As organizational-activational dichotomy was increasingly successfully applied to the study of many sex differences, at all levels of analysis from molecular to behavioral, many of us began to be lulled into the expectation that all
sex differences might be explained by this single theory (as amended by subsequent studies). Few, if any, authors insisted that it explained all sex differences, but hormones became the only factors that were investigated or discussed as proximate signals causing sex differences in the brain. An alternative idea, that the genetic differences between XX and XY cells cause functional sex differences intrinsic to male and female cells, seemed unlikely because XX females in some cases were completely masculine in some phenotypes if they were treated neonatally with testosterone (e.g., Dohler et al., 1984
; Nordeen et al., 1985
). XY males in which the organizational effects of testosterone were blocked, were completely feminine in some cases (Breedlove and Arnold, 1980
). There was no need to invoke other factors.
In the period 1989–1995, several discoveries re-awakened an interest in the direct effects of the X and Y genes (O et al., 1988
; Renfree and Short, 1988
; Beyer et al., 1991
; Reisert and Pilgrim, 1991
; Burgoyne et al., 1995
; Dewing et al., 2003
). Sex differences were found before the gonads differentiated, or before plasma levels of testosterone were reported to be sexually dimorphic. Moreover, in at least one model system, the neural circuit for song in songbirds, manipulations of the type of gonad or level of gonadal hormones failed to sex-reverse the phenotype fully (Arnold, 1996
; Wade and Arnold, 1996
). Because sex differences in some phenotypes were not explained by organizational or activational effects of gonadal hormones, we turned to a consideration of an alternative but old idea, that genetic differences intrinsic to male and female brain cells might be the origin of some sex differences in phenotype (Arnold, 1996
; De Vries et al., 2002
). Male and female zygotes have an identical set of autosomes, on average, which comprise about 95% of the genome. Those common genetic factors make males and females quite similar in their function and behavior. The sex chromosomes, comprising the other 5% of the genome, differ in three main ways in the zygote: (1) males alone have Y genes, (2) the two sexes differ in the copy number of X genes (although the sex-specific effect of this difference is largely eliminated by X-inactivation), and (3) females receive a paternal X imprint that is lacking in the male. As the individual develops, however, three other intrinsic genetic sex differences arise that are not caused by gonadal hormones. (1) When X-inactivation occurs, it utilizes some cellular resources only in females, which may make females more vulnerable than males to some genetic mutations or environmental perturbations at early stages of embryonic development (Chen et al., 2008
). (2) Female tissues are mosaics. One X chromosome is randomly selected to be transcriptionally silenced In each female cell. Thus, about half of the cells activate the maternal X chromosome and express the maternal X alleles or maternal imprint, and the other half cells express the paternal X alleles or imprint. Mosaic (female) tissues might differ in phenotype from tissues that are not mosaic (male), because they may function better in a range of environments (I.e., different alleles are more adaptive in some environments) or have or have muted susceptibility to diseases involving X genes (Arnold, 2004
; Migeon, 2007
). (3) In populations of animals, the representation of some alleles may differ between males and females because some gene variants might be less compatible with survival in one sex. Such population effects could lead to average sex differences in phenotype.
Using rodent models, three approaches provide convincing evidence that these differences in XX and XY genomes directly cause sex differences in non-gonadal cells. Such sex differences, called sex chromosome effects
, could result from any of the differences discussed in the last paragraph, except that there is no allelic variation in inbred mouse strains discussed below. One approach is to interfere directly with the expression of a Y gene to demonstrate that it has a male-specific effect in the brain (Dewing et al., 2006
). The testis-determining gene Sry
is expressed in the adult rodent and human substantia nigra. Antisense oligonucleotides were used to reduce Sry
expression in the brain of adult rats and mice. Loss of Sry
led to reduced expression of tyrosine hydroxylase in the dopaminergic cells of the substantia nigra and striatum, and interfered with motor function. This study (Dewing et al., 2006
) is the first to identify a Y gene that has a direct effect on brain phenotype. A second approach is study mice lacking the steroidogenic factor 1 (SF-1) gene. These mice lack adrenals and gonads, but survive if they are treated neonatally with corticosteroids and then implanted with adrenal tissue (Grgurevic et al., 2008
; Budefeld et al., 2008
). This model compares XX and XY mice that never had gonads. SF-1 knockout XY and XX mice differ in body weight and in distribution of specific immunohistochemically defined cells in the preoptic area, bed nucleus of the stria terminalis, and hypothalamus, indicating that sex chromosome complement affects these phenotypes.
The mouse model used most often to date to study sex chromosome effects is the “four core genotypes” (FCG) model, in which gonadal sex and sex chromosome complement are uncoupled (De Vries et al., 2002
; Arnold and Burgoyne, 2004
; Arnold and Chen, 2009
; Arnold, 2009
). FCG mice comprise XX and XY gonadal males (XXM and XYM) and XX and XY gonadal females (XXF and XYF). In FCG mice the Y chromosome is deleted for Sry
, the testis-determining gene, which is then inserted as a transgene onto an autosome. The autosome becomes testis-determining, and the sex chromosomes are irrelevant to the gonadal sex (testes vs. ovaries) of the animal. This model allows testing of organizational and activational effects of hormones, but more importantly also can be used to test for group differences in XX vs. XY mice that have the same type of gonads. Such differences between XXM and XYM, or between XXF and XYF, are attributed to the differential effects of an XX vs. XY genome. Finally, the model tests for interactions of hormonal and sex chromosome effects, for example if testosterone has different effects in XX vs. XY mice (Arnold and Chen, 2009
The FCG model was first used to examine possible sex chromosome effects on several brain and behavioral traits long known to show sex differences (De Vries et al., 2002
). In each case, previous work had demonstrated that these sexual dimorphisms were caused by organizational and/or activational effects of gonadal steroids. Among the phenotypes measured were male copulatory behavior, and morphological sex differences in vasopressin fibers in the lateral septum, in the spinal nucleus of the bulbocavernosus (SNB), and the hypothalamic anteroventral periventricular nucleus (AVPV). The FCG mice were gonadectomized as adults and treated equally with testosterone (mirroring the classic methods of Phoenix et al.), so that group differences were not attributable to group differences in activational effects of sex steroids. For these phenotypes, studies of FCG mice confirmed in all cases that the sexually dimorphic phenotype differed in mice that developed with testes vs. ovaries (i.e., were caused by organizational effects revealed as differences between in XXM vs. XXF, and in XYM vs. XYF). For most of the phenotypes, there were no differences between XX and XY mice that had the same gonadal sex. The same results were obtained in studies of sex differences in thickness of the cerebral cortex, and in progesterone receptor expression in the preoptic nucleus of the hypothalamus in neonates (Markham et al., 2003
; Wagner et al., 2004
). Thus, the organizational-activational framework accounted completely for the majority of the classic sex differences first studied in the FCG model.
One exception was that septal vasopressin, in addition to being sexually differentiated by organizational and activational effects of sex steroids, shows a small difference between XX and XY mice, with XY mice of either sex showing greater vasopressin fiber density than XX mice of the same sex (De Vries et al., 2002
; Gatewood et al., 2006
). Thus, for this phenotype it appears that sex chromosome factors might sum with hormonal effects to produce sex differences.
Further studies of FCG mice have uncovered convincing new evidence that sex chromosome complement has an important impact on sexually dimorphic phenotypes, in some cases producing large sex chromosome effects (Arnold and Chen, 2009
; Arnold, 2009
). Based on these studies as a group and those mentioned from other approaches reviewed above, there is little doubt that XX and XY cells are intrinsically different, not just because of organizational and activational effects of gonadal hormones. The following is a list of examples.
- When mesencephalic cells are dissociated and cultured from mouse embryos 14.5 days after coitus, the number of dopaminergic neurons that differentiate (I.e., those expressing tyrosine hydroxylase) is greater in cultures from XY mice of either gonadal sex, compared to XX mice (Carruth et al., 2002). This study confirms that XX and XY mesencephalic cells have different properties that are not caused by effects of gonadal steroids (Beyer et al., 1991; Reisert and Pilgrim, 1991).
- When FCG mice are gonadectomized as adults and treated equally with testosterone, the XY gonadal females are more aggressive than XX females, as evidenced by their more frequent attack of a male intruder mouse in the home cage) than XX females, whereas XX and XY males are equally aggressive. XX and XY females also show differences in measures of parenting behaviors and social interactions (Gatewood et al., 2006; McPhie-Lalmansingh et al., 2008).
- In FCG mice that are gonadectomized in adulthood, the expression of prodynorphin mRNA in the striatum is higher in XX mice than XY mice (Chen et al., 2009). This gene encodes the precursors of the dynorphin peptides that are ligands of the kappa opioid receptor. Based on the idea that the robust difference between XX and XY prodynorphin expression might influence the mouse’s response to nociceptive stimuli, we tested gonadectomized adult and gonadally intact neonatal mice in several tests of thermal and chemical nociception (Gioiosa et al., 2008a; Gioiosa et al., 2008b). In all cases, the XX mice showed greater or faster responses than XY mice, irrespective of gonadal sex. The greater responsiveness of adult gonadectomized XX mice suggests that sex chromosome complement contributes to sex differences in response to nociceptive stimuli.
- XX and XY mice also differ in tests of habit formation that are potentially relevant to addiction (Quinn et al., 2007). Human females are reported to increase usage of some drugs more quickly than males, to the point of addiction. Mice learn a task if they receive food reward, which can progress to a “habit” after continued conditioning. A habit is relatively insensitive to the contingencies of reinforcement such as the value of the reinforcer, in contrast to the initial stages of conditioning when reward value is important to the performance of the task. In an experiment in which FCG mice were trained to respond to food reward, XX mice of either gonadal sex progressed to the level of habitual responding (continued responding despite devaluation of the reward) more quickly that XY mice of either gonadal sex (Quinn et al., 2007). Thus, sex chromosome complement may influence the rate at which mice develop a habit.
- Neural tube closure defects influence human female neonates more than males. In some mouse models of neural tube defects, the sexes also differ in the effects of the mutation that interferes with tube closure. In mice with a null mutation of the p53 gene, female embryos develop more anencephaly and exencephaly than do male embryos, and most females lacking p53 die by the day of birth. The sex difference is caused by XX vs. XY differences in the genome, not by gonadal hormones, as was demonstrated in FCG mice (Chen et al., 2008).
- The incidence and progression of autoimmune diseases is sexually dimorphic. Females are more affected than males by multiple sclerosis (MS) and systemic lupus erythematosus (SLE). In mouse models of these diseases, females are often more susceptible than males. The mouse models involve treating mice with an antigen and/or adjuvant that triggers an autoimmune response with properties similar to MS or SLE. When gonadectomized adult FCG mice are used in these mouse models, XX mice fare much worse than XY mice. The progression of the MS-like disease is faster in XX than XY mice of either sex, and in the SLE model XX mice die faster than XY (Smith-Bouvier et al., 2008). Although organizational and activational effects of gonadal hormones also explain some sex differences in the MS-like model (Voskuhl and Palaszynski, 2001; Voskuhl, 2009), it appears that sex chromosome complement also plays a role.
An important issue in comparing FCG mice is whether the sex chromosome effects, which are difference in XX vs. XY groups, could themselves be caused by group differences in the levels of gonadal hormones. In all cases mentioned, the effects are measured in the absence of gonads, so the group differences are not caused by activational effects. It is conceivable, however, that XX and XY males, or XX and XY females, might have received different exposure to gonadal secretions prior to adult gonadectomy. On balance such differences appear to be unlikely, because the mice of the same gonadal sex appear to be equally masculinized (or not) on a number other variables (De Vries et al., 2002
; Markham et al., 2003
; Wagner et al., 2004
; McPhie-Lalmansingh et al., 2008
), and because in some cases there are no organizational effects as measured in the FCG model itself (Gioiosa et al., 2008a
; Arnold and Chen, 2009
). Ultimately, the mechanisms mediating the effects can be established when the gene(s) mediating the effects are identified.
The long term goal of studies of sex chromosome effects is to find the genes responsible, and their mechanisms of action. Several X and Y genes appear to contribute to sex chromosome effects. As indicated above, the Y-linked Sry
gene has been shown to have male-specific effects on the substantia nigra and striatum. The sex chromosome effect on prodynorphin expression is explained by the difference in number of X chromosomes, since XO mice have similar prodynorphin expression as XY mice, which is less than XX (Chen et al., 2009
). Similarly, the sex chromosome effect on neural tube closure in p53-deficient mice is an X-linked effect based on similar evidence from mice with different numbers of X and Y chromosomes (Chen et al., 2008
). The X effects could either be differences in the expressed dose of X genes that escape X-inactivation (i.e., higher expression in XX than XY), or the result of XX vs. XY differences in the expression of X genes that are parentally imprinted (different levels of expression in XX vs. XY because only XX mice receive a paternal X imprint).