What science has to say on the etiology of homosexuality (Part 3): How much influence nurture has on sexuality?

In the final installment of this 3 part series, we shall examine closely the effect nurture has on sexuality. Or more specifically, that question we ask is this: can you actually raise a boy in such a way that he becomes sexually attracted to men when he grows up?

To find out, let's take a look at what happens when boys who were born genetically male (that is they possess a Y chromosome) but unfortunately had gender reassignment surgery to be raised as females due to medical reasons.

The following study from the Archives of pediatrics & adolescent medicine is a classic, in my opinion.

What science has to say on the etiology of homosexuality (Part 2): Insights from Congenital Adrenal Hyperplasia

In the previous post, we have gained an insight as to how steroid hormones can influence the development of certain parts of the brain across a number of different animal species. These differences also seem to be well correlated with behavioural sex or brain sex.

In this post, we will explore a condition found in humans that was mentioned in the first post- Congenital Adrenal Hyperplasia. We will also explore the effects of this condition have on the behaviour of children.

Before we start, here's some background information on Congenital Adrenal Hyperplasia. Congenital Adrenal Hyperplasia is a genetic condition which causes excessive growth of the adrenal glands. Adrenal glands plays a part in regulating the levels of sex hormones, glucocorticoids (the kind of stuff you use to stop an itch) and mineralocorticoids (involved in salt and water balance). So, what are the effects of this condition on the choice of toys in children? Find out by reading the paper from the Journal of Clinical Endocrinology & Metabolism below.

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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 11 5119-5124
Copyright © 2002 by The Endocrine Society

Original Article

Sex-Typed Toy Play Behavior Correlates with the Degree of Prenatal Androgen Exposure Assessed by CYP21 Genotype in Girls with Congenital Adrenal Hyperplasia

Anna Nordenström, Anna Servin, Gunilla Bohlin, Agne Larsson and Anna Wedell

Department of Pediatrics, Karolinska Institute, Huddinge University Hospital (A.N., A.L.), S-141 86 Stockholm, Sweden; Department of Psychology, University of Uppsala (A.S., G.B.), S-751 42 Uppsala, Sweden; and Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital (A.W.), S-171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Dr. Anna Nordenström, Department of Pediatrics, Karolinska Institute, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: anna.nordenstrom@klinvet.ki.se.

Abstract

Previous studies have shown that girls with congenital adrenal hyperplasia (CAH), a syndrome resulting in overproduction of adrenal androgens from early fetal life, are behaviorally masculinized. We studied play with toys in a structured play situation and correlated the results with disease severity, assessed by CYP21 genotyping, and age at diagnosis. Girls with CAH played more with masculine toys than controls when playing alone. In addition, we could demonstrate a dose-response relationship between disease severity (i.e. degree of fetal androgen exposure) and degree of masculinization of behavior. The presence of a parent did not influence the CAH girls to play in a more masculine fashion. Four CAH girls with late diagnosis are also described. Three of the four girls played exclusively with one of the masculine toys, a constructional toy. Our results support the view that prenatal androgen exposure has a direct organizational effect on the human brain to determine certain aspects of sex-typed behavior.

BEHAVIORAL STUDIES in children with congenital adrenal hyperplasia (CAH) are important for several reasons. They provide information that is important for the management and follow-up of patients. In addition, the influence of prenatal and neonatal hormonal factors on sex differences in behavior can be studied in girls with CAH, as these children have been exposed to elevated levels of androgens from early fetal development.

In the 1960s, Money and Ehrhardt (1) reported that girls with CAH often preferred boys’ toys and outdoor sports. Since then, several studies have shown that girls with CAH are behaviorally masculinized (2, 3, 4). Girls with CAH reached higher scores than their sisters in some cognitive parameters, such as three-dimensional rotational spatial ability, a pattern similar to that seen in comparisons of normal boys and girls (5, 6). Women with CAH showed a typical male pattern for measures of personality traits (7). In direct observational studies of toy play Berenbaum et al. (8, 9) showed that girls with CAH played more with boys’ toys and less with girls’ toys than their unaffected sisters. The results regarding the correlation of disease severity with degree of masculinization of behavior have been somewhat contradictory (2, 3, 8, 10). Berenbaum et al. (11) and our group (Servin, A., A. Nordenström, A. Larsson, and G. Bohlin, submitted for publication) have shown that childhood boy-typical interest was strongly associated with the degree of virilization of the genitalia, an indicator of prenatal androgen exposure. It has been argued that the behavioral changes in girls with CAH are the results of the parental treatment triggered by the virilization of genitalia at birth (13). On the other hand, the persistence of sex-atypical interests, activities, and careers in adolescent girls with CAH suggests that they result from the direct effects of androgens on the developing brain rather than social responses, because these girls were brought up as females (14). The vast majority of girls with CAH have a typical female gender identity (15).

CAH constitutes a family of defects in the synthesis of steroid hormones in the adrenal cortex. In more than 90% of the cases it is caused by a defect in the 21-hydroxylase gene (CYP21) (16, 17). The enzyme deficiency results in impaired synthesis of cortisol and aldosterone. The low cortisol level results in increased production of ACTH by the pituitary, which causes hyperplasia of the adrenal glands and increased synthesis of steroid precursors, resulting in high androgen levels. The androgen excess is present from early embryogenesis and results in varying degrees of virilization of the external genitalia in girls depending on the degree of enzyme deficiency. In severe forms, the virilization may result in uncertainty in gender assignment at birth, and the sex of some girls is initially designated as male.

The molecular genetics of 21-hydroxylase deficiency have been studied extensively. More than 95% of the patients are homozygous or compound heterozygotes for any of nine different point mutations or deletion of the CYP21 gene. With very few exceptions there is a good correlation between the CYP21 genotype and disease severity (18, 19, 20). Deletions or mutations that completely abolish enzyme activity are referred to as null mutations. Patients who are homozygous for null mutations have the most severe form of the disease, with salt loss in the neonatal period and severe prenatal virilization of external genitalia in girls. The I2 splice mutation is slightly less severe; some homozygous patients are not affected by salt loss. The I172N mutation is associated with varying degrees of virilization of external genitalia, but only about 10% of patients with this genotype show signs of salt loss. The V281L mutation is even milder and is associated with nonclassical CAH without virilization of external genitalia at birth. Untreated, these patients develop symptoms of androgen excess later in life, such as accelerated growth rate, hirsutism, or infertility. At birth, most children with CAH are diagnosed either because of clinical signs or in neonatal screening programs. The treatment consists of glucocorticoid and mineralocorticoid substitution that decreases/normalizes ACTH levels and thereby androgen production. Corrective surgery on the external genitalia is performed when needed (17).

In this study we wanted to investigate further the possible influence of disease severity, i.e. the degree of fetal androgen exposure, on toy play and toy preference in girls with CAH. For this purpose we took advantage of the possibility of determining the degree of 21-hydroxylase deficiency by CYP21 genotyping. There is a good genotype-phenotype correlation (18, 19, 20), and genotyping is also a more objective way to measure disease severity compared with other methods, such as Prader score or classification according to salt loss. Furthermore, we have previously shown that at birth the level of one of the hormones preceding the enzyme block, 17-hydroxyprogesterone, is correlated to the CYP21 genotype (21). This indicates that the androgen level, to which the fetus is exposed, during intrauterine life is related to the genotype. We measured sex-typed play behavior in a structured play situation. To assess parental influence, toy play was studied when the child was playing alone as well as when a parent was present. A possible influence of postnatal androgen exposure on toy preference was studied in four children with late diagnosis who had been untreated until 3–6 yr of age.

Subjects and Methods

Study population

All families in Sweden with girls with CAH between 1 and 10 yr of age were contacted during 1997–2000 and asked to participate. Five families did not agree to participate. A total of 40 girls with CAH in 35 families were included in the study. CYP21 mutation analysis was performed in 39 of these children. The 4 girls who had been diagnosed prenatally and treated with dexamethasone in utero until term were excluded from this study. Four girls had been missed by the screening and were diagnosed late, at 3–6 yr of age. Healthy girls matched for age ±2 months on a case by case basis were used as controls.

Mutation analysis

CYP21 mutation analysis was carried out using allele-specific PCR from genomic DNA prepared from venous blood samples (22). This detects the 95% of alleles that carry any of the common pseudogene-derived mutations. Additional rare alleles were characterized by direct DNA sequencing (23). The genotypes were divided into four groups with respect to the severity of the mutation of the allele with the mildest mutation: null, I2 splice, I172N, and V281L (see Tables 1 and 2). We were able to obtain a sample for CYP21 mutation analysis from all but one child.

View this table: [in this window] [in a new window] Table 1. CYP21 genotypes of the 31 girls with CAH diagnosed in the neonatal period

View this table: [in this window] [in a new window] Table 2. CYP21 genotypes of the four girls with CAH diagnosed later in childhood

Toy play

In a structured toy play situation, 10 different toys that had previously been defined as masculine, feminine, or neutral for children in the presently employed ages were used (9, 24, 25, 26, 27). Feminine toys included a doll with a blanket and feeding bottle, Barbie and Ken dolls, a teapot with four cups, and a female doll’s head with brush, comb, and mirror. Masculine toys were a bus, a garage with four cars, a constructional toy (Lincoln logs), and two fighting figures. Neutral toys were a sketchbook and a deck of cards. The toys were arranged in a standard order in a semicircle on the floor in the homes of the children, with every other toy being masculine and feminine and the neutral toys in between. The child was asked to sit in the middle of the semicircle and was videotaped for 7 min when playing alone and for 7 min when playing with her parent. The play order, alone vs. with a parent, was alternated. The families with a girl with CAH could choose whether the mother or the father would participate (two fathers participated). The control families were matched for these factors. The tapes were then scored for the number of seconds that the child played with the different types of toys. Play was defined as the child touching the toy. The person who scored the tapes was blind to the status of the child on the tape. At the end of the visit the children were given a toy to keep as a present. They were able to choose between a doll (feminine), a car (masculine), and a ball (neutral).

Statistical analysis

The girls with CAH, regardless of disease severity, and the controls were compared with respect to toy play using the Mann-Whitney U test. The relationship between toy play and genotype was analyzed by means of the Spearman rank order correlation coefficient. The groups were graded according to enzyme activity, with the null genotype group being the lowest and the controls the highest. The girls with late diagnosis were compared with respect to toy play with other girls of the same age, both controls and girls with CAH and early diagnosis, using the Kruskal-Wallis and Mann-Whitney U tests. Intraindividual comparison of toy play with and without a parent was analyzed using Wilcoxon’s signed ranks test. Fisher’s exact test was used to compare the choice of toy to keep as a present. The choice of doll vs. one of the other toys and the choice of car vs. one of the other toys was tested for the CAH girls with early diagnosis (as a group) compared with the controls. The choice of doll vs. one of the other toys was tested for the CAH girls with late diagnosis compared with girls with CAH and early diagnosis of the same age as well as with girls with the same mutations and early diagnosis regardless of age. SPSS computer program 10.1 (SPSS, Inc., Chicago, IL) was used for all the statistical analyses. The level was set at 0.05.

The study was approved by the ethical committee of the Karolinska Institute (Stockholm, Sweden). Informed consent was obtained from all participants.

Results

The 31 girls with early diagnosis and start of treatment in the neonatal period were divided into 4 genotype groups (Table 1). Three of the groups were of equal size, and 1 was smaller, comprising only 3 children. The mean Prader scores for the different genotype groups are also listed in Table 1. In addition, there were 4 girls with late diagnosis and treatment (Table 2). Two of them belonged to the I172N genotype group and had started treatment at 3 yr of age. The other 2 belonged to the V281L genotype group and had been treated since 6 yr of age (Table 2).

We compared toy play for CAH and control girls when the children were playing alone. As expected, there was a significant difference in toy play for masculine toys between the girls with CAH as a group and the controls (P = 0.017). In addition, we found a significant correlation between the degree of disease severity as measured by CYP21 genotypes and the amount of time the CAH girls spent playing with masculine toys (r = -0.39; P = 0.002; Fig. 1A). The correlation was also significant when the controls were excluded (r = -0.41; P = 0.024). For play with neutral toys the correlation was significant, but less striking (r = 0.27; P = 0.036), and the coefficient when the controls were excluded was r = 0.34; P = 0.064 (Fig. 1C). The correlation was not significant for play with the feminine toys (Fig. 1B; r = 0.20; P = 0.129 with the controls included and r = 0.12; P = 0.53 with the controls excluded from the calculation). In conclusion, the milder the disease, i.e. the higher the enzyme activity and therefore the lower the androgen level, the less time was spent with the masculine toys, whereas neutral toys were increasingly preferred. As shown in Fig. 1, the girls with CAH in all of the genotype groups played more with the masculine than with the feminine or neutral toys. The control girls also played more with the masculine toys than with the feminine ones (Fig. 1 and Table 1).

View larger version (13K): [in this window] [in a new window] Figure 1. Amount of time (seconds) that the CAH girls played with masculine toys (A), feminine toys (B), and neutral toys (C) in relation to the CYP21 genotype groups. The box plot shows the median values and the 10th, 25th, 75th, and 90th percentiles. The extreme values are denoted with an asterisk, and the outliers are denoted with a circle.

Figure 2 shows the difference in the amount of time that the girls with early diagnosis in the different genotype groups spent playing with the different types of toys alone compared with when a parent was present. Positive values thus represent time spent with the toys when the child was alone, whereas negative values indicate toys that were chosen more often when the parent was present. For the control girls the influence of the parents was negligible, the median difference with or without a parent was 10 sec for masculine toys. As a group, the CAH girls played somewhat less with the masculine toys when the parent was present (the median difference was 39 sec), but this difference was not significant (P = 0.12). When considering the different genotype groups separately, the girls seemed to be increasingly diverted from masculine toys by the parents with decreasing disease severity.

View larger version (19K): [in this window] [in a new window] Figure 2. The mean difference in the amount of time that the girls in the different genotype groups played with the different types of toys when they played alone compared with when they played with a parent (bars represent time playing alone minus time playing with parent). Positive values thus represent preference for toys when the child was alone, whereas negative values indicate toys that were chosen more often when the parent was present.

The results of the study of choice of toy to keep as a present are shown in Fig. 3. The girls in the null group chose a car more often than a ball, whereas the girls with I2 splice chose a car or a ball equally often. Some of the girls with I172N chose a doll. None of the three girls with V281L and two of the control girls chose a car. The observed difference between girls with CAH and controls in choice of car and choice of doll was significant (P = 0.001).

View larger version (18K): [in this window] [in a new window] Figure 3. Choice of toy to keep as a present in the different genotype groups.

There were four girls in this study with late diagnosis (Table 2). The two girls diagnosed at 3 yr of age with the I172N mutation played more with the masculine toys than the girls with the same mutation but early diagnosis and treatment, as shown in Fig. 1A and Tables 1 and 2. They both played exclusively with the constructional toy. In the mildest group, mutation V281L, the two girls with late diagnosis (at age 6 yr) showed different toy play patterns. One girl, studied at 8 yr of age, played in a way similar to that of the girls with the same mutation treated early on. The other girl, studied at age 10 yr, played with the constructional toy the whole time when she played alone. To substantiate these observations, the four girls with a late diagnosis were compared with girls of the same age, 8–10 yr, both controls (n = 12) and girls with CAH diagnosed in the neonatal period, including the severe forms (n = 8). Play with the constructional toy was analyzed separately. The girls with late diagnosis played significantly more with the constructional toy (median, 420 sec; range, 64–420 sec) than control girls of the same age (median, 13 sec; range, 0–316 sec; P = 0.01). There was a statistical trend that the girls with late diagnosis played more with the constructional toy than the other girls with CAH of the same age (median, 420 sec; range, 64–420 sec vs. median, 218 sec; range, 0–420 sec; P = 0.1). In addition, we compared the choice of toy to keep as a present between the different groups of girls of this age. Three of the girls with late diagnosis chose a doll, and the fourth chose a ball. The controls were more likely to choose a ball (car, n = 1; ball, n = 10; doll, n = 1). The CAH girls with early diagnosis chose a car and a ball equally often (car, n = 4; ball, n = 4). The difference in choice of doll between the patients with late and early diagnosis was significant (P = 0.018). The choice of doll as a toy to keep as a present was also compared for the patients with the I172N or V281L mutations regardless of age or late (n = 4) vs. early diagnosis (n = 12). The girls with late diagnosis chose a doll more often (P = 0.052).

Discussion

We found that girls with CAH played more with masculine toys than controls, which is in line with the findings of previous studies (9, 3, 28). In addition, this study is the first to correlate behavior to CYP21 genotype, which is known to reflect the degree of disease severity and thus the degree of fetal androgen exposure. We found a dose-response relationship between disease severity (i.e. degree of fetal androgen exposure) and degree of masculinization of toy play and preference. This finding supports a biological basis for the differences in play behavior between CAH girls and unaffected girls. It has been argued that the masculinization of play behavior that has been seen in girls with CAH can be attributed to parental influence due to expectations of a more masculine behavior in these girls triggered by the virilization of external genitalia at birth (13). Our results do not support this view. When a parent was present, there was no difference in toy play for the girls with the most severe form of CAH, and the girls with less severe forms of CAH played less, rather than more, with masculine toys.

The possible effects of prenatal vs. postnatal androgen exposure on behavior have been discussed. Hormones are considered to affect behavior in different ways (29), namely by having an early organizational effect that takes place during certain critical periods of development and/or a later activational effect, for instance during puberty. Maze learning in rodents, for example, is dependent on organizational influences of androgens (29). In this study the results in girls with CAH diagnosed and treated at an early age favor a prenatal, organizational effect of androgens during the development of the central nervous system. On the other hand, even though there are few observations, our results for the girls with CAH diagnosed late indicate that postnatal androgens may have effects as well. The girls with late diagnosis all had less severe forms of CAH and can be assumed to have been exposed to lower levels of androgens in utero. They were diagnosed at 3 or 6 yr of age and therefore had an overproduction of androgens during the first years of life. Three of these girls played with the Lincoln logs the whole time when they played alone. This result was significantly different from the result for the controls in the same age group. In addition, there was a statistical trend that the girls with late diagnosis played more with the constructional toy compared with CAH girls of the same age but with early diagnosis (P = 0.1). These results are intriguing because they contrast with the results in the choice of toy to keep as a present. It was striking that 3 of the CAH girls with late diagnosis preferred the doll, and only 1 chose the ball to keep, while among the girls with the same mutations, I172N or V281L, and early diagnosis 2 of 12 chose a doll. This raises questions concerning the possibility that prenatal and postnatal androgen exposure may affect different aspects of cognitive development.

In a study by Berenbaum et al. (11) a much less marked effect of postnatal than prenatal androgen exposure in masculinizing play behavior was shown. This is in agreement with the results of our studies. However, in their study the effect on spatial ability or play with constructional toys was not studied specifically. The possibility of a postnatal effect of androgens on central nervous system development has implications for treatment during the first years of life. It has been argued that the glucocorticoid dose should be kept low during the first 1–2 yr to minimize the negative effects on growth, as no effect of androgens on growth or skeletal maturation has been observed during this period (30). However, if elevated androgen levels have an effect on the developing brain, perhaps this regimen should only be used in boys with CAH.

We recognize that additional factors, such as upbringing and cultural influences, play vital roles in such complex human characteristics as behavior. We believe, however, that girls with CAH will benefit from an increased understanding and acceptance of their preferences as well as from the acknowledgment that parental expectations, if anything, tend to counteract them.

In conclusion, we have found evidence supporting the idea that prenatal androgen exposure has a direct organizational effect on the human brain so as to determine certain aspects of sex-typed behavior. In addition, our data raise questions concerning possible postnatal effects of androgens. Further studies are needed on this subject to clarify the possible sensitive periods and levels of androgens mediating these effects.

Acknowledgments

We gratefully acknowledge Sheri Berenbaum for her valuable contribution in planning this study. We are grateful to the children and their families for their cooperation.

Footnotes

This work was supported by the Swedish Medical Research Council, Grants 4792 and 12198, the Novo Nordisk Foundation, the Märta and Gunnar Philipson Foundation, the Samariten Foundation, and the Frimurare Barnhuset Foundation.

Abbreviation: CAH, Congenital adrenal hyperplasia.

Received September 21, 2001.

Accepted July 30, 2002.

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What science has to say on the etiology of homosexuality (Part 1)

I shall start a series of blogposts examining what science has to say on the etiology of homosexuality. I will not attempt to interpret the findings myself. Instead, I hope readers will be able to figure it out on their own.

I'll start off with this nice paper that i found in Nature, on the topic of sexual differentiation of the vertebrate nervous system. This paper explores the possible determiants in brain sex (as opposed to genetic, gonadal and phenotypic sex). Perhaps it might give us a better idea of how sexuality in humans develop. Much better than reading unsubstantiated claims on the net that "if you truly do not wish to see your children turn to gay relations, you shouldn't be promoting that gay sex is natural and acceptable."

Nature Neuroscience 7, 1034 - 1039 (2004)
Published online: 27 September 2004; | doi:10.1038/nn1325

Sexual differentiation of the vertebrate nervous system

John A Morris, Cynthia L Jordan & S Marc Breedlove

Neuroscience Program, Michigan State University, East Lansing, Michigan 48824, USA.

Correspondence should be addressed to S Marc Breedlove breedsm@msu.eduUnderstanding the mechanisms that give rise to sex differences in the behavior of nonhuman animals may contribute to the understanding of sex differences in humans. In vertebrate model systems, a single factor—the steroid hormone testosterone—accounts for most, and perhaps all, of the known sex differences in neural structure and behavior. Here we review some of the events triggered by testosterone that masculinize the developing and adult nervous system, promote male behaviors and suppress female behaviors. Testosterone often sculpts the developing nervous system by inhibiting or exacerbating cell death and/or by modulating the formation and elimination of synapses. Experience, too, can interact with testosterone to enhance or diminish its effects on the central nervous system. However, more work is needed to uncover the particular cells and specific genes on which testosterone acts to initiate these events.The steps leading to masculinization of the body are remarkably consistent across mammals: the paternally contributed Y chromosome contains the sex-determining region of the Y (Sry) gene, which induces the undifferentiated gonads to form as testes (rather than ovaries). The testes then secrete hormones to masculinize the rest of the body. Two of these masculinizing testicular hormones are antimullerian hormone, a protein that suppresses female reproductive tract development, and testosterone, a steroid that promotes development of the male reproductive tract and masculine external genitalia. In masculinizing the body, testosterone first binds to the androgen receptor protein, and then this steroid-receptor complex binds to DNA, where it modulates gene expression and promotes differentiation as a male. If the Sry gene is absent (as in females, who receive an X chromosome from the father), the gonad develops as an ovary, and the body, unexposed to testicular hormones, forms a feminine configuration. The genitalia will only respond to testicular hormones during a particular time in development, which constitutes a sensitive period for hormone action: hormonal treatment of females in adulthood has negligible effects on genital morphology¹.

Of the two gonadal hormones that masculinize the body, it is testosterone that also masculinizes the brain. Scientists first demonstrated this by exposing female guinea pigs to testosterone in utero, which permanently interfered with the animals' tendency to show female reproductive behaviors in adulthood². Treating adult females with testosterone had a transient effect, or none at all, on these behaviors. Early exposure to steroids such as testosterone also masculinizes brain structures. In this review, we will contrast the various mechanisms by which testosterone masculinizes the central nervous system, discuss the unknowns that remain and relate these findings to human behavior.

Apoptosis and sexual dimorphism in the nervous system
Lesions of the entire preoptic area (POA) in the anterior hypothalamus eliminate virtually all male copulatory behaviors³, whereas lesions restricted to the sexually dimorphic nucleus of the POA (SDN-POA) have more modest effects, slowing acquisition of copulatory behaviors⁴. The volume of the SDN-POA in rats is several-fold larger in males than in females (Fig. 1). Treating female rats with testosterone just before and just after birth causes the SDN-POA in adulthood to be as large as in normal males⁵, whereas castrating male rats at birth results in a smaller, feminine SDN-POA in adulthood. Thus, sexual differentiation of this nucleus resembles that of the genitalia—male hormones early in life permanently masculinize this brain region.

Figure 1. Sexual dimorphisms in the brain. (a,b) The sexually dimorphic nucleus of the preoptic area (SDN-POA) is larger in male rats (a) than in females (b) because the testes secrete testosterone during the perinatal sensitive period. After that time, testosterone has little effect on SDN-POA volume. (c,d) In contrast, the volume of the rat posterodorsal medial amygdala (MePD), which is about 1.5 times larger in males (c) than in females (d), retains its responsiveness to testosterone throughout life. (e,f) In zebra finches, the robustus archistriatum (RA) nucleus is crucial for song production and has a greater volume in males (e) than in females (f). Like the rat SDN-POA, exposure to steroid hormones early in life is essential for the RA to develop a masculine phenotype. For the RA, however, the steroids may not originate from the testes, but are rather synthesized locally in the brain itself. SCN, suprachiasmatic nucleus; 3V, third ventricle; ot, optic tract. All scale bars = 250 m. Full Figure and legend (430K)

One difference is that it is not testosterone itself, but a metabolite of testosterone, that masculinizes the SDN-POA. The enzyme aromatase, which is abundant in the hypothalamus, converts androgens (such as testosterone) into estrogens (such as estradiol). Estrogen then interacts with estrogen receptors, not androgen receptors, to induce a masculine SDN-POA. Naturally occurring cell death seems to regulate sexual differentiation of the SDN-POA (Fig. 2). There are more dying cells in the SDN-POA of neonatal females than males⁶, and treating newborn females with testosterone reduces the number of dying cells, which presumably leads to a larger SDN-POA⁶. In contrast, hormone manipulations in adulthood, after the period of naturally occurring neuronal death, have no effect on the volume of this nucleus⁷.

Figure 2. The conversion of testosterone into estrogen. In male rats, the testes secrete testosterone, which is converted into an estrogen in the brain by the enzyme aromatase. Estrogen then binds to estrogen receptors (ER) to modulate gene expression such that the SDN-POA differentiates in a masculine fashion. Steroid reduces naturally occurring cell death, resulting in more neurons surviving in males than in females. Full Figure and legend (51K)

The ferret SDN-POA also has a greater volume in males than in females due to perinatal stimulation of estrogen receptors in males, but in this species, cell death does not seem to be involved in sexual differentiation⁸. Unfortunately, Nissl stain does not reveal an SDN-POA in mice⁹, but cells containing androgen receptors demarcate a sexually dimorphic area in the mouse POA that resembles the rat SDN-POA¹⁰. If the areas are homologous, then scientists may be able to use genetic tools available in mice to discern the molecular events involved in masculinization of this nucleus.

Sexually dimorphic brain regions are not always larger in males; the anteroventral periventricular nucleus (AVPV), part of the hypothalamic system regulating ovulatory cycles, is larger in females than in males, in both mice and rats¹¹. As with the SDN-POA, sexual differentiation of the AVPV is due to the actions of aromatized metabolites of testosterone on estrogen receptors, which modulate apoptosis early in life; hormone manipulations in adults have no effect. In contrast to the SDN-POA, masculinization of the AVPV is due to testosterone-induced apoptosis, resulting in a smaller nucleus in adult males than in females¹¹. The opposing responses of SDN-POA and AVPV to testosterone indicate that the molecular makeup of the cells targeted by hormone in these two systems must differ.

The spinal nucleus of the bulbocavernosus (SNB) also relies on apoptosis for sexual differentiation. These motor neurons in the lumbar spinal cord innervate striated muscles that attach to the base of the penis¹². Male rats have more and larger SNB motor neurons than do females, and similar dimorphisms have been found in humans¹³ and green anole lizards¹⁴. Rats of both sexes have SNB motor neurons and their target muscles before birth, but these components die in females around the time of birth unless they are exposed to testosterone^{15, 16}. The effect of testosterone relies exclusively on androgen receptors. Although adult SNB motor neurons possess androgen receptors, the primary effect of testosterone is to prevent death of the target muscles, which then secondarily spare SNB motor neurons from apoptosis (Fig. 3). Thus, testosterone does not act directly on SNB neurons to keep them alive. For example, SNB motor neurons with dysfunctional androgen receptor genes are nevertheless spared from apoptosis by testosterone treatment¹⁷.

Figure 3. Before birth, the SNB system is present in both male and female rats, and motor neurons have established a functional neuromuscular junction. However, the muscles and their motor neurons die shortly after birth unless exposed to (or treated with) testosterone. In males, testosterone acts primarily to prevent the muscles from dying and this action secondarily prevents death of the motor neurons. One hypothesis is that testosterone induces the muscles to produce a trophic factor that preserves the muscles and either the same factor or an additional factor preserves the motor neurons. Full Figure and legend (75K)

Testosterone also acts on AVPV neurons, causing them to release a chemoattractant that establishes a sexually dimorphic innervation pattern¹⁸. Thus, steroids can induce one cell population to transynaptically masculinize another. Moreover, the same hormone that promotes neuronal apoptosis in the AVPV can prevent it in other systems (SDN-POA, SNB). Mice that overexpress the anti-apoptotic gene Bcl2 show reduced sex differences in both the SNB and the AVPV¹⁹, suggesting that the Bcl2 gene mediates sexual differentiation in both structures, but that testosterone modulates expression of this gene in opposite directions to either promote or prevent apoptosis.

Sexual differentiation of the brain in adulthood
Arginine vasopressin fibers innervate the septal region in rats and contribute to pair bonding and parental behavior in a variety of rodents (see accompanying review²⁰ in this issue). The extent of this innervation is greater in males than in females, but testosterone during development is not sufficient to fully masculinize the region; the hormone must also be present in adulthood²¹. The posterodorsal medial amygdala (MePD), which receives input from olfactory and pheromonal centers and is important for male sexual arousal, depends even more on adult testosterone. MePD volume is about 1.5 times larger in males than in females in rats and mice (Fig. 1c,d)^{22, 23}, and testosterone manipulations in adulthood can completely reverse this sex difference. Castration of adult male rats causes MePD volume and cell soma size to shrink to feminine proportions within 30 days after surgery and concurrently reduces male arousal to airborne cues from receptive females^{22, 24}. Conversely, treating females with testosterone for one month enlarges the MePD to masculine size²².

Testosterone also affects the SNB system in adulthood: castration of adult males, though it does not affect the number of motor neurons or muscle fibers, nevertheless causes both to shrink²⁵, leading to a loss of spinal reflexes of the penis that are crucial for reproduction³. Testosterone treatment prevents this shrinkage by acting on androgen receptors^{26, 27}, and this modulation of the SNB system probably reflects the seasonal breeding strategy of the ancestors of laboratory rodents, in which the reproductive system regressed each fall. For example, both the SNB system and the volume of MePD in male Siberian hamsters wane as reproductive systems are suppressed by short winter-like periods of light exposure in the laboratory, and will wax full again when the animals return to a reproductive condition^{28, 29}. Furthermore, MePD volume and cell soma size respond to exogenous testosterone in castrated hamsters only if they are kept in long summer-like periods of light exposure³⁰, so it appears that day length can regulate whether the brain will respond to testosterone.

Songbirds open up new possibilities
In several species of songbirds, males sing more than females, and the forebrain regions controlling song, including the higher vocal center (HVC) and the nucleus robustus archistriatum (RA), are larger in males than in females (Fig. 1e,f)³¹. In canaries, testosterone treatment of adult females, while insufficient to fully masculinize HVC and RA morphology, nevertheless enlarges these nuclei enough to induce the females to sing³². This remarkable level of plasticity in adult canaries includes the capacity to produce new, functional neurons³³, and it probably evolved in response to a reproductive strategy of seasonal breeding in which males compete, via the size of their song repertoire, for the most attractive females.

Zebra finches, in contrast, form longer-lasting pair bonds and retain reproductive capacity year-round. Accordingly, castration of adult male finches reduces singing only modestly, and testosterone treatment of adult females cannot induce them to sing nor their brain regions to grow³⁴. The first studies of sexual differentiation of the zebra finch brain indicated a story very similar to that of mammalian dimorphisms. Treatment of newly hatched female zebra finches with estrogen, followed by testosterone treatment in adulthood, masculinized the females in terms of both singing and the volume of RA and HVC³⁵. Furthermore, there are more dying cells in these regions in developing female zebra finches than in males, suggesting that estrogen prevents apoptosis in developing males, which results in a larger HVC and RA in adulthood³⁶.

But a perplexing problem emerged in zebra finches that does not fit the mammalian mold—neither castration early in life nor any pharmacological blockade of steroid receptors prevents these nuclei from developing a masculine phenotype in genetic males³⁷. This difficulty in preventing masculinization of the brain of males gave rise to an alternative view: perhaps the genetic sex of the brain was directing masculinization independent of the gonads³⁸. Experimental support for this idea came when researchers examined isolated slices of young zebra finch brain and found that differentiation proceeded according to the genetic sex: fibers from HVC entered and innervated RA more extensively in slices from males than from females. What's more, the brain slices produced detectable levels of estrogen in the medium, with slices from males producing more steroid than slices from females³⁹. So it appears that a male genotype causes the zebra finch brain to locally produce steroid hormones, which then masculinize the birdsong system (Fig. 4). Consistent with this idea is a rare, spontaneously mosaic zebra finch, genetically male on one side and genetically female on the other, that had a larger song system on the male side than the female side⁴⁰. There was even some indication that steroids derived from the male side of the brain might have diffused to masculinize the opposite side, as the brain regions of this animal's female side were slightly larger than that of control, wholly female brains.

Figure 4. In newly hatched zebra finches, axons from HVC reach the vicinity of their target in RA, but do not actually enter RA in significant numbers unless estrogen receptors are stimulated. For example, treating young females with estrogen promotes axonal ingrowth into RA and a large, masculine RA in adulthood. But interfering with gonadally produced estrogen in males does not prevent masculine development, so it has been hypothesized that the genetically male zebra finch brain may produce its own estrogen to induce HVC ingrowth and masculine development. Explants of zebra finch brain confirm this idea, as slices from males release more estrogen into the medium than do slices from females39. Full Figure and legend (63K)

The emerging recognition that genetic sex may directly masculinize the songbird brain has given rise to speculation that perhaps genetic sex also directly masculinizes the mammalian brain⁴¹. However, examination of mice in which genetic sex has been dissociated from gonadal sex indicates that the gonads direct sexual differentiation of mammalian neural structure and behavior. XY mice with a defective Sry gene develop ovaries, whereas XX mice carrying a transgene for Sry on an autosome develop testes. For the most part, these mutants confirm that it is the presence of testes, not the sex chromosome composition, that ensures a masculine central nervous system in terms of behavior, the SNB and other neural dimorphisms^{42, 43}. In a few instances, XX mice with testes, while more masculine than normal females, are not as masculine as XY male controls⁴³. However, XX testes are demonstrably abnormal in some regards (e.g., they do not produce sperm), so it is possible that the XX testes may not produce normal male levels of testosterone throughout all the sensitive periods for various brain regions, which would cause some regions to be under-masculinized. It is technically difficult to measure fetal testosterone levels in these animals, which would be required in order to rule out this possibility. To show that a gene affects neural sexual differentiation independently of the gonads, the ideal experiment would be to compare the brains of XX and XY mice that are genetically prevented from developing any gonads at all. If genes on the sex chromosomes directly drive brain sexual differentiation independent of the gonads, the brains of XY mice should be more masculine than those of the XX mice. If testes fully control masculinization of the mammalian brain, then the central nervous systems of gonadless mice, whether they carry XX or XY chromosomes, should be equally and fully feminine.

Neural sexual dimorphisms are remarkably diverse. Some rely solely on perinatal actions of testosterone (SDN-POA, AVPV), and some require both perinatal and adult testosterone (septal vasopressin, SNB, RA). Yet others require testosterone only in adulthood (MePD). In some cases, testosterone acts on only estrogen receptors (SDN-POA, AVPV) or activates both androgen receptors and estrogen receptors (septal vasopressin, MePD, RA). In other cases, only androgen receptors act perinatally (SNB). The zebra finch song system offers a distinctive twist: the brain itself seems to produce the 'testicular' hormone. But the commonality in all of these systems is that a single hormonal signal, testosterone, induces the central nervous system to take on a male phenotype—in the absence of testosterone, the nervous system differentiates as a female.

Where does testosterone first act?
In none of the cases described so far do we know the primary cellular targets on which testosterone acts to masculinize the nervous system during development. Although we know that exposing newborn female rats to estrogen prevents apoptosis of neurons in the SDN-POA, we do not know where estrogen acts to accomplish this. Is it directly on SDN-POA neurons themselves, or on other cells that then spare the SDN-POA neurons? That testosterone could act on distant target cells to spare SDN-POA neurons is entirely plausible because we know that testosterone acts on target muscles to spare SNB motor neurons. Likewise, no studies narrow down the list of possible targets for testosterone to masculinize the AVPV, MePD, RA or vasopressinergic innervation of the septum. Even for the SNB system, where we know that the developing motor neurons are not the site of action of testosterone, we do not know which cell types within the target muscles (muscle fibers, fibroblasts, Schwann cells) are directly modulated by the hormone.

However, we do know where testosterone acts to masculinize some systems in adulthood. Local implants of testosterone in the brain masculinize the morphology of ipsilateral birdsong regions in white-crowned sparrows⁴⁴ and in prepubertal zebra finches⁴⁵, so apparently testosterone acts somewhere within the forebrain in these cases. In genetically mosaic rats, only SNB motor neurons possessing a functional androgen receptor gene expand their somata or suppress peptide expression in response to adult testosterone treatment^{26, 46}. These cell-autonomous responses of SNB motor neurons are the only demonstrations to date of steroids directly altering the morphology or gene expression of neurons in vivo.

There are several genetic strategies that could provide information about the sites where steroids act to masculinize the developing nervous system. Once we understand whether testosterone masculinizes a system by activation of androgen receptor, or via aromatization and activation of either of two estrogen receptors, then it should be possible to conditionally knock out the gene for the relevant steroid receptor in particular classes of cells. For example, if the SDN-POA develops in a masculine fashion in males in which the estrogen receptor genes have been knocked out in astrocytes, but fails to differentiate properly in males missing estrogen receptor genes in neurons, then we will know that estrogen is acting specifically on neurons, perhaps even in the SDN-POA itself, to masculinize the nucleus. Similarly, if the SNB system is spared in genetic males that possess a functional androgen receptor only in muscle fibers, then the steroid must act on muscle fibers themselves to spare the muscles and their motor neurons.

Which downstream genes does testosterone modulate?
Because we do not know the target cells that first respond to testosterone or its metabolites during development, we know very little about the changes in gene expression that begin the process. In the SNB system, testosterone may alter the expression of trophic factor genes to spare the muscles and their innervating motor neurons. For example, injecting ciliary neurotrophic factor (CNTF) into the perineum of newborn rats spares the SNB system in normal females⁴⁷ and in males with a dysfunctional androgen receptor gene⁴⁸, indicating that CNTF is the molecular messenger dispatched by testosterone to maintain the system. However, male mice lacking the CNTF gene nevertheless develop a masculine SNB⁴⁹. On the other hand, male mice lacking the gene for the CNTF receptor alpha subunit fail to maintain the SNB system⁵⁰, suggesting that some ligand(s) other than CNTF itself normally stimulates CNTF receptor alpha to maintain SNB muscles and motor neurons⁵¹.

Although no genes have been implicated in the masculinization of the SDN portion of the POA, testosterone does induce the formation of prostaglandin E₂ (PGE₂), which then masculinizes another aspect of the POA, the formation of dendritic spines. These responses are also important for the development of male copulatory behaviors⁵². It is interesting that pharmacological manipulations of PGE₂ that affect spine formation in newborns (blocking PGE₂ in males stunts spine formation, whereas providing exogenous PGE₂ in females promotes spines) has no effect on the volume of the SDN-POA. This suggests that testosterone simultaneously starts several balls rolling: one that regulates expression of genes to produce PGE₂ (ref. 53), which then promotes spine formation, and another that regulates some other gene(s), which then acts to enlarge SDN-POA volume.

Sexual differentiation of the human brain
If steroids have such a pivotal role in masculinizing the brains of nonhumans, do they also have the same function in our own species? Do men and women behave differently because males are exposed to more testosterone prenatally? Do they behave differently because both sexes are indoctrinated into gender roles by family and society at large? The complexity of human behavior, which is powerfully shaped by social influences, makes it difficult to answer this question. On the surface, testosterone is responsible for sex differences in human behavior, if only indirectly—testosterone provides male fetuses with genitalia that provoke other people to raise them like boys. The deeper question is whether prenatal testosterone also masculinizes the fetal brain directly, without relying on other humans to shape the brain through experience.

Roughly 1 in 2,000 girls is exposed to slightly elevated levels of testosterone prenatally. Congenital adrenal hyperplasia (CAH) causes the fetal adrenal glands to produce androgens such as testosterone that slightly masculinize the genitalia and thus could potentially masculinize the fetal brain. Although these girls are more likely than other girls to engage in male-typical play⁵⁴, as adults most women with CAH are heterosexual. On the other hand, women with CAH are more likely to report a homosexual orientation than are other women⁵⁵. However, because it is possible that the parents or the girls themselves may have ambivalent feelings about their 'true' gender, it is difficult to know whether these slightly masculinized behaviors are the result of prenatal testosterone inducing the brain to rebel against socially prescribed gender roles.

Another human syndrome suggests that if testosterone masculinizes the human fetal brain, it does so by acting on androgen receptors, not through aromatization and action on estrogen receptors. Genetically male (XY) individuals with a dysfunctional androgen receptor gene develop testes that secrete testosterone, but the body exterior, without a functional androgen receptor, develops a feminine phenotype. People with complete androgen insensitivity are usually undetected at birth because they appear to be girls, are raised as girls and as adults self-identify as women^{56, 57}. The feminine behavior of these women could be due to either the brains' inability to respond to fetal testosterone, or their rearing as females. Thus, this syndrome does not tell us whether social influences or prenatal steroids inculcate sex differences in human behavior. But if, as in rats, testosterone masculinized the brain of fetal humans by being converted to estrogens and activating estrogen receptors, then we would expect these people with women's bodies to display male behaviors, because their estrogen receptor genes are intact.

An important but elusive question is whether prenatal steroids masculinize the human brain with respect to sexual orientation. Does the testosterone that masculinizes the human body also masculinize the fetal brain so that, in adulthood, the person will be attracted to women? One strategy has been to find somatic markers that correlate with prenatal androgen and ask whether they also co-vary with sexual orientation. Eyeblink patterns⁵⁸, otoacoustic emissions (clicks emanating from the ears)⁵⁹ and measures of fingers⁶⁰ and limbs⁶¹ all indicate that lesbians, on average, are exposed to more prenatal androgen than heterosexual women. However, none of these studies indicate a simple relationship between prenatal testosterone and sexual orientation—there is considerable overlap between lesbians and heterosexual women for each marker, which makes it clear that testosterone is not the sole factor at work. Nevertheless, crude as these measures are, it is impressive that such a wide range of characteristics, reported by a wide number of laboratories, all indicate that the more prenatal testosterone a girl is exposed to, the more likely she is to self-identify as lesbian in adulthood. Examining these same markers in men provides conflicting results, sometimes yielding evidence that gay men are exposed to less prenatal testosterone than straight men, sometimes finding the opposite and sometimes finding no differences between the two groups. While still controversial, it is possible that both lower-than-average and higher-than-average testosterone exposure before birth can increase the likelihood that a boy will develop a homosexual orientation.

Of course, animal models have helped by framing the questions that scientists have asked about the origins of human sexual orientation. For example, the models tell us that if we want to seek a prenatal influence on human sexual orientation, we should pay attention to testosterone and its target tissues in the brain. Studies of the rat SDN-POA inspired examination of the human POA, where a nucleus was found that was smaller in women than in men, and smaller in gay men than in straight men⁶². A similar difference is seen in sheep: a nucleus in the POA is smaller in male sheep that prefer to mount other rams than in males that prefer to mount ewes⁶³. For neither humans nor sheep do we know whether the differences in the POA preceded the development of sexual orientation or arose after the establishment of orientation, but taken together, these studies make it seem likely that this brain region is important for determining sexual orientation. A better understanding of how these and other sexual dimorphisms in the nervous system arise will help us appreciate why men and women behave differently, and why most of us find one of the sexes, and only one, so very enchanting.

Received 12 July 2004; Accepted 13 August 2004; Published online: 27 September 2004.