Four core genotypes (FCG) mice
One of the most widely applied models are FCG mice, in which the sex chromosome complement (XX versus XY) is independent of gonadal sex. The mice were produced by a two-step genetic manipulation. The Sry gene was deleted (a small spontaneous deletion that probably removed only the Sry gene, although deletion of other regulatory regions cannot be ruled out completely), and then a functional Sry transgene was inserted onto an autosome, driven by its own endogenous promoter (20–23). Thus, testis determination was transferred from the Y chromosome to an autosome; the Y chromosome no longer determines gonadal sex, and the number of X chromosomes is no longer correlated with gonadal sex. The four genotypes are XY gonadal males (XYM), XX gonadal females (XXF), XX gonadal males (XXM) and XY gonadal females (XYF). Comparisons of phenotypes in mice with different gonads (XXF versus XXM, XYF versus XYM) reveals the effects of gonadal secretions. More accurately, this comparison is between mice with and without the Sry transgene, which is the only gene that differs between the comparison groups. Most effects of Sry are indirect, the result of differences in gonadal secretions, which is why we can adopt the shorthand statement that these comparisons test for the effects of gonadal secretions and are called sex effects. It is important to remember, however, that some effects of Sry are not mediated by gonadal secretions (19). By contrast, comparisons of phenotypes of mice with the same type of gonad but different sex chromosome complement (XXF versus XYF, XXM versus XYM) yields information about the differential effects of an XX and XY genome. These are called ‘sex chromosome effects’. Note that gonadal hormones are not eliminated in this model. Instead, XX and XY mice are compared that developed with ovaries, and XX and XY mice are compared that developed with testes. Because FCG mice have been used so far mostly to determine whether sex chromosome effects occur, they have typically been studied after gonadectomy in adulthood, so that, at the time of testing, there were no group differences induced by the acute (activational) effects of gonadal hormones. However, FCG mice gonadectomised as adults experienced the organisational effects of hormones, so that group differences in effects of gonadal secretions before the time of gonadectomy are not controlled by this design. This issue is discussed further below. The removal of gonads of adult FCG mice prior to testing is critical, however, if the goal is to detect sex chromosome effects that are not mediated by differences in gonadal secretions. Many or most sex differences in phenotype are caused by activational effects of hormones. In a recent global study of gene expression in liver, for example, the majority of sex differences in gene expression were abolished by gonadectomy (24).
To date, FCG mice have been analysed for numerous neural and non-neural phenotypes, including brain morphology, behaviour, brain and liver gene expression, and phenotypes related to sex differences in disease (23). The earliest studies focused on brain and behavioural phenotypes representing some classic sexually dimorphic traits. These dimorphisms had previously been shown to be caused by organisational actions of testosterone or oestradiol. Among the dimorphic traits were male copulatory behaviour, neurone number in the spinal nucleus of the bulbocavernosus and anteroventral periventricular nucleus of the hypothalamus (AVPV), thickness of the cerebral cortex, and progesterone receptor expression in the neonatal hypothalamus (22, 25, 26). These traits all were found to show sex effects (i.e. they differed in gonadal males versus females) in adult FCG mice gonadectomised and treated equally with testosterone, but they showed no sex chromosome effects (i.e. they were not different in XX versus XY). The results confirmed the long-standing conclusion that many sex differences are caused by organisational effects of gonadal hormones. In subsequent studies (27), FCG mice of the four groups were found not to differ on general measures of numerous traits (i.e. no sex or sex chromosome effects), including open field activity, elevated plus maze tests of anxiety, tests of olfaction, and threshold response to footshock. The lack of group differences suggests that the groups show no marked differences in physiology in the systems tested.
Studies of FCG have revealed several sex chromosome effects in which XX and XY mice differed, irrespective of gonadal sex. Among these are XX versus XY differences in the number of tyrosine hydroxylase nerones in dissociated cell cultures of embryonic mouse mesencephalon (XY > XX) (28); the density of vasopressin fibers in the lateral septum of adult mice (XY > XX) (22, 29); the response to thermal and chemical nociceptive stimuli in adult mice (XX > XY) (30); the response to thermal nociceptive stimuli in neonatal mice (XX > XY) (31); the learning of habits (XX > XY) (32); sniffing and grooming of an intruder mice (XX < XY) (27), and gene expression in several tissues (24, 33–36). XX mice also show greater susceptibility to disease in mouse models of autoimmune diseases and neural tube closure defects (37–39). In other experiments, the XX versus XY difference was found only in one sex. For example, when FCG were gonadectomised as adults and treated equally with testosterone, XXF showed fewer aggressive responses to an intruder mouse than the other three groups (29). XXF mice tested after gonadectomy without hormone treatment were also found to differ from the three other groups in their parental behaviour (i.e. more pup retrieval) and they showed less asocial digging behaviour in response to a cage intruder (27, 29). Thus, sex chromosome effects have been found under a variety of conditions, involving different behavioural systems. A major conclusion from these studies and FCG studies discussed below is that XX and XY mice of the same gonadal sex differ in phenotype. These differences in XX and XY cells in the FCG model probably also operate in normal XY males and XX females because the groups compared have XX and XY genomes that have the same three genetic differences (X dose, X imprint, Y dose) found in comparisons of normal XY males and XX females.
The study of Gioiosa et al. (30) on sex differences in nociception serves to illustrate the types of conclusions that can be drawn from studies of FCG mice. Pain is perceived and experienced in men and women differently (40–43), and sex differences are also found in mouse models of pain. FCG mice were tested in a standard test, in which the mouse is placed on a hot plate. The amount of nociception is inversely proportional to the latency with which the mouse licks its feet. In FCG mice gonadectomised as adults, XX mice responded more quickly to the thermal stimulus than XY mice, irrespective of their gonadal sex. In a test of acute nociception on naive mice, an injection of morphine increased latencies but did not differentially influence XX and XY mice. In a second test on the development of tolerance to morphine, mice were injected twice daily for 6 days with morphine or saline, with or without an N-methyl-d-aspartate antagonist, which is thought to block the development of tolerance. On the seventh day, mice were tested on the hotplate, then injected with morphine and tested at various intervals after morphine injection. In this test, XX mice showed dramatically shorter latencies to respond relative to XY mice, before or after morphine, and the effect of morphine was greater in XY mice than XX mice (Fig. 1). Thus, we conclude that thermal nociception differs in XX and XY mice. Because the sex chromosome complement of these mice mimics the difference in the genome between normal XY males and XX females, the results suggest that X- or Y-linked genes act to produce sex differences in the tissues that mediate the nociceptive response. The tests of FCG mice do not determine whether the genes are X- or Y-linked, nor do they resolve the sites of action of the sex chromosome effect. The chromosome of origin of the effect can be resolved in future studies that vary the number of X chromosome and Y chromosomes independent of each other (e.g. see subsequent section on ‘Attributing sex chromosome effects to X or Y genes’). Moreover, the mechanisms mediating the effect can be resolved once the responsible gene(s) is/are identified. Because of the dominant hormonal theory of sexual differentiation, an important question is whether the group differences in XX versus XY mice could be mediated by group differences in gonadal secretions. Although a gonadal hormonal explanation remains possible, several considerations make it unlikely. First, because the groups did not differ in their levels of gonadal secretions at the time of testing, the group differences were not caused by acute (activational) effects of gonadal hormones. The groups could have differed in the levels of gonadal hormones prior to gonadectomy, which could have had lasting effects. For example, it is conceivable that even though XXM and XYM both have testes, XXM might experience levels of testosterone different from those experienced by XYM during some perinatal critical period, which might influence the adult hotplate response. That idea also is unlikely, because the hotplate response was no different in gonadal males and females under the conditions of testing (Fig. 1); thus, the response appears to be insensitive to the presence of testicular versus ovarian secretions prior to gonadectomy. Under those conditions, smaller within-sex differences in levels of gonadal hormones, prior to gonadectomy, would be unlikely to account for XX versus XY differences in the response. Moreover, XXM and XYM have been found both to be fully masculine on numerous traits, and to differ from XXF and XYF who are similar to each other and fully feminine on those same traits. These results suggest that same-sex FCG mice generally experience similar levels of gonadal hormones during early critical periods (22, 25, 26). Thus, for some traits, studies of FCG mice have led to the confident conclusion that X and Y genes act directly on non-gonadal tissues to produce sizable sex differences in the trait.
Figure 1. Robust sex chromosome effect on nociception. Four core genotypes (FCG) mice were gonadectomised as adults and then tested for the development of tolerance to morphine. Mice were injected twice daily with morphine or saline, plus an N-methyl-d-aspartate antagonist or saline. On the seventh day, mice were tested on a hotplate at time zero and then injected with morphine and tested after 30, 60 and 90 min. The graph shows the latency for the mice to lick or shake their paws. All data are averaged across drug treatment groups. XX mice of either gonadal sex showed much lower latencies than XY mice (*P < 0.00001), and the presence of testes or ovaries prior to gonadectomy had no effect. Reprinted from Hormones and Behaviour (30), copyright 2008, with permission of Elsevier, Inc.
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Some of the largest sex chromosome effects detected by the FCG model are found in disease models. Many diseases differ in incidence or progression in men and women. Autoimmune diseases, such as multiple sclerosis (MS) and systemic lupus erthematosus (SLE), affect women more than men. The same sex differences can be detected in mouse models of these diseases. Experimental autoimmune encephalomyelitis (EAE) is a mouse model of MS that in some strains affects females more than males. Although androgens and oestrogens protect females from EAE, in the absence of gonadal hormones, XX FCG mice show greater neural histopathology and faster progression of EAE than XY FCG mice, irrespective of their gonadal sex (37, 38). In a model of SLE, XX mice show greater kidney pathology and die sooner than XY mice, again irrespective of gonadal sex (37). These sex chromosome effects are large and robust.
Mice lacking gonads
In the FCG model, the effect of an XX versus XY genome is compared both in a testicular hormonal environment, and in an ovarian hormonal environment. The value of the model stems in part from the power of comparing XX and XY under two different gonadal environments because any differences between XX and XY are more likely to be direct sex chromosome effects (e.g. as opposed to group differences caused by hormones) if they can be observed under different hormonal conditions. A disadvantage of the FCG model is that the effects of gonadal hormones have not been removed. Another mouse model solves this problem by observing mice that lack gonadal secretions altogether. This is possible in mice with a null mutation in the gene steroidogenic factor 1 (SF1, also called AD4BP) (44, 45). SF1 has been shown to be critical for development of the gonads, adrenals, ventromedial nucleus of the hypothalamus (VMH) and anterior pituitary. Mice without SF1 lack gonads and adrenals, and die soon after birth because of the lack of adrenal secretions. These mice can be rescued by neonatal injections of glucocorticoids and then implantation of adrenal tissue. Sex differences found in such SF1 knockout mice clearly do not require gonadal secretions, and therefore are likely attributable to direct sex chromosome effects (XX versus XY) on non-gonadal tissues (46, 47). Gonad-independent sex differences are found in body weight of mice, and in the expression of neuronal nitric oxide synthase in several limbic brain regions [preoptic area, bed nucleus of the stria terminalis (BST) and AVPV]. Moreover, sex differences are found in the expression of calbindin in the VMH. In other traits, mice without gonads did not show sex differences that are normally present in males and females that are gonadectomised before puberty (external genitalia, calbindin expression in the POA and BST, and aggressive behaviour), indicating that the sex differences in these phenotypes require the presence of gonads before puberty and hence are likely the result of sex differences in gonadal secretions.
The use of mice without gonads is an important approach that helps tease apart the direct genetic effects and hormonal effects that cause sex differences in phenotypes. One caveat in the use of SF1 knockout mice is that the SF1 mutation itself could create sex differences that are not found in normal mice. In adult SF1 knockout mice, the structural organisation of the VMH is disrupted (48–50). Thus, it is conceivable that the disruption of the VMH, or some other effect of the SF1 knockout, affects males and female differently. Still, such sex-specific effects do not require the gonads, and provide evidence that XX and XY systems are not just differentiated by gonadal secretions (46). Moreover, a similar caveat applies in one form or another to all mice with disrupted sex chromosomes because, in each case, one can imagine a situation in which the disruption in the genome could conceivably create differences between XX and XY that do not occur normally. As always, therefore, the conclusions based on studies of any one model should be confirmed using other approaches.
Mice with X chromosome monosomy (XO)
Recently, several studies have examined the behavioural phenotype of gonadally female mice with one X chromosome. XO mice showed greater fear reactivity than XX mice, as measured by the time that they spent in the open arms of an elevated plus maze (51), and also showed impaired discrimination in a demanding visual task requiring high levels of attention (52). In these tests, the parent of origin of the X chromosome of XO mice had no apparent effect. For fear reactivity, the difference between XO and XX was not caused by the different numbers of pseudoautosomal regions (PARs) because XY*X mice (which have one X chromosome but two PARs; Fig. 2 and Table 1) were similar in fear reactivity to XO (51). It was concluded that the likely difference in fear reactivity between XO and XX was caused by the difference in the expressed dose of a non-PAR X gene that escapes inactivation. A different result was found in tests of visuospatial attention. In that case, the mice with one X chromosome plus two PARs differed from XO mice but were similar to XX mice, suggesting that haploinsufficiency of a PAR gene (probably steroid sulphatase) was responsible for the attention deficit of XO mice (52). In all of these tests of X monosomic mice, XO and XX mice were tested gonadally intact, so hormonal mediation of the X monosomy effect is possible, although some studies are not in agreement (51, 52). The X dosage effect found in fear reactivity should also operate in the comparison of XX and XY mice, so the results could be relevant to the main question of this review. However, FCG XX and XY mice tested after adult gonadectomy did not show a similar difference in fear responding (27), suggesting that the presence of the Y chromosome might mitigate the X dosage effect. In addition to its relevance to sex differences, the comparison of XO versus XX difference is quite relevant as a model for understanding differences between XX and XO women.
Figure 2. Chromosome maps of variants of the X and Y chromosomes. Progeny of XY* males, mated with XX females, are: XX, XY*, XO, XY*X, XXY*. The normal X and Y chromosomes, and the recombinant sex chromosomes Y*, Y*X and XY*, are shown. The pseudoautosomal region (PAR) is separated into proximal to distal segments A, B, C. XPB, X-PAR boundary; YPB, Y-PAR boundary; NPX, non-PAR region of the X chromosome; MSY, male-specific (non-PAR) region of the Y chromosome. Adapted with permission (39) and based on previous studies (68, 69).
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Table 1. Sex Chromosomes of the Progeny of XY* Fathers.
| ||Sry||NPX||MSY||PARAB||PARC||Near-PAR X||Xm||Xp|
Comparison of XO mice with a maternal versus paternal X chromosome imprint has revealed that the parent of origin of the X chromosome influences cognitive functions (53). XmO mice (i.e. those inheriting their X chromosome from the mother) differed from XpO mice (i.e. with a paternal X imprint) in a test of reversal learning. Mice with a maternal X imprint were less able to alter their responses when the reward contingency changed. The X gene Xlr3b shows parent of origin effects on its expression and is expressed in brain regions important for reversal learning, and is thus a candidate gene accounting for the behavioural differences between XmO and XpO mice. These parent of origin effects are a model of behavioural differences caused by parent of origin in women with Turner’s syndrome (XO) (54). It will be interesting to determine whether the differences in imprinting of this gene contributes to sex differences in comparisons of XX and XY mice.