Article | Perinatal vinclozolin alters sex differentiation exposed during gestation and the neonatal period via maternal dosing with the gavage procedure. Although many play behavior studies have focused on the role of androgens during the neonatal period, perhaps because of the ease of working with newborn versus fetal rats, the available evidence suggests that the critical period for the differentiation of play begins late in gestation and continues through PND10 (Meaney et al. 1983; Ward and Stehm 1991). Data on the effects of prenatal morphine suggest that the onset of the critical period for play is GD16 (Niesink et al. 1999). A survey of the developmental toxicology literature indicates that the reduction of erec- tions measured in the present study is one of the most sensitive outcomes observed to date in a perinatal Vz study. Earlier work found that reduced AGD in male neonates and nipple retention occurred after exposure to maternal doses as low as 3.125 mg/kg, whereas at least 50 mg/kg was required to affect ventral prostate weight and increase the incidence of hypospadias (Gray et al. 1999a). Although behavior analysis was not an objective in these earlier investigations, during copulation, exposed males mounted but were unable to achieve vaginal penetration (Gray et al. 1994). In adult male rats, a number of manipulations can produce similar effects, including castra- tion (Leipheimer and Sachs 1993), lesions of the medial preoptic area (Everitt 1990), or microinjection of dopamine antagonist drugs (Pfaus and Phillips 1991). In developing males, prenatal exposure to antiestrogens (Matuszczyk and Larsson 1995) also appears to impair cop- ulatory performance without disturbing sexual motivation. All of these procedures produce structural or functional changes in the erectile system (Hull et al. 1992; Monaghan et al. 1993; Warner et al. 1991). The Vz-exposed males showed a selective reduction of low-intensity (E1) erections. In this regard, the exposed offspring resemble males castrated as adults, which also show an early reduction of E1 erections (Leipheimer and Sachs 1993). The behavior of the exposed offspring is also reminiscent of male rats that have been administered serotonin receptor agonists (Mas et al. 1985) or agents that block the synthesis of nitric oxide (Hull et al. 1994). Both of these treatments reduce erections and increase seminal emissions. Perhaps the most parsimonious explanation of the differential regulation of penile erections versus seminal emissions has been offered by Hull and others. In a series of drug microinjection studies, this group has demonstrated that pharmacologic stimulation of dopamine D2 receptors in the medial preoptic area decreases the frequency of erections while increasing seminal emissions (Bazzett et al. 1991). Stimulation of D2 recep- tors in the paraventricular nucleus also facili- tates seminal emission (Eaton et al. 1991; Pehek et al. 1989). On the other hand, stimu- lation of D1 receptors in the medial preoptic area has the opposite effect and occurs at much lower doses (Hull et al. 1992). Because the functional integrity of dopamine systems in this part of the brain is maintained by cir- culating testosterone (Du and Hull 1999), an environmental antiandrogen such as Vz might disrupt the development of these complex interactions. As mentioned above, animal studies indi- cate that fetal males are far more sensitive to environmental antiandrogens than adults. Results from maternal stress studies shed some light on the likely developmental mechanisms affected by environmental antiandrogens. Maternal stress during the last week of preg- nancy lowers the surge of plasma testosterone that is normally present in male rat fetuses during GD18 and GD19 (Ward and Weisz 1984). Attenuation of the GD18–19 surge is associated with impaired sexual behavior in adulthood (Dunlap et al. 1978; Ward and Reed 1985). This testosterone surge also exerts an organizational effect on the muscle and spinal cord mechanisms that control penile erections in adulthood (Grisham et al. 1991). Perinatal androgens serve to rescue SNB motor neurons from programmed death (Sengelaub et al. 1989), a process that could be blocked by an antiandrogen like Vz. In the present study, animals were exposed to Vz from GD14 through PND3 in order to com- pare our results with previous perinatal Vz investigations. However, because differentia- tion of spinal cord motor neurons continues until PND10 (Mills and Sengelaub 1993) and the weight of the adult BC muscle is the most sensitive to Vz exposure during the GD16–17 period (Wolf et al. 2000), it is likely that the toxic window for Vz on erectile function spans the GD16–PND10 period. Thus, it appears that the critical periods for masculinization of erectile function and play behavior in the male rat are the same. As of yet, no one has exam- ined the effects of environmental antiandrogen exposure during this entire perinatal sensitive period. It may be the case that social play and erectile functions are responsive to even lower doses of Vz, if an exposure were to span the GD16–PND10 period. No previous studies explicitly link Vz to human erectile dysfunction. However, Vz and other antiandrogenic fungicides are used in agriculture, they may be responsible for the recently noted link between pesticide exposure and erectile dysfunction in otherwise healthy men. Speciﬁcally, pesticide-exposed men had a significantly higher incidence of complete impotence, showing little to no change from baseline ﬂaccidity on measures of penile rigid- ity, tumescence, frequency, and duration (Oliva et al. 2002). Occupational exposure to stilbene has also been associated with an Environmental Health Perspectives • VOLUME 113 | NUMBER 6 | June 2005 increase in self-reported impotence and decreased libido (Quinn et al. 1990; Whelan et al. 1996). Stilbene is a component of textile ﬁnishing agents and detergents, and it is struc- turally similar to the synthetic estrogen diethylstilbestrol. Both of these clinical studies examined the effects of exposure during adult- hood. The long-term effects in men after peri- natal exposure are unknown. It is estimated that children 1–6 years of age are exposed to 0.167 mg Vz/kg body weight/day (U.S. EPA 2000b). Given this chronic exposure estimate, a 2-year-old boy who weighs 13 kg [Centers for Disease Control and Prevention (CDC) 2000] would consume an average of 2.17 mg Vz/day, whereas a 6-year-old with a body weight of 21 kg would consume an average of 3.51 mg Vz/day. Both of these estimated daily intakes exceed the ED10 benchmark doses associated with altered juvenile play behavior and erec- tile dysfunction in our animal model. Typically, the U.S. EPA would divide the 95% lower bound by 100 to calculate a refer- ence dose. If this practice were applied to the juvenile play behavior or erectile data, the average daily intake of Vz would exceed the reference doses based on these data by more than two orders of magnitude. It should also be pointed out that humans are exposed to multiple compounds on a chronic basis, whereas this study examined only Vz, which was administered during a limited period of development. The cumulative effects of chronic exposure to multiple compounds and their metabolites are unknown. Lastly, our benchmark doses should be interpreted as conservative estimates because they are based on maternal doses. The actual amount of Vz and/or metabolite that entered our fetal or neonatal subjects is unknown, although the level was certainly lower than the applied maternal dose. In conclusion, the results of this study demonstrate that the effects of perinatal expo- sure to an environmental endocrine disruptor can be observed throughout the life span, pro- vided that age-appropriate, sex-specific end points are examined. Low doses of Vz adminis- tered during the GD14–PND3 period signiﬁ- cantly increased social play behavior in juvenile male rat offspring. These results are interesting in light of recent findings in humans that higher prenatal levels of PCBs have been asso- ciated with less masculinized play behavior in Dutch schoolboys and more masculinized play in schoolgirls (Vreugdenhil et al. 2002). Even lower doses of Vz reduced the erectile response in adult male offspring. Because men who work with agricultural chemicals are more likely to experience erectile dysfunction (Oliva et al. 2002), it is quite possible that some of the relevant agrochemicals are antiandrogenic fungicides. 705