Androgens and male behavior

Molecular and Cellular Endocrinology 198 (2002) 31 /40 www.elsevier.com/locate/mce

Androgens and male behavior Louis J.G. Gooren a,*, Frank P.M. Kruijver b a

Department of Endocrinology, Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands b Graduate School of Neurosciences, Netherlands Institute for Brain Research, Amsterdam, The Netherlands

Abstract Sexual differentiation into a male or a female includes sexual differentiation of the brain. The paradigm of mammalian sexual differentiation is that in the presence of androgens (normally produced by the fetal testis) a male brain differentiation occurs, while in the absence of androgens (normal in females) a female brain differentiation follows. In the human there is a sex-dimorphism in gender identity/role, sexual orientation, sexual functioning, and in non-sexual functions, such as spatial ability, and verbal fluency. Inasmuch these properties can be studied in other mammals the effects of androgens are solidly demonstrable. In the human the evidence for androgen effects is equally plausible, evident from observations in subjects with errors in the process of sexual differentiation and in morphological studies of brain structures presumably related to these properties. But clinical observations show compellingly that other, largely unidentified, factors may modulate, or even override the effects of androgens. # 2002 Published by Elsevier Science Ireland Ltd. Keywords: Androgens; Brain; Sexual differentiation; Gender identity; Sexual orientation

1. Introduction From the beginning of this century on it has become apparent from studies in rats, mice and other lower mammals, pioneered by Steinach, that their sexual differentiation is not completed with the differentiation of the external genitalia into either male or female, the traditional criterion to label them as male or female. Also the brain undergoes a sexual differentiation which can be demonstrated neuroanatomically. It expresses itself in sex-dimorphic sexual behavior (such as copulatory positions) but also in sex-dimorphic nonsexual behavior such as aggression, defense of territory, and caring for the young. The paradigm of this step in the sexual differentiation process of lower mammals is similar to the previous ones: in the presence of androgens (normally produced by the testis of the male fetus) a male brain differentiation follows, while in the absence of androgens (as is the normal situation in females) a female brain differentiation results. In other words, the

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same hormones that determine the nature of the genitalia, will at a later stage of development also program the brain. This process has been termed the organization, the ‘wiring’ of the brain to prepare it for future sexual/reproductive behavior and also non-sexual behavior in agreement with the gonadal/genital status (McEwen, 1983; Arnold and Gorski, 1984). This programing laid down during the fetal period or shortly thereafter in lower mammals becomes activated by the hormones of puberty (Feder, 1984). In laboratory experiments it has been possible to transform this step in the sexual differentiation on the basis of the fact that it is androgen dependent in lower mammals. It appeared possible to induce a male copulatory pattern in laboratory animals with a female gonadal/genital differentiation and, vice versa, to induce a female copulatory pattern in a male rat by depriving it from its androgenic stimulus precisely at the window of time of the sexual differentiation of the brain (Arnold and Gorski, 1984; Feder, 1984). These observations in experimental animals have led to speculations that these mechanisms also operate in the human, and that phenomena such as transsexualism and homosexuality might result from variations in the (hormonal) program-

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L.J.G. Gooren, F.P.M. Kruijver / Molecular and Cellular Endocrinology 198 (2002) 31 /40

ing of the brain with regard to male/female (non) sexual functioning (Do¨rner, 1988). It has, however, been difficult to relate clinical syndrome with their hormonal abnormalities straightforwardly to complex human behaviors as gender dysphoria/transsexualism and homosexuality (Gooren, 1990; Gooren et al., 1990). In the human one relies for this information on the socalled experiments of nature and medicine: genetic and endocrine disorders that spontaneously occur in the human fetus or in the offspring of drug hormone-treated pregnancies which must then be related to behaviors that manifest themselves many years later in life. In view of the many intervening variables it has been difficult to obtain reliable information on the relationship between hormonal influences occurring early in life on the one hand, and complex behaviors such as gender identity/ sexual orientation, sexual and nonsexual behavior on the other hand (Gooren, 1990; Gooren et al., 1990). In the present paper, the role of androgens in four aspects of male behavior will be analyzed. 1) Gender identity, the self-experience of being male or female or in-between. 2) Sexual orientation: the experience of being attracted to partners with genital morphology of the opposite or the same sex, or to both. 3) Sexual behavior, in particular sexual interest. 4) Non-sexual behavior: patterns of behavior not exclusively male or female but with different thresholds for their manifestation in one sex or the other. Examples are sex differences in cognition such as spatial ability or verbal fluency. These are often not demonstrable at an individual level but only as group differences between the sexes. And further, androgen effects on mood.

2. Gender identity/role and sexual orientation Since times immemorial parents have assigned their newborns to that sex that the morphology of the external genitalia (as a rule unequivocally) indicated. This time-honored practice impresses as reasonable since babies appearing boys and girls at birth generally grow up to become normally functioning adult men and women. In other words, no major (paedagogic) effort has consciously to be made to raise baby boys to men and baby girls to women. Manhood and womanhood seem to be intrinsic, biologically determined, properties of boys and girls awaiting their completion by the hormones of puberty, which stress the sex differences and herald the erotosexual interaction between the two sexes. The very existence of phenomena as transsexualism and homosexuality indicates that a gender identity in agreement with other sex characteristic, and a

heterosexual orientation are not foreordained, but apparently the outcome of a developmental process, of which the mechanisms presently are virtually unknown. Transsexuals, in spite of having the normal biological characteristics of one sex, experience themselves as members of the opposite sex. Homosexuality is distinct from transsexualism in that homosexuals experience no discomfort with their bodily state of being male or female, but are able to interact erotosexually only with others of the same morphological sex. By and large the determinants of gender identity and sexual orientation are not known. Some researchers view both transsexualism (Do¨rner, 1988) and homosexuality (Do¨rner, 1988; Gladue et al., 1984) as crossing-overs of the sexual differentiation process of the brain prenatally/perinatally, inferring an analogy with the process of brain differentiation in lower mammals. It is presumed that the latter results in sexual behavior and neuroendocrine structures/functions of the opposite sex in afflicted subjects. The paradigm of brain development in the sexual differentiation process of lower mammals is similar to the previous ones: in the presence of androgens (normally produced by the testis of the male fetus) a male brain differentiation occurs, while in the absence of androgens (as is the normal situation in females) a female brain differentiation follows. It is presumed that transsexuals and homosexuals alike are neuroendocrinologically different from heterosexuals with regard to the feedback response of luteinizing hormone (LH) to an oestrogenic stimulus (as a telltale of an earlier cross-sex brain differentiation), inferring that this feedback response is of the female-type in male homosexuals and transsexuals, and, vice versa. The underlying extrapolation is that in lower mammals androgen exposure pre/perinatally leads to male brain programing and loss of the capacity to respond with an estrogen positive feedback mechanism of LH, and vice versa, non-exposure to androgens (as is normal in females) induces a female brain programing with preservation of the capacity of an estrogen positive feedback of LH (Do¨rner, 1988). Endocrinologically better designed research shows that transsexuals and homosexuals are neuroendocrinologically indistinguishable from heterosexuals with regard to the dynamics of the hypothalamic /pituitary/gonadalaxis (Gooren, 1990; Gooren et al., 1990). In addition, in females of the primate species exposure to supraphysiological androgen levels does not block the capacity to respond with an estrogen positive feedback of LH, as shown in girls with congenital adrenal hyperplasia (Reiter et al., 1975). So, there is ample evidence that the above indicated combination of androgen-induced masculinization of the brain coupled with loss of the capacity to respond with an estrogen positive feedback on LH secretion is not present in primate species.

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3. Homosexuality When rather precise mechanisms to determine hormone levels in peripheral blood became available, a number of studies found that homosexual men had lower testosterone levels than heterosexuals and that homosexual women had higher testosterone levels than heterosexual women. Other studies failed to confirm these findings (for review Gooren et al., 1990). Similar to the situation in transsexuals, the evidence that homosexuals have had an abnormal endocrine milieu prenatally/perinatally (too little androgen exposure in males and an unduly high androgen exposure in females) is debatable and largely by inference (for review: Gooren et al., 1990). It is truly exceptional that transsexuals or homosexuals have gone through an atypical endocrine episode prenatally or perinatally, either on the basis of an endocrine abnormal pregnancy or an own endocrine disease like androgen insensitivity or congenital adrenal hyperplasia, or other diseases. Family and twin studies in men (Bailey and Pillard, 1995) and DNA linkage analysis in men (Bailey and Pillard, 1995; Hamer et al., 1993) suggest that familial and genetic factors are relevant in (some) men with a homosexual orientation, but not in women. The route by which genotype could influence sexual orientation/ behavior remains to be specified. Some epidemiological findings suggest that a late birth order and a higher brother to sister ratio in the family correlate with a homosexual orientation in men; again a finding that is not easy to interpret in terms of biological mechanisms (Blanchard, 1997). The hypothesis advanced is that that the late birth order, with many male siblings born earlier, could lead to a progressive immune response of the mother to androgens and/or Y-linked minor histocompatibility (H /Y) antigens which, by maternal transfer of these immune antibodies to the fetus, could impair brain masculinization of the fetus (Blanchard, 2001). Why this mechanism would selectively impair only certain presumed androgen dependent processes, such as the brain programing, and not others, like formation of the genitalia is not explained by this hypothesis. There are vast numbers of homosexual men in whom the above features cannot be identified. Some women have a prenatal endocrine history atypical for their sex, such as exposure to diethylstilbestrol or suffer from CAH (Ehrhardt et al., 1985; Dittman et al., 1990). Some long-term follow-up studies in these women have shown that the incidence of a partial or complete lesbian erotosexuality is elevated, although the majority of subjects with an identical history did not develop a homoerotic sexuality (for review: Gooren et al., 1990; Meyer-Bahlburg, 2001). Critical analysis shows that at, present, no simple cause- and -effect relationship between the prenatal endocrine milieu and the postnatal sexual orientation can be established. In the human

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these endocrine abnormalities are apparently just one of various plausible factors that shape a person’s future sexual development while, for the larger part, these factors remain enigmatic. To date, at best, some correlations between a prenatal hormonal variable and sexual orientation can be established (Money, 1988; Wisniewski et al., 2000). Another approach is to analyze effects of androgens on the brain structures possibly related to the above mentioned categories. Research on sex differences in brain morphology has indeed yielded some interesting results. A sexually dimorphic nucleus (SDN) was described in 1985 in the preoptic area of the human hypothalamus (Swaab and Fliers, 1985). This nucleus is twice as large in men as in women. In the brains of a sample of homosexual men who died of acquired immunodeficiency syndrome, the SDN was of the same size as in control heterosexual men (Swaab and Hofman, 1990; Swaab et al., 1995), refuting the global hypothesis by Do¨rner that male homosexuals would have a female hypothalamus (Do¨rner, 1980). Recent brain studies in homosexual men did not corroborate this hypothesis either (Kruijver et al., 2000, 2001). The SDN of the hypothalamic preoptic area (SDN / POA (in humans also termed: interstitial nucleus of the anterior hypothalamus 1 [/INAH-1]) has been shown to be sexually dimorphic in the rodent and human brain (Gorski et al., 1978; Bleier et al., 1982; Swaab and Fliers, 1985; Swaab and Hofman, 1988; Hofman and Swaab, 1989). But the sexual dimorphism in volume and neuron number of this structure in the human has not been confirmed by others (Allen et al., 1989; LeVay, 1991; Byne et al., 2000). However, the stronger androgen receptor immunoreactivity (AR-ir) (Ferna´ndez-Guasti et al., 2000), estrogen receptor alpha immunoreactivity (ERa-ir) and estrogen receptor beta immunoreactivity (ERb-ir) in the SDN /POA in men compared with women (Kruijver et al., 2002; Kruijver et al., submitted) is consistent with its reported sexual dimorphism (for a recent review on this topic and the other three interstitial nucleı¨ [INAH-2-4] see also Swaab et al., 2001). The SDN /POA has been implicated in certain aspects of male sexual behavior (DeJonge et al., 1989; reviewed by Swaab, 1997) but for the human solid functional data are not available. An enlarged suprachiasmatic nucleus (SCN) in volume and neuron number was found in homosexual men versus heterosexual men and women (Swaab and Hofman, 1990), and further the INAH-3 was reported to be smaller in volume in homosexual men and heterosexual women versus heterosexual men (LeVay, 1991). On the basis of these and other structural findings (Allen and Gorski, 1992; Kruijver et al., 2000) the male homosexual brain, therefore, seems to be not similar to the female differentiation in the majority of the brain areas so far

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studied, but would fit the category proposed by Magnus Hirschfeld of ‘a third sex’ (Swaab and Hofman, 1995). Recently the smaller INAH-3 in male homosexuals could, not be re-confirmed, neither by its volume nor by its neuron number (Byne et al., 2001). This group found that the INAH-3 shows only a trend to have a smaller volume in homosexual men. Byne et al. (2000, 2001) were not able to replicate any of the sex differences in the brain found by others, except for INAH-3, and more studies of the human hypothalamic anterior-preoptic area (POA) and other areas are required to further establish morphological brain differences on the basis gender and sexual orientation (cf. Swaab et al., 1992). Interestingly, INAH-3 could sometimes morphologically be identified by ERa-ir in 6 mm thick coronal sections of the human hypothalamus (Kruijver et al., 2002; Kruijver et al., submitted). Further, also the human SCN expresses androgen, estrogen and progesterone receptors (Ferna´ndez-Guasti et al., 2000; Kruijver and Swaab, 2002; Kruijver et al., 2002; Kruijver et al., submitted). The presence of sex hormone receptors in INAH-1, INAH-3 and the SCN seems at least to corroborate the idea that the interaction between sex hormones and the developing brain may have played a potential role in the establishment of the various morphological differences in relation to gender and sexual orientation (Kruijver et al., 2002; Kruijver et al., submitted). In this respect, it comes somewhat as a surprise that the human SDN /POA or INAH-1 becomes only gradually sexually dimorphic between 4 years of age and puberty (Swaab et al., 1992) when the differences in levels of sex steroids between the sexes are minimal. However, prenatal and perinatal peaks in testosterone levels in males may have programmed this nucleus to develop its sexual dimorphism only later in life (Swaab and Hofman, 1995). A similar pattern of a relatively late structural sexual dimorphic development can be found in another limbic brain area, i.e. the bed nucleus of the stria terminalis, which has a sex reversed volume and neuron number in the transsexual brain (cf. Zhou et al., 1995; Kruijver et al., 2000).

4. Transsexualism Transsexualism is the condition in which a person with apparently normal somatic sexual differentiation is convinced that he or she is actually a member of the opposite sex. It is associated with an irresistible urge to be hormonally and surgically adapted to that sex. Transsexualism cannot be explained by variations in chromosomal patterns, or by gonadal, genital or actual hormonal anomalies (Gooren, 1990). Neither is there an indication that genetic factors play a role; there is no familial clustering (Gooren, 1990).

In the presence of androgens (normally produced by the testis of the male fetus) a male brain differentiation occurs, while in the absence of androgens (as is the normal situation in females) a female brain differentiation follows. Whether this hormonal mechanism is decisive in the formation of gender identity in the human has seriously been questioned (Gooren, 1990). Follow-up studies of subjects with a congenital endocrine disease or abnormal hormone exposure during pregnancy have been unable to link androgen exposure to the development of a male gender identity and absence of androgen exposure to the development of a female gender identity (Gooren, 1990). This has led to a theory that the main factor in the formation of gender identity is assignment of sex postnatally or shortly after birth followed by a consequent rearing in that sex (Money and Ehrhardt, 1972). Wilson (1999) has convincingly argued for a significant contribution of prenatal androgen exposure to the formation of gender identity, mainly on the basis of findings in subjects with 17bhydroxysteroid dehydrogenase 3 and steroid 5a-reductase 2 deficiencies who, not rarely, later in life appear to have a male gender identity after having been originally assigned to the female sex on the basis of the appearance of their female looking genitalia. The phenomenon of transsexualism seems to question the absolute necessity of a proper androgen stimulus for the formation of male gender identity and, conversely, its absence for the formation of a female gender identity. In transsexuals there is at present no well documented evidence of an abnormal endocrine milieu prenatally that could acceptably explain their condition. The endocrine evidence that androgens are significant for the formation of male gender identity is convincingly presented by Wilson (1999), but he indicates also that it is unlikely to be the sole determinant. In cases of transsexualism the other determinant(s) could be so powerful that they override the effects of androgens (or its absence in the case of females) on the formation of gender identity. Research on the brains of male-to-female transsexuals has found that regardless of sexual orientation the sexual differentiation of one brain area that belongs to the limbic system (the bed nucleus of the stria terminalis central part or BSTc) shows a female pattern in volume and neuron number (Zhou et al., 1995; Kruijver et al., 2000) and a male pattern in volume and neuron number in the first ever studied brain so far of a female-to-male transsexual (Kruijver et al., 2000). These sex reversed findings of the BSTc in the transsexual brain are, thus, related to gender identity and may lead to a concept of transsexualism as a form of intersex, where the sexual differentiation of the brain is not consistent with the other variables of sex: chromosomal pattern, nature of the gonad and of internal/external gonads. So it could be argued that transsexualism is a sexual differentiation disorder of the brain. In this regard it may be of interest

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to note that the BSTc is an androgen and estrogen sensitive area evidenced by the expression of ARs and ERs (Ferna´ndez-Guasti et al., 2000; Kruijver et al., 2002; Kruijver et al., submitted). Therefore, it cannot be excluded that at the levels of interaction between sex hormones and the developing brain, the sexual differentiation of the brain might have taken a different course in transsexuals (Zhou et al., 1995; Kruijver et al., 2000). The findings by, that the BST area becomes only gradually sexually dimorphic (not earlier than until young adulthood) might suggest that, in addition to sex hormones, also other, so far unidentified, determinants may be involved in its sexual dimorphic development. While, traditionally, the attention has focused on hormonal factors, genetic factors may play a role as well (Kruijver et al., 2000). An example of a genetic factor is the possible local ‘limbic’ involvement of sex determining genes (Reisert and Pilgrim, 1991) like the SRY gene, which is known to be transcribed in the male brain but not in the female brain of control subjects (Mayer et al., 1998). Failure of expression of e.g. SRY gene function in the BSTc and other connected parts of the limbic system might contribute to the failure of the sexual differentiation process into a male direction in male-to-female transsexuals. Whatever the underlying mechanisms are, morphological evidence argues strongly for a disturbance of the sexual differentiation process in the phenomenon of transsexualism (Zhou et al., 1995; Kruijver et al., 2000).

5. Adult male sexual functioning Androgens are necessary, though not the single factor in normal sexual behavior in men. Clinicians have long been impressed with the influence of androgen replacement on the sexual functioning of androgen-deficient men but its scientific proof is of recent date. Most data have been obtained in studies of androgen deficiency (often on the basis of withdrawal of androgen administration and replacement) (Bancroft and Wu, 1983; Bancroft, 1988; Anderson et al., 1992; Alexander et al., 1998). These studies show consistently that within 3/4 weeks of androgen withdrawal there is a decline in sexual interest and in self-initiated sexual activity (Bancroft, 1988). A sexually active partner may be a factor in prolongation of sexual activity beyond this period (Bancroft, 1988). In men without partners masturbation follows the above pattern of sexual interest. In most men the ejaculatory capacity is profoundly decreased after androgen withdrawal affecting sexual activity in its own right. These changes are reversed within seven to 14 days after reinstating androgen treatment (Bancroft, 1988). The distinction between sexual interest (libido) and erectile function has helped considerably to clarify the

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role of androgens in male function (Rowland et al., 1987). It has been claimed that spontaneous erections, particularly those that occur during sleep nocturnal penile tumescence (NPT), and probably fantasy-induced erections are androgen-dependent, whereas, erections in response to more explicit erotic (e.g. visual or tactile) stimuli are relatively androgen-independent (Bancroft and Wu, 1983). These early studies addressed maximum increase in penile circumference as the only parameter, but more recent work suggest that that androgens do affect penile responses to erotic stimuli with regard to duration of response, degree of rigidity and speed of detumescence (Carani et al., 1996). In men the principal target of androgen appears to be sexual interest or appetite (Bancroft, 1988). Androgen might enhance the persistence of attention to eroticism, which, in turn, might affect sexual behavior. While the main effect of androgens on male sexual functioning and erections is on the central nervous system, there is now also evidence that androgens act on nitric oxide synthase in the corpus cavernosum, essential for smooth muscle relaxation in penile erection (Mills and Lewis, 1999). The blood level of testosterone critical for normal male sexual functioning has been difficult to assess. Testosterone replacement studies have found that critical values differ among individuals, but mostly 60/ 70% of the reference values of eugonadal men were sufficient for restoration of sexual functioning (Gooren, 1987; Bancroft, 1988; Buena et al., 1993). From this it follows that in men with sexual dysfunction and normal androgen levels, additional testosterone is likely to be of no help, although a short-lived beneficial effect from additional testosterone in eugonadal men who complained of lack of sexual interest has been found (Anderson et al., 1992) which was confirmed in men receiving high doses testosterone in a male contraceptive study (Alexander et al., 1998), but the follow-up was limited to 6 weeks. The evidence that long-term high testosterone levels enhance male sexual functions is lacking. In general it has been difficult to establish a relationship in men between circulating testosterone levels (above a certain therapeutic threshold) and levels of sexual interest, responses, or behavior (Buena et al., 1993; Salehian et al., 1995). Administration of an aromatase inhibitor to testosterone-treated orchiectomized monkeys has a negative effect on their sexual interest (Zumpe et al., 1993). In summary, it is certain that androgens are powerful modulators of the biochemistry of peripheral structures related to sexual functioning (prostate, seminal vesicles, and the NO production in the penis) and the brain, thus, modulating behavior. Their effects are strongly intertwined with idiosyncratic aspects of the person concerned, they enhance sexual motivation in men, be it a heterosexual, homosexual or paraphilic man.

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6. Paraphilias and sex offences Persons with a paraphilia are compulsively responsive to and dependent on an unusual and often personally or socially unacceptable sexual stimulus for sexual arousal and orgasm. Paraphilias are extremely variable and range from the socially intolerable, e.g. lust murder or pathological sadism, to the self-destructive, e.g. asphyxiophilia (self-strangulation with asphyxiation), to socially harmless foibles, e.g. arousal by sexually explicit stories or pictures (Money, 1986). There is no known correlation between paraphilic behavior and an endocrine condition, past or present, in either males or females. Comparison of testosterone levels of violent rapists with those of non-violent sex offenders has yielded contradictory results (for review: Gijs and Gooren, 1996). There are some case reports of men with Klinefelter’s syndrome with low testosterone levels and limited virilization, with a negative impact on their physical self-image and self-esteem, showing aggressive behavior towards women. Testosterone treatment virilized their physical appearance and improved their interactions with women (Nielsen et al., 1988). Paraphilias are rare in women and occur predominantly in men. As a result they have been linked to androgens. The socially intolerable paraphilias (such as rape, exhibitionism, and pedophilia) may bring persons in conflict with the law and (forensic) medicine may play a part in pharmacological interventions aiming to help those paraphiliacs who have difficulties to control their self-destructive or socially unacceptable sexual behavior. As in normal persons, in paraphilias testosterone lowers the threshold of occurrence of erotosexual imagery and sexual activity; it has no effect on the contents of the imagery (Money, 1986; Gijs and Gooren, 1996). Therefore, drugs that interfere with androgen production and/ or action may be of benefit, particularly for those paraphilias characterized by intense and frequent sexual desire and arousal. Some forms of paraphilias are not so much characterized by sexual desire but are obsessivecompulsive disorders, impulse control disorders or are acted out in depressive mood states. These forms do not respond well to antiandrogenic pharmacological intervention but can be successfully treated with psychotropic drugs as the modern antidepressants (Gijs and Gooren, 1996).

7. Androgens and cognitive abilities Several studies indicate that men generally outperform women on visuospatial tasks and women outperform men on verbal fluency and perceptual speed tasks (Kimura, 1996; Alexander et al., 1998; Halpern and Hilsdale, 2000). There is little doubt that many addi-

tional factors influence these abilities such as education, experience, environment and cultural background. It is further of note that the scores on the relevant tests to measure these abilities show a considerable overlap between the sexes. There is evidence suggesting that these sex differences are prenatal/perinatal in origin. Adult men with idiopathic hypogonadotropic hypogonadism have impaired visuospatial ability; testosterone administration at the time of puberty does not improve their ability (Hier and Crowley, 1982). Conversely, women with congenital adrenal hyperplasia have a more male type cognitive pattern. This assumption seems credible since prepubertal children already show this sex difference in cognition (Resnick et al., 1986; Karadi et al., 1999) It is inferred that androgens in very early development (prenatally or perinatally) establish certain neural pathways (‘organizational effects’) facilitating these behaviors later in life upon exposure to testosterone (‘activational effects’). This notion seems to be contradicted by our studies in transsexuals undergoing cross-sex hormone treatment. First of all, before cross-sex hormone administration results of cognition tests in male to-female transsexuals were similar to those of men, and, vice versa, results in female-to-male transsexuals were not significantly different from those in women. Administration of testosterone to female-to-male transsexuals improved their visuospatial ability and impaired verbal fluency (Van Goozen et al., 1995). Conversely, antiandrogen
/estrogen administration to male-to-female transsexuals impaired spatial ability and improved verbal fluency. In a more detailed study these results could not be fully replicated. The effects of androgens on improvement of spatial ability were robust, were present after 3 months of androgen administration, had not further increased after 10 months of androgen administration, and were still present when androgen had been withdrawn for 5 weeks (Slabbekoom et al., 1999). In this replication study androgen administration to female-to-male transsexuals did not impair verbal fluency, neither were their effects of antiandrogens
/estrogens on spatial ability and verbal fluency in male-to-female transsexuals. Studies in hypogonadal men receiving androgen replacement have not unequivocally shown that their spatial abilities improve (Hier and Crowley, 1982; Alexander et al., 1998) though some studies have found positive effects (Silverman et al., 1999). Correlational studies between plasma levels of testosterone and cognitive abilities have yielded mixed results: a linear relation, no relation and a curvilinear relation. The latter is the most frequent finding: increasing very low testosterone levels to low-normal improves spatial ability and impairs verbal fluency while an increase of testosterone from low-normal to high has the converse effect: improving verbal fluency and impairing spatial ability (Halpern and Hilsdale, 2000). The explanation

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offered for this observation is that high circulating testosterone levels generate high estrogen levels which subsequently exert these effects on cognitive abilities. It has indeed been found that in healthy elderly men estrogen levels are positively associated with verbal fluency (Carlson and Sherwin, 2000). Recent neuroanatomical distribution studies have revealed that brain areas that associated with cognition, such as the cholinergic nucleus basalis of Meynert complex (NBMC) and mamillary body complex (MBC), are androgen and estrogen sensitive brain areas. These brain areas express ARs and ERs in a gender dependent way which, however, appeared to be dependent on circulating levels of sex hormones rather than on sexual orientation or gender identity (Ferna´ndezGuasti et al., 2000; Kruijver et al., 2001, 2002; Kruijver et al., submitted). These findings seem to underline the potential stimulatory (neuroprotective) dynamic effects of circulating sex hormones on specific brain areas that are involved in cognition. This notion is moreover supported by the finding of a strong decrease of AR-ir in the MBC of five old gonadally intact male subjects (Kruijver et al., 2001), which might be a reflection of decreased circulating levels of androgens during aging (Sternbach, 1998; Seidman and Walsch, 1999). Protective actions of androgens on neurons (Ahlblom et al., 1999; Hammond et al., 2001; Pike, 2001) and on memory loss (Adinof et al., 1993; Carlson et al., 1999) have been described. It may in this regard be of interest to investigate the possible neuroprotective effects of androgens in age-related diseases in men, in a similar way as has been and is done for estrogen replacement therapy in postmenopausal women with reported beneficial effects on physical status, mood, cognition and the prevention of Alzheimer’s disease (McEwen, 1999; Ohkura et al., 1994; Tang et al., 1996), although the latter certainly requires more investigations (cf. Kruijver et al., 2001). Cognition is always subject to a large number of potential confounding factors (e.g. experience, education, training) which should as much as possible be carefully taken into account for when studying the effects of e.g. androgen replacement therapy on cognition in elderly men. In summary, the weight of evidence suggests that androgens play a role in cognitive abilities in men, particularly in the expression of spatial abilities. Relevant studies (with disparate methodologies, tests, and populations studied) do, however, not (yet) provide a clear picture of the effects of androgens on various aspects of cognition and how this can be linked to specific androgen sensitive brain areas. Neuroanatomical mapping studies of the human brain combined with functional MRI studies and cognitive tests may be very powerful approaches in the nearby future to address specific androgen mediated effects on cognition in health and disease.

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8. Androgens and mood Testosterone-deficient men show irritability, dysphoria, and fatigue, and testosterone replacement improves energy levels, mood and quality of life (Wang et al., 1996; Rubinow, 1996). Subsequently, the question has been addressed whether men with a psychiatric diagnosis of depressive illness have lowered plasma testosterone levels (Seidman and Walsch, 1999). The findings have not been consistent. Lowered testosterone levels might be secondary to a hyperactivity of the hypothalamo-pituitary-adrenal (HPA-) axis leading subsequently to a suppression of the hypothalamo-pituitary-testicular axis with a lower output of LH and testosterone (Schweiger et al., 1999). Administration of testosterone to men with depressions and with low plasma levels of testosterone have not universally led to an improvement of psychiatric symptoms. This may be due to different operational definitions of the depressive state of these patients and to the different treatment modalities. A recent randomized placebocontrolled trial did not find an effect of testosterone superior to that of placebo; the latter was followed by a 40% positive response (Seidman et al., 2001). Clearly, more placebo-controlled trials are needed to fully assess the role of various androgens in the regulation of mood. Androgen replacement therapy in elderly men might moreover improve or restore age related disturbances in circadian SCN function, which has been implicated in the regulation of mood (Zhou et al., 2001; Kruijver and Swaab, 2002). Therefore, it seems of importance that future androgen replacement therapy trials are designed to monitor mood as well as SCN related functions, such as sleep and HPA-axis activity (see also Kruijver and Swaab, 2002).

9. General conclusions Similar to the situation in other mammals, the role of androgens in the sexual differentiation process of the human is manifest. With regard to the effects of androgens on the sexual differentiation process of the brain and later in life on sexual functioning the situation is complex. Effects on androgens can be identified, both in clinical studies of subjects whose sexual differentiation process has not followed the normal track and very clearly in morphological studies of sex-dimorphic structures of the brain. In the latter, the abundance of androgen (and estrogen) receptors argues in favor of effects of androgens (and of estrogens) on these structures. But whatever the prenatal/postnatal androgen effects on the brain are, clinical observations show that other factors are likely to be superimposed on these androgen effects. In lower mammals androgens have a robotlike on- and -off effect on sexual functioning.

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While androgen effects are still recognizable in primates, they are no longer the sole factor in their sexual functioning. However, what these other factors are is not yet very clear. Clinical studies offer leads, but these leads are, scientifically speaking, at a descriptive level; the human situation usually allows no thorough testing of assumptions based on these leads. With regard to gender identity development effects of androgens have been convincingly demonstrated in subjects whose sexual differentiation process has not been normal (for review: Wilson, 1999), and also morphological studies point to androgen/estrogen sensitivity of sex dimorphic brain structures, which is particularly evident in the sex-reversed BSTc in the transsexual brain (Zhou et al., 1995; Ferna´ndez-Guasti et al., 2000; Kruijver et al., 2000, 2001). The development of gender identity in some intersexed subjects challenges the idea of androgens as sole factors. The latter is particularly true for transsexuals. Apparently, a male gender identity may develop without evidence of an androgenic stimulus in female-to-male transsexuals. Conversely, the probable effects of androgens on gender identity formation might in male-to-female transsexuals be overridden by as yet unknown factors. The above reasoning applies largely also to sexual orientation. Morphological studies of the human brain to date do not point to androgens as a strong factor at the receptor level, but clinical observations support a role for androgens. Similar to the situation in transsexuals, there is no evidence of a difference in androgen exposure/ action in homosexual subjects compared with their heterosexual counterparts, and it remains mysterious what factors can override plausible androgen factors in the development of sexual orientation. Androgens exert a profound effect on sexual interest, less so on erectile function, though the two are interrelated. There is no demonstrable effect on the idiosyncrasies of sexual likes and dislikes (Money, 1986). The latter applies also to parphilias. A number of nonsexual mental functions are influenced by androgens, possibly by prenatal programing effects but also by actually circulating testosterone levels. Men perform better on tasks requiring spatial abilities and women outperform men on verbal fluency. Particularly spatial abilities appear to be influenced by circulating androgen levels. It is of note, however, that these sex differences are group differences. Education, training and experience are all factors affecting spatial ability and verbal fluency. Testosterone replacement in hypogonadal men has mood elevating effects. Men with clinical depressions are often found to have lowered testosterone levels. Testosterone administration to these men did not produce unequivocally positive effects, maybe because their low testosterone levels were secondary to their state of depression rather than at the root of their mood state.

The final conclusion must be that in the human androgens act on the brain in an interface of nature and nurture, which is not only the case in humans but also in other primates (Wallen, 2001). A contribution of androgens to the establishment of gender identity and sexual orientation, and to behavior and cognition is very plausible, but the human experience confronts us with enigmatic factors which probably can override or compete with androgen mediated effects during sexual brain development. With the focus on sex steroids, neurogenetic studies may have been undervalued in this regard. Both refined clinical observations and morphological studies of the brain and their relations with sex steroids will advance our knowledge.

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