Article | Perinatal vinclozolin alters sex differentiation SNB/BC system. Vz exposure during the peri- natal (Wolf et al. 2000, 2004) or peripubertal period (Monosson et al. 1999) significantly reduces the weight of the BC and levator ani muscles in adult males. Other antiandrogens such as procymidone, prochloraz, and linuron also affect the development of the BC muscle (Lambright et al. 2000; Ostby et al. 1999; Vinggaard et al. 2002). What are the functional implications of an underweight BC muscle that has been affected by Vz exposure? Gray et al. (1994) have shown that adult male rats exposed to perinatal Vz will mount sexually receptive females but are unable to achieve vaginal penetration, suggest- ing that there is an underlying erectile dys- function. Other environmental antiandrogens, such as p,p´-dichlorodiphenyldichloroethylene (p,p´-DDE), have already been shown to reduce erectile functions in rats (Brien et al. 2000). Female rats can detect subtle behavioral deficits and prefer to copulate with healthy, dominant males (McClintock et al. 1982). Antiandrogens could therefore affect the reproductive success of a wide range of animal species by altering male copulatory behavior. For instance, female guppies prefer males with high rates of sexual display, and Vz exposure has been shown to significantly reduce male guppy courtship display (Baatrup and Junge 2001; Bayley et al. 2002). Most functional investigations of environ- mental endocrine disruptors have focused on the effects of perinatal exposure in adult off- spring and have ignored the developmental tra- jectory of the effects of antiandrogen exposure. Juvenile play is a sexually dimorphic behavior that is an important precursor to adult sexual behavior (Pellis et al. 1992) and dominance relationships (Pellis and Pellis 1992). Males typically engage in more bouts of play and per- form more behaviors during bouts than females (Thor and Holloway 1983). Even though they are prominent at different times in the life span, juvenile play and copulation are interconnected. During play, rats perform numerous crawl-over behaviors with same-sex partners. There is a shift of interest in male pups in their preferred play partners during the postnatal day (PND)36–40 period, from male to female (Meaney and Stewart 1981). Older, sexually mature but naive males will perform crawl-overs with females until they achieve a mount with a successful vaginal intromission. After the ﬁrst intromission, mounting behavior increases and crawl-overs decrease. Adult males that do not have the opportunity to experience normal play during development show exces- sive play components but little normal copula- tory behavior in the presence of sexually receptive females (Gerall et al. 1967; Goldfoot 1977; Gruendel and Arnold 1960; Hard and Larsson 1968). Thus, early environmental fac- tors can affect important neonatal or juvenile social interactions, culminating in aberrant behaviors in adulthood (Dunlap et al. 1978). The neural mechanisms for play are poten- tial targets for environmental antiandrogens because they are organized in part by androgens during the perinatal period. Administration of the androgen receptor antagonist flutamide during the ﬁrst 10 postnatal days demasculin- izes male rat play behavior (Meaney et al. 1983). Other manipulations such as prenatal protein deprivation (Almeida et al. 1996), perinatal genistein exposure (Flynn et al. 2000), prenatal polychlorinated biphenyl (PCB) exposure (Vreugdenhil et al. 2002), or maternal stress during gestation can also demasculinize play behavior (Ward and Stehm 1991). Recently there has been some interest in the effects of perinatal Vz exposure on social play behavior in juvenile subjects. Hotchkiss et al. (2003) exposed neonatal rat pups to 200 mg/kg Vz on PND2 and PND3. This acute, high-dose exposure had long-term consequences for male rats. Juveniles per- formed significantly fewer chase and pin behaviors during play sessions with a same-sex partner. Female pups were not exposed to Vz in this study, although it is well known that female play can also be altered by neuro- endocrine manipulations (Hines 2003; Nordenstrom et al. 2002; Servin et al. 2003). One study that used a chronic, low-level dietary exposure to Vz found that female rats were more sensitive than males (Flynn et al. 2001). In the present study, pregnant rats were exposed to low doses of Vz through the last third of gestation and for several days after parturition. Play behavior was examined in Table 1. Mean values for parturition end points for control and Vz-exposed rats. Sperm-positive females assigned to groupa Sperm-positive females that delivered a litter Gestation length (days) Pups per litter Male Female Postnatal mortality Exposure group Control Vz 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 14 8 16 15 7 12 7 13 13 7 22.1 22.0 21.9 22.2 22.3 6.3 4.2 6.1 4.7 6.1 6.3 5.7 6.3 5.2 7.1 1 3 3 2 7b aThe number of sperm-positive females differs because several females did not copulate or the sperm plug was not detected. bFive pups were lost from one litter and two from a second litter. Environmental Health Perspectives • VOLUME 113 | NUMBER 6 | June 2005 juvenile male and female offspring. Erectile function in adult males was assessed using the ex copula penile reﬂex procedure. Materials and Methods Breeding and exposure. Long-Evans hooded rats (Harlan, Indianapolis, IN) were allowed to acclimate to the University of Southern Maine Vivarium quarters for 2 weeks before breeding. All rats were fed standard pellet chow (Teklad Global 18% Protein Rodent Diet; Harlan Teklad, Madison, WI) ad libitum and were maintained on a 12-hr light/12-hr dark cycle in a barrier facility room with an ambient tem- perature of 68 ± 2°F and 40–60% humidity. Groups of three females were housed with stud males, and vaginal smears were examined each morning for the presence of sperm. We regarded a sperm-positive smear as gestation day (GD)0. Pregnant rat dams were placed individually into polycarbonate shoebox cages and assigned to an exposure condition accord- ing to a randomized block design. Each block consisted of ﬁve groups: 0, 1.5, 3, 6, or 12 mg Vz/kg maternal body weight. Vz (Crescent Chemical Co. Inc., Islandia, NY) was dis- solved in corn oil, and the appropriate volume (~ 0.5–1.5 mL) was administered via gavage from GD14 through PND3 to coincide with the period of sexual differentiation in the rat (Miller et al. 1988). Vz was not administered on the day of parturition (PND0). We chose the doses in order to examine a range below the U.S.EPA’s LOAEL of 11.5 mg/kg/day (U.S. EPA 2000a). The adverse developmental event that is associated with the LOAEL is the retention of nipples and areolas in immature male offspring. We recorded maternal body weights daily during the gestational period. Cages were inspected each morning and afternoon for the presence of litters. Litter size, sex distribution, pup weights, and AGDs were recorded on PND1 and every 4 days thereafter. Using a randomized procedure, litters were culled to eight offspring on PND4, maintaining equiv- alent sex distributions when possible. After weaning on PND21, offspring were housed with same-sex littermates in plastic cages with filter bonnets. All animal procedures com- plied with approved institutional animal care protocols and in accordance with National Institutes of Health guidelines (Institute of Laboratory Animal Resources 1996). Animal care and welfare were supervised by a veteri- narian and a Registered Laboratory Animal Technologist certified by the American Association of Laboratory Animal Science. The exposure and rearing procedure yielded a total of 51 viable litters (Table 1). From this cohort, we assigned 11, 11, 8, and 6 pairs of same-sex littermates from the 0-, 3-, 6-, and 12-mg/kg groups, respectively, to the play procedure. Only those litters with at least 701