Arch Sex Behav (2008) 37:9–18 DOI 10.1007/s10508-007-9259-z ORIGINAL PAPER: MINOT SPECIAL ISSUE Intrauterine Position Effects on Anogenital Distance and Digit Ratio in Male and Female Mice Peter L. Hurd Æ Allison A. Bailey Æ Patricia A. Gongal Æ Reginia H. Yan Æ John J. Greer Æ Silvia Pagliardini Published online: 14 December 2007 Ó Springer Science+Business Media, LLC 2007 Abstract Anogenital distance (AGD) and the ratio of the Introduction second (index) to fourth (ring) digit lengths (2D:4D) are two widely used indicators of prenatal androgen exposure. The Prenatal exposure to male sex hormones can have a life-long former is commonly used in rodent models, while the latter is influence on behavior in both mice and humans (Cohen- principally used in human studies. We investigated variation Bendahan, van de Beek, & Berenbaum, 2005; Ryan & in these two traits in C57BL/6J mice to test the hypothesis Vandenbergh, 2002). Humans exposed to higher levels of that variation in these two traits reflect a common underlying androgens in utero are more aggressive (Berenbaum & variable, presumably testosterone exposure. AGD is a sex- Reinisch, 1997; Reinisch, 1981) and girls exposed to high ually dimorphic trait used to sex young rodents. This distance prenatal androgens show more male-typical play behavior, typically increases and becomes more male-like in females lower heterosexual interest, and more masculine core sexual pups when their uterine neighbors are male. 2D:4D is sex- identity (Hines, Brook, & Conway, 2004). Mice show com- ually dimorphic in a number of species, including humans parable behavioral effects as a function of intrauterine and other great apes. Lower digit ratios may be associated position (IUP). Female mice gestating between two males are with greater exposure to androgens during fetal development exposed to greater androgen concentrations and show more in humans. We found the expected sexual dimorphism in masculinized aggressive and sexual behavior than those AGD, but no significant sex difference in 2D:4D, and no gestating between two females (Clark & Galef, 1995; Ryan & correlation between 2D:4D and AGD. Gestating next to Vandenbergh, 2002; vom Saal, 1984, 1989; vom Saal, Grant, males increased a pup’s 2D:4D ratio, but it had no effect McMullen, & Laves, 1983). Anogenital distance (AGD), on AGD. The lack of correlation between 2D:4D and AGDs which is used tosex young rodents (maleshave larger AGDs), in this mouse strain suggests that these two measures do is also influenced by IUP: Females with more male neighbors not reflect a common influence of androgen exposure. The have longer AGDs (Ryan & Vandenbergh, 2002). possible roles of temporal and localized effects of masculin- Not all mouse strains show an IUP effect on AGD. For ization are discussed. example, CF-1 and ICR mouse strains do not exhibit any IUP effect (Nagao et al., 2004; Simon & Cologer-Clifford, Keywords Intrauterine position Á Digit ratio Á 2D:4D Á 1991). Androgens are also not the only hormones implicated Anogenital distance Á Masculinization in AGD variation. In CD-1 mice, AGD can be masculinized by increased estrogen exposure (Gupta, 2000). Similarly, female uterine neighbors and estradiol exposure have a masculinizing effect on prostate morphology (Even, Dhar & vom Saal, 1992; vom Saal, 1989; vom Saal et al., 1997; Timms, Petersen, & vom Saal, 1999). The aromatization P. L. Hurd (&) Á A. A. Bailey Á P. A. Gongal Á hypothesis (MacLusky & Naftolin, 1981; Naftolin & Mac- R. H. Yan Á J. J. Greer Á S. Pagliardini Lusky, 1984) predicts that masculinization may result from Department of Psychology, University of Alberta, Edmonton, AB, Canada T6G 2E9 estrogen exposure, as well as androgen exposure when e-mail: [email protected] masculinization is driven by estrogen locally aromatized 123 10 Arch Sex Behav (2008) 37:9–18 from testosterone in the target tissue. Differentiation of shown to influence AGD in mice (Gupta, 2000; Ryan & sexually dimorphic nuclei within the brain appears to be due Vandenbergh, 2002). to such a mechanism (Lephart, Lund, & Horvath, 2001; The global androgenization hypothesis predicts that MacLusky, Walters, Clark, & Toran-Allerand, 1994; Svare, neighboring males will masculinize both AGD and 2D:4D Kinsley, Mann, & Broida, 1984). and, therefore, that AGD and 2D:4D will correlate within Anogenital distance is not the only morphological feature sexes. We make no prediction on the direction of the cor- suggested to be affected by prenatal androgen exposure. The relation, given the uncertainty over the direction of sexual ratio of second (index) to fourth (ring) finger lengths has also dimorphism in this trait in animals. The localized effects been suggested to reflect in utero androgen exposure (Man- hypothesis suggests that there is no single ‘‘true’’ level of ning, 2002). This digit ratio, 2D:4D, is sexually dimorphic in androgen exposure, and thus the masculinizing effects of great apes (McFadden & Bracht, 2005; Peters, Tan, Kang, IUP may vary between these two traits. Teixeira, & Mandal, 2002), lab mice (of the genus Mus; Brown, Finn, & Breedlove, 2002; Manning, Callow, & Bundred, 2003; but not in a larger study by Bailey, Wahlsten, Method & Hurd, 2005), wood mice (of the genus Apodemus; Leoni, Canova, & Saino, 2005), zebra finches (Taeniopygia guttata; Subjects Burley &Foster, 2004; but not ina larger studyby Forstmeier, 2005) and pheasants (Phasianus colchicus; Saino, Rubolini, C57BL/6J mice were bred at the University of Alberta. The Romano, & Boncoraglio, 2007; but not in a different line; timing of pregnancies was determined from the appearance Saino, Rubolini, Romano, & Boncoraglio, 2005). Sexual of sperm plugs in the breeding cages, and the embryonic age dimorphism in human digit ratio has been noted since at least of mice was confirmed by measuring crown-rump length the late 1800s (Peters et al., 2002). (Kaufman, 1994). Pregnant mice at the 18th day of gestation More recent scientific attention has focused upon the (sperm plug appearance = day 0) were anesthetized with relationship between digit ratios and psychological traits halothane (1.25–1.5% delivered in 95% O2 and 5% CO2) (Manning, 2002). Digit ratio correlates with various psy- and pups were delivered by Cesarean section. The position chological traits, including sexual orientation (Brown, Finn, from the uterine horn was recorded and the fetuses were Bradley, & Breedlove, 2002; Hall & Love, 2003; Lippa, immediately fixed in 4% paraformaldehyde in phosphate 2003; McFadden & Shubel, 2002; McFadden et al., 2005; buffer. Mice were sexed by inspection of the gonads during Rahman & Wilson, 2003; Williams et al. 2000), sex role dissection. The 95 pups (44 males and 51 females) from nine strength (Csatho´ et al., 2003), and aggression (Bailey & litters were distributed across IUPs as listed in Table 1.By Hurd, 2005; Benderlioglu & Nelson, 2004). Lower 2D:4D using all pups, rather than restricting comparisons only has been suggested to result from greater exposure (Lutch- between those with either two male or two female neighbors, maya, Baron-Cohen, Raggatt, Knickmeyer, & Manning, we could separate the effects of neighboring males and 2004) or sensitivity to (Manning, Bundred, Newton, & females and gain additional statistical power. Since any pup Flanigan, 2003) androgens during fetal development. Female had a maximum of two uterine neighbors, having two of one dizygotic twins having male co-twins have more masculine sex means they will have none of the other; while the number left hand 2D:4D than those with female co-twins (van Anders, of neighboring male and female pups was necessarily Vernon, & Wilbur, 2006). inversely related, r(93) =-.68, p < .0001, the two mea- Because both of these morphological traits (AGD and sures were different enough, due to pups with only one 2D:4D) have been suggested to reflect the degree to which an neighbor, that we were able to discriminate between individual’s central nervous system has been masculinized the effects of neighboring males and females on pup mor- (Manning, 2002; Ryan & Vandenbergh, 2002), we should phology. Procedures for animal care were approved by the expect AGD and 2D:4D to show similar patterns of variation Animal Welfare Committee at the University of Alberta. across IUPs. Our recent study of inbred mouse strains found significant variation in digit ratio between strains, but no significant difference between the sexes (Bailey et al., Table 1 Distribution of 44 males and 51 females across the six 2005). We hypothesized that these results were due to intrauterine positions masking of sex differences by IUP effects, and that con- trolling for IUP would magnify sexual dimorphism in digit Pup’s sex Intrauterine position (sex of neighboring pups) ratio. In order to test this hypothesis, we analyzed AGD and 0M 0F 0M 1F 0M 2F 1M 0F 1M 1F 0M 2F hind paw 2D:4D ratios in 95 C57BL/6J mice by uterine Male 1 7 10 6 15 5 position. We tested for the effect of pup sex and neighboring Female 1 10 5 11 17 7 pup sex, because these are the IUP variables previously 123 Arch Sex Behav (2008) 37:9–18 11 Measures paw side. All analyses were performed on data from the right rear paws. AGD The GIMP ‘‘measure’’ tool was used to measure all images. Each digit (2D and 4D) was measured from the To measure AGD, each fetus was photographed ventrally middle of the base to the tip of the digit, excluding the nail. using a Leica MS5 stereoscope with a Canon Powershot S50 To find the base, we drew a line from the point at which the digital camera. Fetuses were placed ventral side up in a middle digit (3D) joined the target digit (2D or 4D), forming holder to ensure the area between the gonads and anus was a junction with the basal mound. The baseline ran from the flat and consistent between subjects. The hind legs were then top of the basal mound to the outside edge of the digit.