Sex differences in juvenile mouse social behavior are influenced by sex chromosomes and social context

INTRODUCTION

Juvenile social behaviors have been examined in several ways: preference for social novelty (Mcnaughton et al., 2008; Moy et al., 2004; Moy et al., 2009), conditioned place preference (Panksepp & Lahvis, 2007), and direct social encounters with peers (Mcfarlane et al., 2008). While young mice, like rats, exhibit play fighting, social encounters are primarily non-contact interactions; mice do not display the same variety of play fighting behaviors as juvenile rats (Pellis & Pasztor, 1999). In addition, male rats engage in more play fighting (also called rough and tumble play) than females (Auger & Olesen, 2009; Meaney, 1988), and female rat play is also qualitatively different from male play (Pellis et al., 1997; Pellis & Pellis, 1990). In contrast, out-bred CD-1 female mice display more play behavior than males (Laviola & Alleva, 1995; Terranova et al., 1993). In inbred strains play studies have only included males (Mcfarlane et al., 2008; Moy et al., 2007), thus it is unknown if commonly used inbred mice display sex differences in play.

In rats, sexual dimorphisms in play fighting are organized by the exposure of the male brain to androgens during neonatal development (Beatty et al., 1981; Meaney & McEwen, 1986; Meaney & Stewart, 1981). In testicular feminization mutant rats, which lack functional androgen receptors, play behavior is comparable to females (Meaney et al., 1983). Male rats treated as neonates with anti-androgens (Casto et al., 2003; Meaney et al., 1983) show reduced play fighting. Conversely, female rats exposed perinatally to either estradiol or dopamine receptor agonists (Olesen et al., 2005) demonstrate increased play. These results suggest that both androgen and estrogen receptors contribute to sex differences in play fighting behavior in rats (Auger & Olesen, 2009; Pellis, 2002).

Nothing is known about the mechanisms that regulate social play in mice. Moreover, we cannot assume that mice and rats are organized in the same manner (Bonthuis et al., 2010). We use the four core genotype (FCG) mouse model to separate the actions of gonadal hormones and sex chromosome genes on sexually dimorphic behaviors (Arnold & Chen, 2009). In this mouse several adult social behaviors (Gatewood et al., 2006; McPhie-Lalmansingh et al., 2008), differ based upon sex chromosome complement. We hypothesized that sex chromosome genes might also play a role in shaping juvenile social interactions. To test this we assessed sex differences in social interactions in non-sibling C57BL/6J (B6) mice on the day of weaning, postnatal day 21 (PN21). We used a dyadic social interaction test which was developed and previously used to investigate strain differences in juvenile male social behaviors in the context of modeling autism, a sexually dimorphic disease (Mcfarlane et al., 2008). Because autism is a sexually dimorphic disease we are interested in whether sex chromosome genetic differences may be one of its causes. In Experiment 1 sex differences were observed in B6 mice. Next we tested FCG mice to assess the independent roles of gonads and sex chromosome genes. Finally, we asked how tests with siblings, instead of strangers, would affect social interactions.

METHODS

RESULTS

DISCUSSION

Here we report sex differences in juvenile social play behaviors as measured by same-sex dyadic interactions in B6 mice. When paired with a non-sibling, females tended to be less social than males, spending more time exploring the test cage. Females also displayed more anogenital sniffing and solicited play more frequently. Males, on the other hand, spent the majority of their time in close physical contact with their partner. Our results are markedly different than those in rats. Male rats show distinct play behaviors that differ both quantitatively and qualitatively from the play of females (reviewed in (Auger & Olesen, 2009). In both play tests with pairs (Panksepp, 1981), as well as observations made in group-housed males (Meaney & Mcewen, 1986; Meaney & Stewart, 1981), male rats show more social play and play-fighting behaviors than do females. However, it is possible that group observation tests, using whole litters, detect more sex differences than dyadic tests (Auger & Olesen, 2009). Although we found sex differences in a paired test, it may be important in the future to compare male and female mice using group (litter) observations.

Because sex differences were apparent in normal inbred mice, we asked if these differences could be attributed to sex chromosome genes and/or gonads by testing FCG mice in the same test. Again, XX females were less social than other genotypes, but they did not differ statistically from XY males on any social or nonsocial behavioral measure. Additionally, in contrast to our findings in normal B6 mice, our FCG “normal” females and males (XX females and XY males) spent more time investigating their partner than hetero-chromosomal mice (XY females and XX males). In addition, XY males solicited play more than mice in any other group. Overall, the interactions between gonadal sex and sex chromosomes were responsible for differences, suggesting that sex chromosome genes are not acting alone to influence these behaviors. Since we found no differences in circulating hormones on PN21, developmental hormones are likely interacting with sex chromosome genes to differentiate social interactions. From these experiments, we cannot concluded which hormone(s) during development are influencing mouse juvenile interactions, but the data on play in rats suggest that both androgens and estrogens are involved (Auger & Olesen, 2009; Pellis, 2002). Future studies in which hormones are manipulated during the neonatal period will help clarify how sex differences in these behaviors arise.

In the last experiment, we asked whether the familiarity of partner had an impact on social behavior. Interestingly, sibling pairs of XX females were more social than any other group, and they spent less time exploring and investigating than XY females and XX males. XX females spent the majority of time sitting side-by-side with their sisters. As was the case in Experiment 2, there were no differences between XX females and XY males. XY males behaved the same whether they were tested with a sibling, as compared to a non-sibling, and did not investigate or play often with their sibling partner. While it is not necessarily surprising that the juveniles would interact differently with siblings than non-siblings, it is interesting that the XX females were the only group to show significant differences in social behaviors when paired with siblings. Adult XX females, tested in a resident-intruder paradigm with a bulbectomized male, display longer latencies to follow the intruder, sniffed the intruder less, and exhibited more digging and avoidance behavior than XY females (McPhie-Lalmansingh et al., 2008). Therefore, XX females may be more responsive to environmental and social surroundings than mice of other genotypes. Social isolation (Terranova et al., 1993), litter sex ratios (Laviola & Alleva, 1995), and spacing of litters (Branchi et al., 2009) have all been examined as factors that influence juvenile mouse social behavior, but, to our knowledge, ours is the first study to demonstrate that juvenile social interactions with siblings differ from those with non-siblings.

This is the first examination of the role of sex chromosome genes in juvenile behaviors. Our lab has reported that other social behaviors are influenced by interactions of sex chromosome genes with gonadal sex in adult mice. Gonadectomized XX females are significantly slower than XY females to follow an intruder in a social interaction test (McPhie-Lalmansingh et al., 2008). In addition, gonadectomized XX females implanted with testosterone are less aggressive than XY females (Gatewood et al., 2006), who behave similarly to testosterone treated XX and XY males. Taken together these data support the idea that changes to sex chromosome complement, along with gonadal hormones during development, affect mouse social behavior.

It is not clear why the patterns of the sex differences we noted in B6 mice were different in the FCG mice, but there are several obvious differences between these lines. One interesting distinction is the origin of the Y chromosome in the FCG. The FCG autosomes and X chromosomes are completely crossed in the B6 background; their Y chromosome, with the spontaneous Sry mutation, is from the 129/Sv strain. Thus, the XY B6 male is not genetically identical to the XY FCG male. This could produce differences in male offspring behavior (reviewed in Curley & Mashoodh, 2010), as strain differences in social behaviors have been previously shown (Moy et al., 2009). Recently, paternal Y chromosomes from different strains have been shown to influence the behavior of female offspring (Nelson et al., 2010), which in the FCG could also affect XX versus XY female behavior. In addition, it is possible that the FCG breeders provide qualitatively and/or quantitatively different parental care as compared with B6 breeders. The difference between the pairs is restricted to the post-natal period since in both cases XX B6 females are the dams. Variations both in parent-offspring contact (Parent & Meaney, 2008) as well as sibling interactions could have significant influences on juvenile social behavior (Curley et al., 2010; Laviola & Alleva, 1995).

Our findings show that B6 mice differ from CD-1 strain in which females were more social and explored less than males (Terranova et al., 1993). However, we used different testing procedures; for example the CD-1 study included repeated tests throughout the juvenile period and some mice were housed alone after weaning. Importantly, our findings that B6 females solicited play more frequently than males are consistent with this study and other subsequent studies by the same group (Laviola & Alleva, 1995). Whereas we are the first to directly compare play behavior in non-siblings vs. siblings, other experiments using males only and a testing procedure similar to ours noted differences in amount of investigation by B6 mice depending on whether they tested with siblings (Yang et al., 2007) or non-siblings (Mcfarlane et al., 2008).

In rodents, as in humans, juvenile play behaviors may be important for the development of social skills and other adaptive behaviors in adults (Panksepp et al., 1984). There are already several genes implicated in the development of social play, including the X-linked MeCP2 gene (Guy et al., 2001; Kurian et al., 2008), which is involved in Rett Syndrome in humans, Kdm5c an X-inactivation escapee which has been linked to X-linked mental retardation, (Xu & Andreassi, 2010), and the genes for vasopressin receptors 1a (Bielsky et al., 2005) and 1b (Scattoni et al., 2008), which are implicated in autism spectrum disorders (ASD). It is important to note that there are sex differences in ASD: this disorder is about 4 times more common in males than females (Auyeung et al., 2010; Giarelli et al., 2010), and it is not known what the cause of this dimorphism is. While future studies are needed to understand how sex differences in social behavior develop, our results show that sex chromosome genes, along with strain differences in Y chromosome genes, can influence juvenile social behavior. Thus, sex chromosome genes may be important contributors to the development of ASD and other neurobehavioral diseases.

Supplementary Material