Genetic, hormonal, and metabolomic influences on social behavior and sex preference of XXY mice

Abstract

XXY men (Klinefelter syndrome) are testosterone deficient, socially isolated, exhibit impaired gender identity, and may experience more homosexual behaviors. Here, we characterize social behaviors in a validated XXY mouse model to understand mechanisms. Sociability and gender preference were assessed by three-chambered choice tasks before and after castration and after testosterone replacement. Metabolomic activities of brain and blood were quantified through fractional synthesis rates of palmitate and ribose (GC-MS). XXY mice exhibit greater sociability than XY littermates, particularly for male mice. The differences in sociability disappear after matching androgen exposure. Intact XXY, compared with XY, mice prefer male mice odors when the alternatives are ovariectomized female mice odors, but they prefer estrous over male mice odors, suggesting that preference for male mice may be due to social, not sexual, cues. Castration followed by testosterone treatment essentially remove these preferences. Fractional synthesis rates of palmitate are higher in the hypothalamus, amygdala, and hippocampus of XXY compared with XY mice but not with ribose in these brain regions or palmitate in blood. Androgen ablation in XY mice increases fractional synthesis rates of fatty acids in the brain to levels indistinguishable from those in XXY mice. We conclude that intact XXY mice exhibit increased sociability, differences in gender preference for mice and their odors are due to social rather than sexual cues and, these differences are mostly related to androgen deficiency rather than genetics. Specific metabolic changes in brain lipids, which are also regulated by androgens, are observed in brain regions that are involved in these behaviors.

Fig. 1.

A: three-chambered social approach task. An unfamiliar stranger mouse and a novel object (plastic brick) are each enclosed in separate aerated enclosures which are then placed in the left and right main chambers, respectively. The separate enclosures are weighed down to prevent escape. The subject (test) mouse is in the middle chamber and can enter either chamber through a single doorway in each dividing wall. Time spent in each chamber (B) and locomotor activity (C) did not significantly differ between XXY and XY mice during the habituation phase (i.e., when all chambers were empty) when assessed by ANOVA. NS, not significant.

Fig. 2.

Sociability test (experiment 1). Relative time preference of XXY (open bars) or XY (filled bars) subject for a stranger mouse or an inanimate object is shown. Data are means and SE of difference in time spent in the chamber containing the stranger mouse vs. time spent in the chamber containing the novel inanimate object, stratified by the sex of the stranger mouse (OVX female, left; male, right). Positive differences indicate preference for the mouse, whereas negative differences indicate preference for the object. Significant nonzero differences were assessed by one-sample Student's t-test and are indicated by * or ** for P < 0.05 or P < 0.01, respectively. Whether these time differences differed between XXY and XY mice was assessed by two-sample Student's t-test as shown by P values.

Fig. 3.

Sociability test after castration (left) and then after testosterone replacement (right) (experiment 2). Relative time preference of XXY (open bar) or XY (filled bar) subject mouse for a novel male stranger mouse or an inanimate object. Data are means and SE of the difference in time spent in the chamber containing the mouse vs. time spent in the chamber containing the novel inanimate object, stratified on whether the subject mouse was castrated (left) or castrated and then testosterone treated (right). Hence, positive differences indicate preference for the mouse, whereas negative differences indicate preference for the object. Significant nonzero differences were assessed by one-sample Student's t-test and are indicated by * or ** for P < 0.05 or P < 0.01, respectively. Whether these time differences differed between XXY and XY mice was assessed by two-sample Student's t-test as shown by P values.

Fig. 4.

Castrated XXY mice prefer male mice over OVX female mice when mice of different sex are presented at the same time (experiment 3). Data from intact (left) castrated (middle), and then testosterone-treated (right) mice are shown. Mice had to choose between a male mouse or an OVX female mouse. Data are means and SE of the difference in time spent in the chamber containing the male mouse stimulus vs. time spent in the chamber containing the female stimulus. Hence, positive differences indicate preference for the male mouse stimulus, whereas negative differences indicate preference for the female mouse stimulus. Significant nonzero differences were assessed by one-sample Student's t-test and are indicated by * or ** for P < 0.05 or P < 0.01, respectively. Whether these time differences differed between XXY (open bar) and XY (filled bar) mice was assessed by two-sample Student's t-test as shown by P values.

Fig. 5.

Preference for odors from male mice instead of odors from OVX female mice (top) or from estrous female mice (bottom) (experiment 4). Mice had to choose between odors from male or OVX female mice (top) and then odors from different male or different estrous female mice (bottom). Data from intact (left), castrated (middle), and then testosterone-treated (right) mice are shown. Top: data are means and SE of relative time spent in the chamber containing the male odor, vs. time spent in the chamber containing odors from OVX female mice. Bottom: data are means and SE of relative time spent in the chamber containing the estrous female odor vs. time spent in the chamber containing male odors. Significant nonzero differences were assessed by one-sample Student's t-test and are indicated by * or ** for P < 0.05 or P < 0.01, respectively. Whether these time differences differed between XXY (open bar) and XY (filled bar) mice was assessed by two-sample Student's t-test as shown by P values.

Fig. 6.

Palmitate (A) and ribose (B) synthesis rates in amygdala and palmitate synthesis rates in blood (C) in XXY, XY, and androgen-deprived (AD) XY mice. Mice received ²H₂O tracer for 5 days, after which amygdala and blood samples were collected for GC-MS MIDA. In amygdala (A), which is a region important for sexual preference, the rate of newly synthesized (fractions of newly synthesized, FNS) palmitate was significantly higher in XXY and AD mice than in controls. No significant differences were observed in ribose FNS in amygdala (B) or palmitate FNS in blood (C). Each value represents a mean of 3 replicate experiments in 4 mice ± SE. Nos. in brackets indicate nos. of mice in experimental setting. *P < 0.05.

Fig. 7.

Palmitate (C16–0) turnover in hypothalamus and hippocampus of XXY, XY, and AD XY mice. Mice received ²H₂O tracer for 5 days, after which hypothalamus and hippocampus regions were collected for GC-MS MIDA. In brain regions important for sexual preference, hypothalamus (A), and hippocampus (B), rates of FNS palmitate were significantly higher in XXY and AD mice than in controls. Each value represents a mean of 3 replicate experiments in 4 mice ± SE. Nos. in brackets indicate nos. of mice in experimental setting. *P < 0.05.