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date: 11 December 2018

Sexual Behavior in Males From a Neuroendocrine Perspective

Summary and Keywords

It is well established that testosterone from testicular origin plays a critical role in the activation of male sexual behavior in most, if not all, vertebrate species. These effects take place to a large extent in the preoptic area although other brain sites are obviously also implicated. In its target areas, testosterone is actively metabolized either into estrogenic and androgenic steroids that have specific behavioral effects or into inactive metabolites. These transformations either amplify the behavioral activity of testosterone or, alternatively, metabolism to an inactive compound dissipates any biological effect. Androgens and estrogens then bind to nuclear receptors that modulate the transcription of specific genes. This process is controlled by a variety of co-activators and co-repressors that, respectively, enhance or inhibit these transcriptional processes. In addition, recent work has shown that the production of estrogens by brain aromatase can be modulated within minutes by changes in neural activity and that these rapid changes in neuroestrogen production impact sexual behavior, in particular sexual motivation within the same time frame. Estrogens thus affect specific aspects of male sexual behavior in two different time frames via two types of mechanisms that are completely different. Multiple questions remain open concerning the cellular brain mechanisms that mediate testosterone action on male sexual behavior.

Keywords: testosterone, preoptic area, intracellular metabolism, aromatase, 5α-reductase, estrogens, nuclear receptors, steroid receptor co-activators, nongenomic membrane-initiated effects

Introduction

In a classic experiment completed on domestic fowl (Gallus domesticus), Adolph Arnold Berthold established in the middle of the 19th century that male sexual attributes, including sexual behavior, depend on the action of a diffusible substance produced by the testes (Berthold, 1849). This chemical messenger was identified as the sex steroid testosterone at the beginning of the 20th century; its purification from animal tissues and, soon thereafter, its full chemical synthesis allowed scientists to analyze in detail the morphological, physiological and behavioral effects of this steroid (Morales, 2013). It is now established that in (nearly) all vertebrate species, male sexual behavior is activated by testosterone originating from the testes, as is well illustrated by castration and replacement studies (Nelson, 2011). In nearly all species tested so far that belong to all classes of vertebrates from fishes to mammals, castration markedly reduces or completely eliminates the expression of a wide range of male sexual behaviors; these behaviors are restored within a few days by a treatment with exogenous testosterone (Nelson, 2011). It must be noted, however, that in many species of fishes from diverse families, the main androgen circulating in the blood is not testosterone but rather its metabolite 11-ketotestosterone (11-KT), which is more powerful than testosterone for activating sexual and aggressive behaviors (Godwin & Lamm, 2017).

The rare exceptions to this rule concern species displaying a dissociated pattern of reproduction in which spermatogenesis and steroid secretion take place during the summer months and precede by at least three-quarters of a year the period when copulation actually occurs in the following spring (see Crews, 2005, for the best documented example, the red-sided garter snakes, Thamnophis sirtalis). However, even in these species, the expression of sexual behavior might still depend on endocrine events, including testosterone secretion, but these events take place several months before the expression of the behavior itself (Krohmer, 2013).

Even if part of the effects of testosterone on sexual behavior result from actions in the periphery (changes in the sensitivity to sex-related olfactory, visual or acoustic stimuli, in the effectors of behavior such as penile muscles in rodents or syrinx muscles in songbirds; Nelson, 2011), the most important sites of androgens action on sexual behavior are obviously in the brain and spinal cord. Although the regulatory role of testosterone (or 11-KT) in the activation of male sexual behavior is well established in a wide variety of vertebrate species, the cellular and biochemical mechanisms through which this behavioral activation takes place is still the focus of active research. Interestingly, the response of the brain to androgens is dynamic. It seems to vary in time and from one subject to another since the intensity or frequency of sexual behaviors does not always correlate with the concentration of testosterone in the blood. Many studies have indeed shown that, though significant correlations are often identified between changes in time of hormones and behavior, individual differences in sex steroid plasma concentrations quite frequently do not correlate with differences in sex behavior (e.g., Balthazart, 2017; Balthazart, Deviche, & Hendrick, 1977; Dessi-Fulgheri, Lucarini, & Lupo di Prisco, 1976; Ramenofsky, 1984)). In a foundational study performed before the advent of sensitive assays for hormones, Grunt and Young had already demonstrated that, in guinea pigs (Cavia porcellus), individual differences in sexual behavior disappear following castration (sexual behavior progressively vanished), but they are quickly reestablished when castrated subjects are all treated with a same standard dose of exogenous testosterone (Grunt & Young, 1953). The differences between sexually active subjects (studs) and more sluggish ones (duds) similarly does not seem to be induced by differences in plasma testosterone concentrations, based on studies in a variety of species (rats, Rattus norvegicus: Damassa, Smith, Tennent, & Davidson, 1977; and Portillo, Diaz, Retana-Marquez, & Paredes, 2006; guinea pigs, Cavia porcellus: Harding & Feder, 1976; chicken, Gallus domesticus and Japanese quail, Coturnix japonica: Van Krey, Siegel, Balander, & Benoff, 1983; and green anolis lizard, Anolis carolinensis: Neal & Wade, 2007).

This lack of a clear correlation between testosterone concentrations in the blood and the occurrence of hormone-regulated behaviors calls for an analysis of the cellular mechanisms of testosterone action in the brain and how they regulate the activation of male-typical sexual behaviors (Ball & Balthazart, 2008b). Thus, a neuroendocrine perspective rather than a solely endocrine perspective is useful in trying to understand the causal action of steroid hormones on male-typical sexual behaviors. The central action of testosterone is regulated by a variety of independent factors, including changes in intracellular metabolism and in receptor availability, or the modulation of transcriptional activity by co-regulators. The focus here is on these mechanisms mostly in mammals and birds where the most detailed analyses have been conducted among vertebrate species. Among mammalian species, most of the data are derived from rodent studies, though work in humans is considered to the extent it is available. Not covered are the steroid-dependent changes in neurotransmitters activity that mediate changes in sexual behavior since these are described in a specific section on neurochemistry and behavior.

Appetitive and Consummatory Aspects of Sexual Behavior

Most of the cellular research analyzing the neuroendocrine bases of male sexual behavior has been devoted to the consummatory aspects of the behavior (intromission and ejaculation in species with internal fertilization, ejaculation in other species). However, before they copulate, males must be able to attract a female through a variety of courtship behaviors, approach her, and ensure that she is sexually receptive. These different aspects of the interaction with the female correspond to different phases of sexual behavior that have been described by terms such as attractivity, appetitive sexual behavior (ASB), and consummatory sexual behavior (CSB). CSB is usually followed by a period of variable duration during which the male’s interest in the female is greatly reduced or nonexistent (refractory period).

The distinction between the appetitive and the consummatory phases of motivated behaviors was originally made by Charles Sherrington and the European ethologists of the first generation (Konrad Lorenz and Niko Tinbergen) and was introduced to the field of behavioral endocrinology by Frank Beach in the 1950s (Beach, 1956; for a more detailed discussion, see Ball & Balthazart, 2008a). Less attention, however, has been devoted to the endocrine control of ASB, though specific procedures have been developed to independently quantify these two aspects of sexual behavior in a number of species, including rats and Japanese quail. The few studies performed so far indicate that the endocrine controls and brain mechanisms underlying ASB and CSB largely overlap but nevertheless show some degree of specificity. These studies are also considered here.

Brain Mechanisms

Sex steroids such as testosterone act on the brain to activate male sexual behavior by molecular mechanisms that are to a large extent similar to those mediating morphological effects of steroids in the periphery. Sex steroid hormones and similar lipophilic compounds such as thyroid hormones can more or less freely enter their target cells, where they are exposed to metabolizing enzymes that will eventually transform them into more active or less active metabolites. The native hormone or its metabolites will then bind to specific intracellular receptors. The hormone-receptor complex will then dimerize and move to the cell nucleus, bind to specific hormone-responsive elements of the DNA, and act as a transcription factor, leading to changes in the transcription of new messenger RNA (mRNA) and synthesis of new proteins that will ultimately alter cell function. These effects are by nature relatively slow and take hours to days to develop (McEwen, 1981, 1994).

Many cellular and behavioral effects of testosterone and its metabolite, estradiol, that were identified more recently are, however, too rapid to be produced by these mechanisms. These fast effects result from brain-specific mechanisms involving direct effects on neuronal membranes or direct interactions with intracellular signaling cascades involving, for example, the phosphorylation of various proteins (Cornil, Ball, & Balthazart, 2012). Less is known about these mechanisms, but the current state of knowledge is also summarized here, as are these different aspects of testosterone action in the brain.

Testosterone Metabolism

Metabolic Pathways

In many cases, testosterone is not the chemical signal that will by itself induce the changes in neuronal physiology leading to activation of behavior. When entering a cell, testosterone can be metabolized by a number of specific enzymes that will transform it into another steroid that can be behaviorally very active or completely inactive. Two main enzymes, aromatase and 5α‎-reductase, catalyze the transformation of testosterone into behaviorally relevant metabolites, respectively, estradiol-17β‎ and 5α‎-dihydrotestosterone (5α‎-DHT) (Figure 1) (Balthazart, 1989; Celotti, Massa, & Martini, 1979). Another enzyme, called 5β‎-reductase, catalyzes the transformation of testosterone into 5β‎-dihydrotestosterone (5β‎-DHT)(Massa, Cresti, & Martini, 1977) that has no or only minor effects on the expression of sexual behavior (Adkins, 1977; Harding, Sheridan, & Walters, 1983), although minor effects were detected in young domestic chicks (Balthazart & Hirschberg, 1979; Balthazart, Malacarne, & Deviche, 1981). 5β‎-reduced androgens, however, play a major function in the control of hematopoiesis (Garavini & Cristofori, 1984; Levere, Kappas, & Granick, 1967). Although this enzyme is broadly expressed in all bird species that have been considered and in ectothermic vertebrates, data collected to date suggest that it is present in substantial amount in only a few species of mammals, including hamsters (Callard, Hoffman, Petro, & Ryan, 1979). The significance of this species specificity has never been investigated.

Sexual Behavior in Males From a Neuroendocrine PerspectiveClick to view larger

Figure 1. Main pathways for testosterone metabolism in the vertebrate brain. Three main enzymes irreversibly transform testosterone into behaviorally active metabolites such as estradiol-17β‎ and 5α‎-dihydrotestosterone (5α‎-DHT) or into behaviorally inactive compounds such as 5β‎-dihydrotestosterone (5β‎-DHT). In addition, a multitude of other enzymes reversibly transform these parent steroids into compounds that are for most of them behaviorally less active, including hydroxylated more polar forms (namely, the 5α‎-androstane-3 α‎ or 3 β‎, 17 β‎ diols, 5α‎-3α‎ or 3β‎, 17 β‎-diols) that are more soluble in water and represent initial steps in the excretion but can also have behavioral activity. A selection only of these metabolites is shown based on their potential relevance for male sexual behavior control. See text for additional information.

The transformations catalyzed by these three enzymes are thermodynamically irreversible in conditions compatible with life, but a multitude of other enzymes, including hydroxysteroid dehydrogenases, oxido-reductases, and isomerases, are also present in the brain and transform in a reversible manner testosterone or its metabolites into other steroids with diverse behavioral properties. It is beyond the scope of this short review to consider all these possibilities (see Balthazart, 1989 and Feder, 1981, for detail), but two are highlighted here for their behavioral relevance (Figure 1):

  • The 17β‎-hdroxysteroid-dehydrogenase activity (17β‎-HSD) reversibly transforms testosterone into androstenedione and estradiol-17β‎ into estrone. These metabolites have a behavioral activity that is very similar to that of their parent steroid but cross-react poorly, if at all, with radioimmunoassays or immunoenzymosassays, which are currently the most commonly used detection methods. They will thus in many circumstances escape detection, and so their presence can lead to erroneous conclusions.

  • The 3β‎-hdroxysteroid-dehydrogenase activity (3β‎-HSD) is reversibly catalyzing the transformation of 5α‎-DHT into the corresponding 5α‎-androstane-3β‎, 17β‎-diol (5α‎- 3β‎, 17β‎-diol). Interestingly, this diol, which is a metabolite of 5α‎-DHT, binds to the estrogen receptor and presumably explains why 5α‎-DHT, which is a pure and potent androgen that cannot be converted back into testosterone nor aromatized, is able to activate some estrogen-dependent responses, including some aspects of male sexual behavior (Baum & Vreeburg, 1976).

Behavioral Effects of Testosterone Metabolites

As already mentioned, many, and perhaps most, behavioral effects of testosterone are produced at the cellular level by locally produced metabolites of the parent steroid. Indeed, testosterone is often referred to as a pro-hormone because its functional effects frequently occur after its metabolism to another steroid. A wealth of information coming from mammalian and nonmammalian studies supports the idea that many effects of testosterone in the brain, including in many species the activation of male sexual behavior, require its transformation into estradiol (for review, see Balthazart, 1989; Celotti et al., 1979; Whalen, Yahr, & Luttge, 1985). These data form the basis of the aromatization hypothesis, which states that effects of testosterone in the brain require its aromatization into an estrogen. A stronger version of this hypothesis, called the estrogen-receptor hypothesis, further proposes that testosterone action is mediated by the binding of the estradiol derived from testosterone aromatization to estrogen receptors (Balthazart, 1989; McEwen, 1981).

The idea that testosterone action on male sexual behavior depends on its central aromatization into an estrogen rests on different types of experimental evidence collected in a wide variety of species, including rats, some strains of mice, hamsters, quail, chicken, and ring doves:

  • Aromatase is largely expressed in brain areas that control male sexual behavior, such as the medial preoptic area and bed nucleus of the stria terminalis and amygdala (Roselli, Horton, & Resko, 1985; Schumacher & Balthazart, 1987).

  • Aromatizable androgens such as testosterone or androstenedione activate male copulatory behavior, while non aromatizable androgens such as 5α‎-DHT and the derived diols have no or only weak effects (Adkins, Boop, Koutnik, Morris, & Pniewski, 1980; Harding et al., 1983; Whalen & DeBold, 1974; Whalen & Luttge, 1971).

  • Estrogens themselves are able to restore most aspects of copulatory behavior in castrated males (see Celotti et al., 1979 for review; hundreds of references are available).

  • Antiestrogens such as tamoxifen or nitromifene citrate (CI-628) or ICI-182780 block the testosterone-induced male sexual behavior (e.g., Adkins & Nock, 1976; Alexandre & Balthazart, 1986; Beyer, Morali, Naftolin, Larsson, & Perez-Palacios, 1976).

  • Aromatase inhibitors such as androstatrienedione (ATD), Vorozole™ (also known as R76713 or 6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole) or Fadrozole™ inhibitor completely block the activating effects of testosterone on male sexual behavior (Adkins et al., 1980; Alexandre & Balthazart, 1986; Balthazart, Evrard, & Surlemont, 1990; Christensen & Clemens, 1975). The specificity of these inhibitions has been confirmed by showing that (a) aromatase inhibitors do not block behaviors activated by estrogens and (b) the blockade induced by aromatase inhibitors can be reversed by the concomitant injection of an estrogen.

Together these data clearly support the aromatization hypothesis and the estrogen-receptor hypothesis in the context of the activation of male sexual behavior. This, however, does not mean that androgens themselves (testosterone or 5α‎-DHT) do not affect male behavior. It is indeed clear that in many species:

  • Consummatory behavior in mammals is dependent on nuclei in the spinal cord that innervate muscles to facilitate erections, such as the spinal nucleus of the bulbocavernosus (SNB) of rats that is homologous to Onuf’s nucleus in humans (Matusda et al., 2008). This androgen-sensitive, sexually dimorphic nucleus innervates striated perineal muscles attached to the base of the penis, which are themselves also androgen sensitive (Matusda et al., 2008). Circulating androgens and androgenic metabolites such as 5α‎-DHT acting in adulthood (but not estrogenic metabolites) are necessary to maintain morphological aspects of this nucleus and its muscle targets to ensure successful functioning of male sexual behavior (Verhovshek et al., 2010)

  • Neurons in the upper lumbar spinal cord that express gastrin-releasing peptide project to lower lumbar regions controlling erections as well as ejaculations in male rats (Sakamoto et al., 2008). This system of neurons is vestigial in females and requires androgens during ontogeny to develop the male-like phenotype and retains some androgen responsitivity in adulthood (Sakamoto et al., 2008).

  • In contrast to actions in the spinal cord, 5α‎-DHT has a weak but nevertheless significant effect on copulatory behavior in some species. Actually, in some species, estrogens apparently play little or no role in the activation of male sexual behavior, and this behavior is mostly activated by nonaromatizable androgens such as 5α‎-DHT acting via binding to androgen receptor proteins. This is the case, for example, in guinea pigs, Cavia porcellus (Alsum & Goy, 1974), hamsters, Mesocricvetus auratus (Whalen & DeBold, 1974), rabbits, Oryctolagus cuniculus (Agmo & Södersten, 1975), rhesus monkeys, Macaca mulatta (Phoenix, 1974), and some strains of mice, Mus musculus (Luttge & Hall, 1973).

  • Antiandrogens, though less effective than the antiestrogens, are nevertheless able to significantly diminish testosterone-induced male behaviors in some species (Alexandre & Balthazart, 1986).

  • Finally and most importantly, in multiple species nonaromatizable androgens synergize with low doses of estrogens to fully restore all aspects of male-typical behavior (Adkins et al., 1980; Baum & Vreeburg, 1973; for additional references, see Balthazart, 1989; Martini, 1982). Many experiments have indeed shown that the behavioral activity of low doses of estrogens is markedly enhanced by a concurrent treatment with a nonaromatizable androgen such as 5α‎-DHT. This synergism between androgens and estrogens has been reported in multiple species, even if the precise mechanism underlying this synergism has not been identified so far (see Balthazart, 1989, for an extensive discussion). This question has received little attention in recent years. It has been suggested that the synergism between androgens and estrogens could result from the cross-reactivity of one of the steroids involved with the other receptor (e.g., of R1881 binding to estrogen receptor) or that estrogens decrease the spontaneously very active catabolism of 5α‎-DHT or that 5α‎-DHT increases the action of estradiol via its metabolism to 5α‎- 3β‎, 17β‎-diol and binding of this diol to the estrogen receptors. But none of these explanations seems to be able to explain all observations. A recent study showed that the nonaromatizable androgen 6α‎-fluorotestosterone, which also acts as an aromatase inhibitor, is able to activate male copulatory behavior in rats and that this effect is blocked by two different aromatase inhibitors (ATD and Fadrozole). This suggests that 6α‎-fluorotestosterone activates behavior by some unknown mechanism potentially involving an action of aromatase independent from the production of estrogens (Yahr, 2015). The question of the steroid specificity in sex behavior activation is no longer of great interest to most scientists, who now rather focus on understanding what sort of gene expression results from steroid action to activate behavior. Nonetheless, important questions about steroid specificity remain unanswered. The fact that pro-hormones like testosterone can be metabolized to estrogenic and androgenic steroids and have tissue-specific effects tell us a lot about the different levels of regulation that can occur when considering the hormonal regulation of behavior. Metabolism to a steroid that binds to a receptor with a higher affinity than the parent compound essentially amplifies the effect of a hormone, while metabolism to an inactive compound dissipates any biological effect.

Androgens Versus Estrogens in the Activation of Human Male Sexual Behavior

The relative role of androgens and estrogens in the activation of sexual behavior in male humans is at present a controversial topic. This situation is due in part because direct experimentation is obviously difficult or impossible for ethical reasons. A variety of clinical studies, however, clearly indicate that in hypogonadal men, treatment with exogenous testosterone increases sexual motivation and copulatory activity (Davidson, Camargo, & Smith, 1979; Snyder et al., 2000; Wang et al., 2000). Whether these effects are mediated by testosterone acting as an androgen or by conversion to an estrogen has been a more difficult question to address.

Some studies initially suggested that in men, as in rhesus monkeys, the activation of sexual behavior and sexual motivation depends mostly on the action of androgens acting as such on androgen receptors. One suggestive study was based on the pharmacological manipulation of testosterone and/or estrogens availability in subjects who were functionally castrated by a chronic treatment suppressing gonadotrophin releasing hormone (GnRH) secretion (Bagatell, Heiman, Rivier, & Bremner, 1994). GnRH is produced by the hypothalamus and stimulates the synthesis of the two gonadotropic hormones (luteinizing hormone and follicle stimulating hormone) in the pituitary gland.
These in turn act on the testes to activate the secretion of testosterone. In the Bagatell study, men were chronically treated with a GnRH antagonist (receptor blocker), so that plasma concentrations of testosterone and estradiol dropped rapidly and treated subjects observed a strong parallel decrease of their interest in sex. They had fewer sexual fantasies, masturbated less often, and the frequency of their sexual activities decreased. A fraction of these subjects were simultaneously treated with testosterone, which was apparently sufficient to maintain their sexual motivation and performance (Bagatell et al., 1994). The simultaneous treatment of these testosterone-treated subjects with a relatively weak aromatase inhibitor (Testolactone) that decreased by about 65% the circulating concentration of estradiol had apparently no effect on the behavioral measures. However, this aromatase inhibition was only partial, and more powerful aromatase inhibitors have now become available.

More recent studies indicate, in contrast, a prominent role of estrogens. A case study of a man expressing a mutation of the aromatase gene that resulted in a decrease in aromatase expression and in sexual motivation revealed a major improvement in sexual motivation and performance when the man was treated with estradiol combined with testosterone but not when treated with testosterone alone (Carani et al., 2005; Carani, Rochira, Faustini-Fustini, Balestrieri, & Granata, 1999).

Sexual Behavior in Males From a Neuroendocrine PerspectiveClick to view larger

Figure 2. Effects of testosterone alone or combined with the aromatase inhibitor anatrozole on the sexual motivation (Sexual desire) and sexual performance (Erectile capacity) in men that had been pretreated with the GnrH agonist goserelin acetate in order to decrease circulating concentrations of testosterone and estradiol. As a consequence, these men experienced a major decrease in both aspects of sexual behavior (0 group treated with placebo), but behavior was restored in a dose-dependent manner by testosterone. Aromatase inhibition significantly blocked this behavioral recovery. Data are means ± SEM. Redrawn from data in Finkelstein et al. (2013).

Another large study including several hundred subjects employed a strategy similar to Bagatelle et al. and functionally castrated subjects but by treating them chronically with a GnRH agonist, goserilin acetate (Finkelstein et al., 2013). This chronic treatment desensitizes the GnRH receptors in the pituitary and after a few days produces a similar inhibition of testosterone secretion. Some of the subjects were then treated in parallel with increasing doses of testosterone (198 subjects in total). Another group of 202 subjects was treated with the GnRH agonist in combination with testosterone in association with the aromatase inhibitor anastozole (=Arimidex™) (Figure 2). Sexual behavior was assessed by periodic questionnaires asking participants to compare their sex drive (motivation) and erectile capacity (probability of getting an erection and reaching orgasm) to the same aspects before the study began.

The inhibition of testosterone secretion caused by the GnRH suppression resulted in a decline of both sexual motivation and sexual performance. This decline was counteracted in a dose-dependent manner by treatments with increasing doses of testosterone. However, concurrent administration of the aromatase inhibitor anastrozole markedly inhibited these effects of testosterone. This study, based on very large samples of subjects, clearly indicates that the activational effects of testosterone on both motivational and performance aspects of sexual behavior in the human male are, like in many animal models, mediated largely by the aromatization of the steroid into an estrogen (Finkelstein et al., 2013).

Changes in the Activity of Testosterone Metabolizing Enzymes

A host of studies have analyzed the changes in the activity of testosterone metabolizing enzymes in a variety of physiological situations in multiple species of vertebrates. These studies have demonstrated that the ratio of active versus inactive testosterone metabolites that are produced in the brain and in peripheral structures can be affected by a host of factors including the sex, age, or hormonal condition of the subjects, as well as by other external factors such as stress, photoperiod, and season. These changes in enzymatic activities provide a range of mechanisms that potentially modulate the activation of behaviors (see Balthazart, 1989, for a review in connection with sexual behavior).

It is, for example, well established that in many species, aromatase activity in the preoptic area is significantly higher in males than in females. This is namely the case in rats (Roselli, 1991) and in Japanese quail (Schumacher & Balthazart, 1986). Given that in these species, the activation of male sexual behavior is largely mediated at the cellular level by the estrogens produced by aromatization of testosterone in the preoptic area, this enzymatic sex difference leading to a more active production of estrogens in males than in females must contribute to the explanation as to why male-typical copulatory patterns are difficult, if not impossible, to activate in females (Adkins, 1975; Becker, Breedlove, Crews, & McCarthy, 2002; McCarthy, De Vries, & Forger, 2009; Nelson, 2011). This is not, however, the only cause for the behavioral sex difference since a treatment with exogenous estradiol, which should bypass the rate-limiting enzymatic activity, is still unable to activate male-typical sexual behavior in females (e.g., in quail: Schumacher & Balthazart, 1983).

Similarly, it has been shown that the 5β‎-reductase activity is extremely high in the brain of Japanese quail during the embryonic and early postnatal life. This activity decreases in a progressive manner between embryonic days 9 to 17 (Balthazart & Ottinger, 1984), and this decrease continues during the first 5 weeks after hatching (Balthazart & Schumacher, 1984). In contrast, the 5β‎-reductase activity in the cloacal gland decreases during embryonic life, but it reaches low values at hatching and no further decrease is observed afterward (Balthazart & Ottinger, 1984; Balthazart & Schumacher, 1984). As 5β‎-reduced metabolites of testosterone are inactive androgens, it has been suggested that the decrease of 5β‎-reductase activity with age corresponds to a potentiation of the effects of testosterone at the level of the brain but not in the periphery.
 This hypothesis was tested by comparing the relative potencies of 5α‎-dihydrotestosterone (5α‎-DHT), which cannot be converted to 5β‎-reduced metabolites, and testosterone in their ability to induce crowing in young 7-day-old gonadectomized quail (Balthazart, Schumacher, & Malacarne, 1984). The promotion of cloacal gland growth by these treatments was also assessed as a control since there are no age-related changes in 5β‎-reductase in this organ. Silastic implants of various lengths (2.5, 5 or 10 mm) containing 5α‎-DHT were significantly more effective at stimulating crowing than similar implants filled with testosterone, but, in contrast, there was no difference in their potency at inducing cloacal gland growth. These results thus support the idea that testosterone action is regulated by the changes in activity of the inactivating enzyme 5β‎-reductase (Hutchison & Steimer, 1981).

Rapid Regulation of Enzymes

It was initially thought that changes in the activity of these testosterone-metabolizing enzymes were exclusively the result of changes in the concentration of the enzymatic proteins. It was, for example, clearly established that the treatment with testosterone of castrated rats (Rattus norvegicus), ring doves (Streptopelia risoria), or quail (Coturnix japonica) induces a four- to five fold increase in preoptic aromatase activity (Roselli et al., 1985; Schumacher & Balthazart, 1986; Steimer & Hutchison, 1981). This is paralleled by a similar increase in the corresponding mRNA, suggesting that the change results from an increased transcription of the gene CYP19A coding for aromatase (Balthazart & Foidart, 1993). As such, these effects based on changes in transcription are relatively slow and only become detectable after several hours or even days (e.g., Balthazart, Foidart, & Hendrick, 1990).

Work performed during the last two decades revealed, however, that more rapid adjustments of these enzymatic activities are also possible. This is best documented for aromatase. It was shown that the activity of this enzyme is rapidly (within 5–10 min) downregulated in brain homogenates by exposure to conditions that stimulate phosphorylations (increased concentrations of ATP, Ca++ and Mg++; see (Balthazart, Baillien, Charlier, & Ball, 2003; Comito, Pradhan, Karleen, & Schlinger, 2015; Cornil, et al., 2012)) and this inhibition was blocked by kinase inhibitors confirming that the enzymatic inhibition results from protein phosphorylations (Balthazart et al., 2003). Subsequent studies based on cells transfected with the human aromatase gene confirmed that the phosphorylations that inhibit aromatase activity concern the aromatase protein itself, but additional work employing an experimental strategy of site-directed mutagenesis failed to identify the specific amino acid that controls this process, suggesting that multiple residues are probably implicated. Similar rapid changes in aromatase activity were also described in preoptic explants maintained in vitro after treatments that modify the intracellular Ca++ concentration (Balthazart, Baillien, & Ball, 2001; Balthazart et al., 2003) or after exposure to agonists of glutamate receptors of the kainate or AMPA subtypes (Balthazart, Baillien, & Ball, 2006). Preoptic aromatase activity of male quail is also decreased within 5 minutes following socio-sexual interactions with a female (Cornil et al., 2005; de Bournonville, Dickens, Ball, Balthazart, & Cornil, 2013), but it increases just as rapidly (5 min) after exposure to a chronic restraint stress (Dickens, Cornil, & Balthazart, 2011). These effects are anatomically specific (present in some brain nuclei but not others) and transient: they usually disappear after 30–120 min. They are associated with and presumably caused, at least in part, by an increased extracellular concentration of glutamate in the preoptic area (de Bournonville et al., 2017). These rapid changes in aromatase activity are thus likely to induce similarly rapid changes in estrogen concentration in the preoptic area that might be responsible for their rapid membrane-initiated effects on sexual behavior (Cornil, Ball, & Balthazart, 2006) described later.

These rapid enzymatic changes are not limited to aromatase but also concern another enzyme located upstream in the synthetic pathway producing sex steroids, the 3β‎-hydroxysteroid dehydrogenase/Δ‎5-Δ‎4 isomerase (3β‎-HSD). This enzyme namely catalyzes the transformation of dehydroepiandrosterone (DHEA) into androstenedione, which can then be metabolized into testosterone and subsequently estradiol. It was known that this enzyme plays a key role in the control of territorial aggression during the autumn and winter in song sparrows, Melospiza melodia (Wingfield & Hahn, 1994). In this species, territorial aggression indeed remains elevated during most of the year even during periods when circulating concentrations of testosterone and estradiol are undetectable. DHEA of adrenal origin remains present, however, and is transformed in the brain by the 3β‎-HSD activity into active androgens and estrogens that activate territorial behavior. Accordingly, this behavior is inhibited by a combined treatment with an antiandrogen and an aromatase inhibitor (Soma, Sullivan, & Wingfield, 1999; Soma et al., 2000). The enzyme is present in the brain year round even if its activity slowly increases between the breeding and nonbreeding season (Pradhan et al., 2010; Soma, Alday, Hau, & Schlinger, 2004). Other studies indicate that its activity can be rapidly inhibited by acute restraint stress (Soma et al., 2004) or by estradiol (Pradhan, Yu, & Soma, 2008). Most interestingly, the 3β‎-HSD activity is increased within 30 min by a simulated territorial intrusion (Pradhan et al., 2010). This increase was positively correlated with the time spent in close proximity to the intruder decoy, suggesting that the enzymatic change relates to and is potentially induced by the behavioral interaction. The increased activity is thus potentially producing increased amounts of testosterone and estradiol that could support, in the short-term, expression of aggressive behavior.

Thus, evidence is accumulating to demonstrate that steroid production and metabolism can be modulated within minutes and that these changes in activity are under the control of the activity of various neurotransmitters, in particular glutamate (Balthazart et al., 2006; Do Rego et al., 2009; Remage-Healey, Maidment, & Schlinger, 2008).

Androgen and Estrogen Receptors

The effects of sex steroids on behavior are classically thought to be mediated by the steroid binding to intracellular receptors, which when occupied dimerize and translocate to the nucleus where they bind to specific loci on the DNA (specific response elements or others) and modulate transcription of target genes. These receptors are characterized by their specificity (they only bind one class of steroids), high affinity (binding affinity has a Kd in the low nanomolar range), and limited capacity (they are rapidly saturated in the presence of nanomolar or even femtomolar concentrations of steroids; see Blaustein & Olster, 1989; McEwen & Alves, 1999).

The neuroanatomical distribution of high-affinity binding sites (receptors) for androgens (AR), estrogens (ER), and progestagens (PR) is remarkably consistent among vertebrate species. This distribution was initially mapped in the 1970s using in vivo autoradiographic techniques (Morrell, Kelley, & Pfaff, 1975; Stumpf & Sar, 1976). More recently, this distribution was reanalyzed by immunocytochemical studies employing mono- or polyclonal antibodies against the steroid receptors molecules. These receptors have also been cloned and sequenced in many species, and probes have been synthesized and used to localize the corresponding mRNA by in situ hybridization. All these different experimental approaches have generally produced results that are in good agreement.

Cells that express a high density of receptors for sex steroids are localized mainly in the medial preoptic area, the hypothalamus (anterior hypothalamic area, ventromedial nucleus, tuberal hypothalamus), telencephalic structures that are part of the limbic system (amygdala, lateral septum, bed nucleus of stria terminalis), and specific parts of the mesencephalon (optic tectum). A schematic presentation of this distribution is shown in Figure 3A. A similar distribution has been observed among a wide range of vertebrates ranging from fishes to mammals (Kelley & Pfaff, 1978; Morrell & Pfaff, 1978). Furthermore, the anatomical distribution of androgen-concentrating cells is in general similar to that of the estrogen-concentrating cells. However, differences have been observed in the intensity and number of labeled cells, as well as in their precise distribution within a given nucleus. These species-specific and steroid-specific features are, however, beyond the scope of the present summary.

Sexual Behavior in Males From a Neuroendocrine PerspectiveClick to view larger

Figure 3. Steroid-binding sites in vertebrates in general (A) and in a specialized case, the songbird brain (B). In addition to the expression of receptors in the preoptic area—hypothalamus, limbic system, and optic tectum, including the nucleus intercollicularis (ICo)—songbirds express receptors for androgens, and in some cases for estrogens, in specialized nuclei of the telencephalon that are implicated in song learning and production such as HVC (formerly High Vocal Center, now used as a proper name), RA (nucleus robustus arcopallialis), and MAN (magnocellular nucleus of the anterior nidopallium). In songbirds, androgen receptors have also been identified in the nucleus of the twelfth nerve (XIIts) that innervates the syrinx. Redrawn from Balthazart and Riters (2001), based in part on data in Kelley and Pfaff (1978); Morrell ND Pfaff (1978); and Arnold et al. (1976).

A species-specific distribution has additionally been observed in vertebrate species that produce vocalizations in the context of reproduction (Figure 3B). For example, in songbirds such as zebra finches (Taeniopygia guttata) and canaries (Serinus canaria), a network of neurochemically specialized brain nuclei controls both the learning and the production of song (Nottebohm, Stokes, & Leonard, 1976). Singing behavior is regulated by testosterone and its metabolite estradiol. Accordingly, most nuclei in the so-called song control system (located in areas homologous to the cortex in the avian telencephalon as well as the mesencephalon and brainstem) contain androgen and for some of them estrogen receptors (Arnold, Nottebohm, & Pfaff, 1976) (for more recent reviews, see Ziegler & Marler, 2008). This presence of androgen receptors in telencephalic nuclei outside the limbic system is unusual among vertebrates and is functionally related to singing. Steroids have a variety of effects on song behavior, and these effects have recently been dissected (Alward, Rouse, Balthazart, & Ball, 2017). Testosterone action in the preoptic region activates increases in song rate (Alward, Balthazart, & Ball, 2013), while steroid hormone action in the song control nuclei and at the syrinx (the vocal production organ) effect different aspects of song quality, including the degree of stereotypy and song structure (Alward, Madison, Gravley, & Ball, 2016; Alward, Madison, Parker, & Balthazart, 2016).

Specializations related to the hormonal regulation of vocal behavior have also been described in the midshipman fish (Porichthys notatus) and several species of amphibians such as the African clawed frog (Xenopus laevis) (see Ball & Balthazart, 2009). It should also be noted that the very discrete localization of sex steroid receptors that had initially been detected by autoradiography is actually broader: the more sensitive techniques such as in situ hybridization have shown that many additional brain areas contain cells expressing these receptors at low densities.

Sex steroids action in the brain is related to the control of various functions, including neuroplasticity, cognition, tumor growth, and behavior. Therefore, only subsets of the sex steroids binding sites in the brain are implicated in the control of male sexual behavior. These sites have been identified by a combination of lesion experiments and of stereotaxic implantation of steroids directly in the brain of castrated subjects (Alward et al., 2017; Pfaff, 1980).

The medial part of the preoptic area is clearly a critical and sufficient site for activation by testosterone (or its metabolite estradiol) of male sexual behaviors (Balthazart & Ball, 2007; Tobet & Fox, 1992). This appears to be true in all species that have been tested in all classes of vertebrates from fishes to mammals. Sexually inactive castrated males recover at a rate of sexual activity that is similar or equal to the level seen in gonad-intact, sexually mature males when implanted with testosterone in this brain area. Additional sites are also implicated and form the so-called social behavior network (Goodson, 2005; Newman, 1999; O’Connell & Hofmann, 2011). For example, androgen action in the septum, bed nucleus of the stria terminalis, and amygdala modulate the expression of male sexual behavior, even if the action of androgens in the preoptic area alone is sufficient to activate this behavior in many cases.

The activation of these nuclear receptors by testosterone or estradiol modulates the transcription of a multitude of genes that encode for a variety of receptors (for neurotransmitters, for neuropeptides, for steroids themselves) and enzymes that control the synthesis or catabolism of neurotransmitters and neuropeptides, as well as protein (neuro)hormones themselves. These changes are confined to the anatomically specific sites that express steroid receptors and ultimately result in specific changes in neural activity and behavior.

With the advent of molecular biology techniques, a second estrogen receptor (ER) was also cloned first in rats (Kuiper, Enmark, Pelto-Huikko, Nilsson, & Gustafsson, 1996) and then in humans (Mosselman, Polman, & Dijkema, 1996) and multiple other species including birds (Foidart, Lakaye, Grisar, Ball, & Balthazart, 1999). It was named ERβ‎ to distinguish it from the initial ER now relabeled ERα‎. Targeted disruptions (knock-out or KO) of ERα‎ confirmed that this receptor plays key roles in the control of male sexual and aggressive behavior (Ogawa, Lubahn, Korach, & Pfaff, 1997), even if the importance of the behavioral effects varied from one study to another (Rissman, Wersinger, Taylor, & Lubahn, 1997). ERβ‎ was initially thought to be of lesser importance (Ogawa et al., 1999). These initial KO models were, however, found to be imperfect, and a reanalysis of these questions with better or more complete disruptions of ERα‎ ανδ‎ ERβ‎ have led researchers to reevaluate this conclusion and to suggest that both ER play specific roles in the control of male sexual behavior (Ogawa et al., 2000). Specific behavioral effects of global AR inactivation such as is seen in rats bearing the testicular feminizing (tfm) mutation or in ARKO mice were difficult to evaluate since these subjects lacked the male-typical genitalia, thus preventing them from engaging in normal intromissive behavior (Ono, Geller, & Lai, 1974; Sato et al., 2004).

More recently, new transgenic models that allow investigators to disrupt the expression of sex steroid receptors specifically in the brain, or in neurons or even in neurons within specific brain regions, have become available, and they now permit a more refined dissection of the specific role of each receptor region by region (e.g., Naule et al., 2016; Raskin et al., 2009; Swift-Gallant, Coome, Srinivasan, & Monks, 2016; Swift-Gallant & Monks, 2017).

Progesterone Receptors

Evidence also exists that the activation of male-typical sexual behaviors and aggression by androgens and their metabolites can be regulated by progesterone acting via progesterone receptors (e.g., Phelps, Lydon, O’Malley, & Crews, 1998). The exact role of endogenous progesterone is not clear in all cases, but mice with targeted disruption (i.e. knock-out) of the progesterone receptor are much less responsive to the androgenic facilitation of male-typical sexual behaviors (Phelps et al., 1998). More recent studies in mice with selective ablation, using genetic techniques, of a relatively small set of cells expressing progesterone receptors only in the ventromedial nucleus of the hypothalamus have also indicated that these progesterone-expressing cells are necessary for the hormonal activation of male-typical sexual behaviors (Yang et al., 2013). A circuitry analysis integrating these actions of progesterone with the actions of androgens and their metabolites on the regulation of male-typical sexual behaviors remains to be completed.

Steroid Receptor Co-Regulators

When steroid receptors were discovered and their mechanism of action as transcription factors was initially elucidated, this mechanism seemed relatively simple. The activated receptor was binding to specific sites of the DNA and in this way activated the transcription of specific target genes (Jensen et al., 1968). A number of physiological situations were later discovered where variation in the behavioral response to steroid hormones was not explained by variation in the systemic concentrations of the steroid. This indicated that variation in responsiveness of the brain targets of the steroid had to be considered (O’Malley & Tsai, 1992). These changes in responsiveness are mediated in the case of testosterone by changes in intracellular metabolism and for all steroids by changes in steroid receptor expression. In addition, it has now become clear that the transcriptional activity of steroid receptors can also be modulated by cofactors that constitute a limiting factor in some physiological situations. The first of these cofactors was identified in the laboratory of Bert O’Malley (Onate, Tsai, Tsai, & O’Malley, 1995) and was called steroid receptor coactivator 1 (SRC1). Two additional members of this protein family were later identified and labeled by multiple names until an agreement was reached to call them SRC2 and SRC3 (see Charlier & Balthazart, 2005, for references). The role of these three proteins in the control of male (and also female) sexual behavior has been deciphered to some extent and will be briefly reviewed here. Note, however, that SRCs are only part of a large family of proteins that act as steroid receptor co-regulators (co-activators or co-repressors) that includes hundreds of members (McKenna, Nawaz, Tsai, Tsai, & O’Malley, 1998; McKenna & O’Malley, 2002).

Co-activators of the SRC family enhance target gene transcription by remodeling the chromatin (through histone acetylation and methylation) and recruiting/stabilizing the general transcription machinery (see Charlier & Balthazart, 2005; McKenna & O’Malley, 2002, for review). They are broadly expressed throughout the body, including the brain where expression is densest in nuclei that also express sex steroid receptors (for androgens, estrogens, and progestins) such as the medial preoptic area, the arcuate and ventromedial nuclei of the hypothalamus in rats (Meijer, Steenbergen, & De Kloet, 2000) as well as in quail (Charlier, Lakaye, Ball, & Balthazart, 2002; Niessen, Balthazart, Ball, & Charlier, 2011) (Figure 4). Interestingly, in songbirds such as zebra finch and canary, expression of SRC1 and of another steroid receptor co-activator, CBP (cAMP response element binding protein-binding protein) is additionally very dense in the telencephalic steroid-sensitive song control nuclei such as HVC (Auger & Ball, 2002; Auger, Bentley, Auger, Ramamurthy, & Ball, 2002).

Sexual Behavior in Males From a Neuroendocrine PerspectiveClick to view larger

Figure 4. Expression of the steroid receptor co-activator 1 (SRC1) in the quail brain (A-B) and behavioral effects of the inhibition of its expression by antisense oligonucleotides (C). The first two panels show the dense expression of SCR1 as observed by nonradioactive in situ hybridization in the medial preoptic nucleus (POM) in A and in the bed nucleus of the stria terminalis (BST) in B. The insert in B shows labeled cells at high magnification illustrating that the mRNA is essentially cytoplasmic in location. Magnification bar is 1000 µm in A, 80 µm in B. Panel C shows the decrease in sexual behavior, as illustrated by the frequency of cloacal contact movements in castrated males who received a subcutaneous Silastic™ Implant filled with testosterone (T) before test 1 (T1) and were then injected daily in the third ventricle with the SCR1 antisense (AS) or the scrambled nucleotide (Ctrl) during pretests (PT) starting three days before testosterone implantation and during tests 1 to 7. The insert in C illustrates the decrease in SRC1 expression as observed by Western blot following repeated injection of the antisense to SRC1 (AS) or of a control scrambled nucleotide (Ctrl); actin expression was not affected. Redrawn for data in Charlier et al. (2002, 2005).

The expression of the co-activators is itself regulated by a variety of factors, including thyroid and sex steroid hormones (Charlier & Balthazart, 2005; McKenna & O’Malley, 2002). In particular, ovariectomy in female rats decreases SRC1 expression in the ventromedial nucleus of the hypothalamus, and estrogens increase their expression in peripubertal male hamsters. SRC1 expression is also regulated in a complex manner by testosterone in male quail. As a consequence, numerous sex differences in the expression of these co-activators have also been reported (for a review see Charlier & Balthazart, 2005).

The role of these co-activators in the control of physiology and more specifically male sexual behavior has been studied by two different approaches: analysis of the phenotype in complete knock-out mice models (i.e., targeted disruption of the gene) and analysis of the effects of treatment with antisense oligonucleotides. Surprisingly, full knock-out mice for SRC1 only showed limited changes in their phenotype. For example, SRC1 knock-out male and female mice were fertile (Xu et al., 1998), females were fully feminized, and males had a normal development of androgen-dependent motoneurons of the spinal bulbocavernosus nucleus (Monks, Xu, O’Malley, & Jordan, 2003). However, SRC2 knock-out mice showed major deficits in the male and female reproductive tracts and males were hypofertile (Gehin et al., 2002). The limited effects of SRC1 inactivation presumably relates to an adaptive developmental mechanism by which SRC2 expression is upregulated in the brain of mice with a SRC1 knock-out (Xu et al., 1998).

To avoid this adaptive compensation, the antisense technique was used to specifically decrease SRC1 or SRC2 in specific brain regions; these experiments clearly demonstrated that these co-activators are critical for the activation by sex steroids of female and male sexual behavior. For example, inhibition of SRC1 largely blocked the development of male reproductive behavior and male-typical brain structures in female rats treated with exogenous testosterone (Auger, Tetel, & McCarthy, 2000). The activation of female-typical sexual behavior in adult female rats is also inhibited by antisense treatments decreasing SCR1 expression (Apostolakis, Ramamurphy, Zhou, Oñate, & O’Malley, 2002; Molenda, Griffin, Auger, McCarthy, & Tetel, 2002), a finding in agreement with the observation that neurons expressing estradiol or progesterone receptors also densely express SRC1 and SRC1 associated with these receptors (Molenda-Figueira et al., 2008; Tetel, Siegal, & Murphy, 2007). Similarly, in castrated male quail, the inhibition of either SRC1 or SRC2 expression by daily injection in the third ventricle of locked nucleotide acid-based antisense inhibited the activation by exogenous testosterone of all androgen- and estrogen-dependent aspects of male-typical sexual behavior (Charlier, Ball, & Balthazart, 2005; Niessen et al., 2011). These treatments blocking SRC1 or SRC2 expression also downregulated male-typical neuroanatomical features such as the volume of the medial preoptic nucleus, the number of cells expressing aromatase in this nucleus, and the density of vasotocinergic fibers in this brain area (Charlier et al., 2005; Niessen et al., 2011).

Steroid co-activators of the SRC family thus appear to play an important role in modulating the sex steroid action of male behavior and explaining the pleiotropic effects of these steroids on various physiological and behavioral responses (Charlier, Seredynski, Niessen, & Balthazart, 2012; Tetel, Auger, & Charlier, 2009). It is very likely that other co-activators and co-repressors are playing similar roles. Their function is now beginning to be understood at the cellular and biochemical level, but much work remains to be done to evaluate their implication at the organismal level and in particular in the control of male sexual behavior.

Membrane Initiated Action of Sex Steroids and Behavior

The steroid-induced behavioral changes described so far usually take place after latencies extending from a few hours to several days. Such a time course can explain changes in reproductive behavior that are observed over months during the annual cycle, with animals alternating between a season of active reproduction and a period of sexual quiescence when no sexual behavior is usually observed.

Faster actions of steroids have also been identified at the cellular level, suggesting that these hormones may also act via fundamentally different mechanisms. It was noted decades ago that estradiol is able to alter the excitability of GnRH neurons in culture within seconds of their application (Kelly, Moss, & Dudley, 1976). Similar observations have been made in a large number of experimental models (McEwen, 1994; Ronnekleiv & Kelly, 2002; Schumacher, 1990). Estrogens (and to a lesser extent testosterone) are thus able to exert effects that are too rapid (seconds to minutes) to be mediated through the activation of protein synthesis. These rapid effects are generally initiated by steroids acting at the cell membrane, resulting in the activation of a wide variety of intracellular signaling pathways. In some cases, these changes in intracellular physiology will also result after longer latencies in changes in gene transcription, usually referred to as indirect genomic effects.

Multiple membrane estrogen receptors (ER) that can potentially mediate these rapid effects have been identified. The classical nuclear receptors for estrogens (ERα‎ and β‎) are able to associate with the cell membrane and generate intracellular signals through association with G-protein-coupled receptors (GPCR) such as the metabotropic glutamate receptors (mGluR) (Micevych & Dominguez, 2009). Additionally, novel membrane receptors such as GPR30 and two putative membrane receptors characterized by their binding properties but that have so far not been isolated, Gq-mER or ER-X, have also been proposed as candidates for mediating membrane actions of estrogens (Filardo & Thomas, 2005; Roepke, Qiu, Bosch, Ronnekleiv, & Kelly, 2009; Toran-Allerand et al., 2002). Intracellular signaling pathways activated by these receptors result in changes in calcium concentrations or phosphorylations of enzymes or receptors, leading, for example, to changes in enzymatic activities or receptor uncoupling from their effectors. These changes in cellular (neuronal) function have also an impact at the organismal level and in particular on behavior. During the last two decades, experimental evidence has accumulated, demonstrating that estrogens acutely influence processes such as pain or cognition but also aggressive and sexual behaviors (for a review, see Cornil, Ball, & Balthazart, 2012).

To our knowledge, the first study demonstrating such rapid effects of estrogens on male sexual behavior concerned castrated male rats (Rattus norvegicus) in which it was demonstrated that a subcutaneous injection of 17β‎-estradiol stimulates mounts and anogenital investigations within 35 min (Cross & Roselli, 1999). Subsequent studies demonstrated that a single injection of 17β‎-estradiol facilitates the expression of most aspects of male sexual behavior within 10–15 min in quail (Coturnix japonica: Cornil, Dalla, Papadopoulou-Daifoti, Baillien, & Balthazart, 2006) and mice (Mus musculus: Taziaux, Keller, Bakker, & Balthazart, 2007). The processing of species-specific auditory stimuli (male conspecific song) is also modulated within minutes by estradiol action in the telencephalic auditory areas of one songbird species, the zebra finch, Taeniopygia gutata (Remage-Healey, Dong, Chao, & Schlinger, 2012; Remage-Healey et al., 2008). The existence of such rapid behavioral effects of estradiol seems to be an ancient feature in vertebrates since they are also observed in fishes. Injection of estradiol indeed modulates within minutes the production of courtship vocalization in the plainfin midshipman fish, P. notatus (Remage-Healey & Bass, 2004).

Detailed studies in quail have demonstrated that estrogens produced locally in the brain (neuroestrogens) acutely modulate the expression of male sexual motivation (Seredynski, Balthazart, Christophe, Ball, & Cornil, 2013). The intracerebroventicular (i.c.v.) injection of estradiol markedly increased, within minutes, the expression of two measures of sexual motivation: the rhythmic cloacal sphincter movements and the learned social proximity response in subjects whose endogenous estrogens production had been depleted by a chronic treatment with an aromatase inhibitor. This effect was mimicked by membrane-impermeable analogs of estradiol, indicating that it is initiated via actions at the cell membrane. Conversely, blocking estrogen action or estrogen synthesis by a single i.c.v. injection of an estrogen receptor antagonist or of an aromatase inhibitor, respectively, acutely decreased these measures of sexual motivation within 15–30 min. Interestingly, these treatments did not affect the copulatory performance of the birds at least when tested in a small arena where copulation occurs almost reflexively and the male does not have to search for and chase the female.

Sexual Behavior in Males From a Neuroendocrine PerspectiveClick to view larger

Figure 5. Rapid effects of estrogens on male sexual behavior in castrated male quail. A. The i.c.v. injection of the aromatase inhibitor Vorozole™ inhibits the frequency of rhythmic cloacal sphincter movements (RCSM), a measure of sexual motivation in quail, 30 min later in comparison with injection of the vehicle (Veh). This effect is blocked by the i.c.v. injection 15 min before the test of the ERβ‎ agonist DPN but not by the ERα‎ agonist PPT, and these two compounds have no synergistic effect. B. The stimulation by DPN of the RCSM frequency in males exposed to the VOR-induced inhibition is no longer present when males are injected with the antagonist of the metabotropic glutamate receptor 1 (mGluR1) called LY367385 (LY). LY has no effect by itself on the behavior already blocked by Vorozole. All males were tested repeatedly in a randomized order three days apart after the different injections as well as without injection before (Pre) and after (Post) the treatments to ensure that there was no long-lasting effect of the injections. The range of values during the Pre and Post tests is indicated by the gray area. ** = p > .01 and *** = p < .001 compared to Veh with Vorozole, ### = p < .001 compared to Veh without Vorozole. Redrawn from data in Seredynski et al. (2015).

A follow-up series of studies based on the i.c.v. acute injection procedure showed that a single injection of DPN, a specific ERβ‎-specific agonist, and to a lesser extent 17α‎-estradiol, possibly acting through ER-X, prevented the decrease in sexual motivation induced by an acute injection of the aromatase inhibitor Vorozole™ (Seredynski, Balthazart, Ball, & Cornil, 2015) (Figure 5). In contrast, drugs targeting ERα‎ (the agonist PPT and the antagonist MPP), GPR30 (the agonist G1 and the antagonist G15), and the Gq-mER (the agonist STX) were without effect. The rapid stimulation of sexual motivation by estrogens acting via ERβ‎ seems to be mediated by a transactivation of the metabotropic glutamate receptor type 1a (mGluR1a). The mGluR1a antagonist, LY367385, significantly inhibited the measure of sexual motivation when injected alone, and most importantly it blocked the effects of estradiol or DPN on this response (Seredynski et al., 2015). This type of interaction between a nuclear ER located at the membrane and a mGluR had been previously identified in the control of sexual behavior in female rats (Dewing et al., 2007; Meitzen et al., 2013), even if different receptor subtypes were implicated in this case.

Estradiol has thus evolved specific mechanisms to regulate different components of male sexual behavior (motivation vs. performance) in distinct temporal domains (long- vs. short-term). This division between two types of actions (i.e., motivation mediated by short-term actions of estradiol and performance mediated by long-term actions of estradiol) may generalize to other behavioral systems regulated by estrogens, including possibly the response to and abuse of drugs such as amphetamine and cocaine, the control of auditory processing and singing behavior in songbirds, or aspects of learning and memory. This idea of the complementary actions of estrogens at different time domains has been integrated into the dual action hypothesis of estrogen action presented in detail elsewhere (Cornil, Ball, & Balthazart, 2015).

Conclusions

The neuroendocrine mechanisms that control male sexual behavior have been intensively investigated during the past 150 years, especially since purified testosterone became available at the beginning of the 20th century. The mechanisms of intracellular action of sex steroids have been to a large extent elucidated, and the mechanisms that have been identified help explain the seasonal variations as well as, in a more limited number of cases, the individual differences in behavior. However, many specific questions remain open, and the diversity of actions in different species are yet to be discovered. Two main groups of new questions have, however, emerged.

First, it has been established that the transcriptional activity of sex steroids requires the recruitment of co-activators and co-repressors. We now know that the family of proteins is very large (more than 300 members are listed in the Nuclear Receptor Signaling Atlas (NURSA); see https://www.nursa.org/nursa/molecules/index.jsf; McKenna et al., 2009), and only a few of them (SRC1-2 and CBP) have been studied in the context of behavior control. A whole new field of investigations thus remains open. A related question that is also wide open is to identify the genes that are being transcribed by steroid binding to their cognate receptors and to describe their functional consequences.

On another front, it has been demonstrated that many effects of testosterone on male sexual behavior are mediated by its metabolite estradiol produced directly in the brain (neuroestrogens). Slow long-term genomic actions of estrogens on behavior are reasonably well understood, but it has more recently become clear that estrogens also participate in the control of male behavior through faster mechanisms initiated by their interaction with membrane receptors. It is currently difficult to assess the importance of these rapid effects of steroids on behavior and to determine how widespread they are since few examples only have been studied in some detail. How these two types of steroid actions interact is also not clear as well as why they have evolved to control complementary aspects of behavior in two different time domains?

Discovery of the rapid actions of estrogens on reproductive behaviors suggests that, in this context, estradiol displays most, if not all, functional characteristics of a neurotransmitter or at least a neuromodulator and thus can regulate short-term changes in behavior. This notion is further supported by the discovery of rapid regulations of the activity of the estrogen-synthesizing enzyme, aromatase. Additional research on the functional significance and the cellular mechanisms underlying such rapid effects of steroids is now required to evaluate their overall importance in the control of reproduction.

Acknowledgments

Writing of this chapter and fractions of the research described in it were supported by grant MH 50388 from the National Institutes of Health.

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