Newman, 1999), we also measured the serum levels of androgens in all experimental groups. We hypothesized that changes in the AR could mediate the long-term alterations in sexual behavior produced by copulation. The AR-ir in these brain areas was compared among sexually sated rats, animals that had one ejaculation and sexually experienced subjects that did not copulate.

1. Methods

1.1. Behavior

Sexually active male Wistar rats were used. Animals were at random divided into three groups of 24 subjects each: (a) sexually active controls, (b) males that performed one ejaculation and (c) sexually exhausted rats. For the sexual behavior tests, males were placed in cylindrical arenas and exposed to sexually proceptive and receptive females. Females were brought into sexual receptivity by the sequential administration of estradiolvalerianate (4 μg/rat) and progesterone (2 mg/rat) (Rodríguez-Manzo and Fernández-Guasti, 1994). Sexual behavior observations were conducted 2 h after the onset of darkness. Mounts, intromissions and ejaculations were identified as previously described (Larsson, 1956 and Meisel and Sachs, 1994). Within the males that were allowed to copulate, a group was separated from the female immediately after the first ejaculation, while the group of sexually satiated animals, was allowed a 4 h period of ad libitum copulation with a single female ( Rodríguez-Manzo and Fernández-Guasti, 1994). The control males were introduced for 15 min into the observation arena and returned thereafter to their home cages. All groups were sacrificed 24 h after the aforementioned procedures. Eight animals of each group were used for immunohistochemicalprocessing and the remaining 16 subjects in each group for serum androgendetermination.

1.2. Immunohistochemistry

Males were deeply anaesthetized with sodium pentobarbital (100 mg/rat, i.p.) and intracardially perfused with 0.1 M phosphate buffer-saline (PBS; pH=7.6) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (300 ml per animal). Brains were removed and postfixed in 4% paraformaldehyde. Before cutting, brains were rinsed with PBS 0.1 M. Sections of 100 μm were cut on a Vibratome using ice-cold Tris buffered saline (TBS; 0.05 M, 0.9% NaCl, pH=7.6). TBS was used to rinse the sections for at least 1 h (three times with TBS) between all steps of the immunohistochemical procedure. All antibodieswere diluted in 0.05 M TBS-containing 0.5 M NaCl and 0.5% Triton-X100 (pH=7.6). Theantibody PG21 (kind gift of Drs G. Prins and G. Green) was used to identify the androgen receptor. PG21 is a rabbit polyclonal antibody directed against a synthetic peptidecorresponding to the first 21 amino acids of the rat androgen receptor (Prins et al., 1991). This antibody was used at a concentration of 1:500.
Free-floating sections were incubated with PG21 and left for 1 h at room temperature and subsequently at 4 °C overnight. After rinsing in TBS buffer, all sections were incubated with a goat anti-rabbit biotinylated second antibody (Vector Laboratories, Burlingame, CA) diluted 1:200 for 1 h. Sections were then washed in TBS buffer and incubated for 1 h with an avidin-biotin complex (ABC) (Vector Laboratories, Burlingame, CA) diluted 1:800. Sections were incubated for 10 min in 0.05 M Tris–HCl containing 0.05% 3,3’diaminobenzidine (Sigma Chemicals, St Louis, MO), 0.0001% hydrogen peroxide, and 0.3% nickel ammonium sulphate as chromogen. After developing, sections were mounted on chrome-alum-coated slides, dehydrated and cover-slipped using Entellan.

1.3. Quantitative analysis

Quantification of the androgen receptor density (ARD) was carried out with the aid of image analysis software (Global LAB Image, SP0550 for PC). Briefly, a portion of an image containing the region of interest was chosen. The magnitude of that image portion was kept constant for all the measurements. A particular gray scale range was set that marked only the stained particles. With the aid of a particle tool, the areas of the stained particles were summed and the total area of the image portion divided that value. Thus, ARD was expressed as proportional area, i.e., the sum of the areas of the stained particles (cell nuclei) divided by the total evaluated area. Data were statistically analyzed by means of a one-way ANOVA followed by a Tukey t test.

1.4. Serum androgen determinations

The animals were sacrificed by decapitation and the trunk blood was collected in chilled plastic tubes. After centrifugation (1250 g, 15 min, 10 °C) plasma samples were stored at −20 °C. Total plasma androgen concentrations were measured by radioimmunoassay following protocols and reagents provided by the WHO Matched Reagent Program ( Sufi et al., 1999). The intra- and inter-assay coefficients of variation were 5.69 and 5.28%, respectively. A one-way ANOVA was used to determine statistical differences.

2. Results

Examination of the stained brain sections revealed that, in general, androgen receptor immunoreactivity (AR-ir) was restricted to cell nuclei in all groups analyzed. The differences found in the experimental subjects, when compared with the control group, were always in the direction of a decrease in the nuclear mark without a concomitant increase in cytoplasmic staining.
Fig. 1 shows the quantitative analysis of the ARD, expressed as proportional area (sum of stained areas/evaluated area), in the brain regions examined. In the MPN a highly significant decrease in ARD was found in both copulating groups, sexually satiated rats and in those that ejaculated once, when compared with the control group (one-way ANOVA for MPN, F=40.841, df=2, P<0.001). However, the reduction in ARD observed in the MPN of sexually exhausted rats was remarkably pronounced and significantly different to that of males that had one ejaculation (P=0.006, Tukey t test). In the nAcc, a statistically significant reduction in ARD was found in both copulating groups as compared to control animals, without differences between animals that ejaculated once and those that copulated to exhaustion (one-way ANOVA for nAcc, F=25.547, df=2,P<0.001). ARD in the VMN was significantly reduced only in the group of sexually exhausted animals when compared both with control animals and males that had one ejaculation (one-way ANOVA for VMN, F=16.888, df=2, P<0.001). Finally, in the BST no differences in ARD were found among groups (one-way ANOVA for BST, F=0.689, df=2,P=0.515, ns).
Quantification of ARD, expressed as proportional area (sum of the areas of the ...
Fig. 1. 
Quantification of ARD, expressed as proportional area (sum of the areas of the stained particles/evaluated area), in the MPN, nAcc, VMN and BST of sexually experienced animals (SEXP), males that ejaculated once (1EJ) and sexually exhausted rats (EXH). Tukey’s post hoc test ∗∗P<0.01; ∗∗∗P<0.001.
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Measurements of serum androgen levels rendered similar values in the different groups: 2.69±0.56 ng/ml for sexually experienced controls, 2.15±0.48 ng/ml for animals that had one ejaculation and 2.90±0.70 ng/ml for sexually exhausted rats. The one-way ANOVA revealed no statistically significant differences among groups (F=0.930, df=2, P=0.414, ns).
Fig. 2 shows representative photomicrographs of the AR-ir in the MPN and BST of male rats after the different sexual behavior conditions. Notice the pronounced differences in AR-ir among groups in the MPN and the similarities in the BST. The reduction in sexually exhausted animals was drastic. A less pronounced decrease was observed in rats that executed one ejaculation as compared with control males.
Representative photomicrographs showing androgen receptor immunoreactivity in ...
Fig. 2. 
Representative photomicrographs showing androgen receptor immunoreactivity in the MPN and the BST of control sexually experienced males (SEXP) and rats that ejaculated once (1EJ) or copulated to exhaustion (EXH). Third ventricle=3V, fornix=fx.
Figure options

3. Discussion

The main finding of the present study is that sexual behavior produces a decrease in ARD in some of the brain areas associated with the control of male sexual behavior, but not in others. Interestingly, in the MPN this reduction depends upon the quantity of sexual behavior displayed, since after sexual satiation fewer cells appear marked as compared with those stained in rats that ejaculated once. In the nAcc the reduction in AR-ir is similar in both groups of copulating rats, while in the VMN a reduction is only seen in sexually exhausted rats. No differences in AR-ir are found among groups in the BST.
It is well documented that the AR-ir in various brain areas depends upon the circulating levels of androgens in males and females of several species, including humans (Handa et al., 1996, Kruijver et al., 2001 and Wood and Newman, 1999). Thus, in contrast with the intense nuclear AR-ir observed in intact male rats, a few days after castration AR-ir is pale in the nucleus and occasionally present in the cytoplasmic compartment of all rat brain areas studied (Handa et al., 1996, Krey and McGinnis, 1990 and Zhou et al., 1994). Treatment with testosterone or non-aromatizable androgens, but not with estrogens, restores the nuclear AR-ir within a very short time that varies between 15 and 30 min (Handa et al., 1996, Krey and McGinnis, 1990, Lu et al., 1998, Wood and Newman, 1993 and Zhou et al., 1994). These data, taken together, indicate that the occupation of the androgen receptor rapidly changes in response to variations in circulating testosterone (Krey and McGinnis, 1990). On these bases, since in the present study the levels of circulating androgens are unmodified in animals ejaculating once or copulating to exhaustion, the reduction in AR-ir found after either one ejaculation or copulation to satiety does not seem to rely on these bases.
Another possibility to explain the decrease in AR-ir in brain areas of males that copulated is the re-compartmentalization and subsequent dilution of AR to the cytoplasmic fraction that would be accompanied by a decrease in the ability of the antibody to recognize the unoccupied androgen receptor. These factors would result in a diminished nuclear immunostaining. However, others, and we have indeed shown substantial cytoplasmic androgen receptor staining in various brain areas, under physiological conditions (Fernández-Guasti et al., 2000 and Michael et al., 1995) and following castration (Iqbal et al., 1995, Wood and Newman, 1993 and Wood and Newman, 1999) demonstrating that AR-ir can be detected in the cytoplasmic fraction. Although some authors consider that PG21, the antibody used here, does not bind to the unoccupied AR (Gréco et al., 1996),Lu et al. (1998) showed that PG21 effectively recognizes free AR. Thus, Western blot analyses yielded ratios of AR band densities that mirrored the immunohistochemical data and concluded that the loss of AR-ir in gonadectomized animals reflects a steep decline in AR protein levels rather than the inability of the PG21 antibody to detect free AR. This fact together with the absence of AR-ir in the cytoplasm of the neurons analyzed here, suggests that the reduced AR-ir found in some of the brain areas of rats that copulated is neither explained by a re-compartmentalization nor by the inability of the antibody to recognize the unoccupied AR.
It has been demonstrated that males exposed to supraphysiological levels of circulating androgens show a denser AR-ir in various tissues including the brain (Kadi et al., 2000,Lu et al., 1998 and Wood and Newman, 1993) than animals with normal levels of androgens. A slowing of the receptor degradation with the consequent extension of the AR-complex half-life in animals treated with androgens could explain the increased immunoreactivity (Kempainen et al., 1992). Recently, Kadi et al. (2000) showed that long-term strength training as well as administration of androgen-anabolic steroids increases the AR-ir in human muscle cells. These data indicate that external stimuli, even those that are not directly related with steroid hormones administration or withdrawal, may modify the density of nuclear AR. Moreover, this study reported that strength training increases AR-ir selectively in the trapezius, but not in the vastus lateralismuscles. Thus, the regulation of AR content following training is muscle selective. These results agree with present findings showing specific changes in AR-ir in the MPN, nAcc and VMN—and not in the BST—after sexual activity. The fact that after one ejaculation changes in AR-ir were only found in the MPN and nAcc, suggests that the regional variation also depends upon the amount of sexual behavior. Recently, it has been published ( Deak et al., 1999) that inescapable shock exposure increases the occupancy of mineralocorticoid and glucocorticoid receptors in the hypothalamus. The changes in corticosteroid receptor activation are evident 24 h after the shock exposure, they occur independently of the presence of endogenous steroid hormones and may be responsible for some of the long-term behavioral changes observed following acute stress. Similar mechanisms may be the basis of our findings concerning sexual activity and the AR.
Although all the brain areas studied participate in the control of masculine sexual behavior, important differences—of either neural or neuroendocrine nature—might account for the differential effect of sexual behavior on the AR-ir in the MPN, nAcc, VMN and BST. In this sense, it has been proposed that little or no processing of copulatory cues occurs in neurons of the BST and that this nucleus primarily relays information from the amygdala to nuclei within the mPOA (Emery and Sachs, 1976 and Meisel and Sachs, 1994). Additionally, while in the MPN of the male rat there is an almost absolute correlation between AR positive neurons and those that express FOS-ir after mating, in the BST the expression of FOS-ir occurs in neurons containing various types of steroid-receptors. These data suggest that different populations of androgen sensitive neurons (Gréco et al., 1998) probably coexist within regions critical for reproduction, influencing different physiological and behavioral phenomena. Thus, for example, two different androgen-dependent behaviors, mating and aggression, activate neurons in similar regions, but each behavior is associated with a distinctive pattern of neuronal response (Walker-Kollac and Newman, 1995).
The reduction in ARD in the nAcc of the two groups of copulating rats appears interesting. On the one hand, dopaminergic transmission in this area seems to play an important role in male rat sexual behavior. According to several authors (Damsma et al., 1992, Mas et al., 1990 and Pleim et al., 1990) dopamine release increases in the nAcc both during preparatory sexual activity and during copulation. On the other hand, recent evidence regarding the mPOA indicates that the presence of testosterone is permissive for both pre copulatory dopamine release and copulation (Hull et al., 1997). Taking these pieces of evidence together, it could be thought that testosterone might play a similar permissive role in the nAcc. In favor of this hypothesis is the finding showing that castration decreases dopamine content in the nAcc (Mitchell and Stewart, 1989). The reduction in ARD found in this area could be related to the deficient copulatory performance and sexual inactivity seen 24 h after one ejaculation (Larsson, 1956) and after copulation to exhaustion (Rodríguez-Manzo and Fernández-Guasti, 1994), respectively. Needless to mention, specific experiments should be undertaken to address this issue.
As previously suggested the VMN, even rich in AR-ir, does not seem to importantly participate in the neural control of masculine sexual behavior. This idea is sustained by the present finding showing that one ejaculation does not modify the ARD in this brain area. However, the decreased AR-ir in the VMN observed after sexual satiation together with the lack of variation in the circulating levels of androgens, suggests that such reduction is not the result of decreased androgen levels and that this brain region might participate in the neural control of sexual satiation. In favor of this idea is the recent demonstration of a very marginal FOS-immunoreactivity in this nucleus after mating (Coolen et al., 1996). The activation of neurons in this area might accumulate after several ejaculations and, in this way, play a significant role after exhaustion. Various experiments should be undertaken to test these ideas.
Notwithstanding masculine sexual behavior relies primarily on the presence of androgen secretion, other factors may contribute to its expression. These factors vary between species and, in some cases, are sufficient to maintain copulation. For example, male hamsters exposed to short photoperiods exhibited deficits in sexual behavior that are independent of steroid hormones since they occur in gonadally intact and castrated-testosterone-treated animals (Miernicki et al., 1990). Interestingly, it has been demonstrated (Bittman and Krey, 1988 and Wood and Newman, 1993) that AR-ir decreases in response to short photoperiod possibly associated to low levels of testosterone. Furthermore, males of the mouse strain B6D2F1 continue to ejaculate even 25 weeks after castration when the plasma levels of androgens and the hypothalamic nuclear steroid receptors are drastically reduced by castration (Clemens et al., 1988). Other interesting data consider the lack of correlation between the decline of sexual activity in old subjects and the normal levels of androgens secreted by their testes (Chubb and Desjardins, 1984 and Phoenix and Chambers, 1986). A mirrored case consists of the marginal effects on sexual behavior of experienced rats after drastically reducing the circulating levels of testosterone (Taylor et al., 1985). The clearest example of hormone-independent processes in the regulation of male sexual behavior is given in humans and other primate species where experience, motivational systems and social contexts surpass the endocrine-based actions (Wallen, 2001). All these data, taken together, indicate the existence of a ligand-independent process in the control of male sexual behavior. The operation of this process is not understood but at least in the rat it seems to involve memory and olfaction (Larsson, 1956 and Meisel and Sachs, 1994). The exploration of these processes linked or independent of the brain AR downstream activation seems interesting.
Present results suggest that a drastic decrease in AR in specific brain regions might play a role in the inhibition of sexual behavior observed in sexually sated males. Similarly, the modified sexual activity that is observed after an animal attained an ejaculation (Larsson, 1956) could be interpreted on the basis of an analogous mechanism. At present the reasons underlying the diminished AR-ir in the MPN, nAcc and VMN of animals that copulated remain unclear. However, after excluding a putative serum androgen decline associated to copulation and on the basis that PG21 recognizes free cytoplasmic AR, it is possible to propose that such reduction is due to a loss and/or to a diminished production of AR in these brain areas.

Acknowledgements

We thank Dr Mariann Fodor, Dr Ana Elena Lemus and Rosendo del Angel for technical assistance and Víctor Flores for animal care. These experiments were partially supported by a grant to A.F.-G. from Conacyt (grant no. 971017).

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Corresponding author. Tel.: +52-54-83-28-56; fax: +52-54-83-28-63

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