Dopamine Activates Masculine Sexual Behavior Independent of the Estrogen Receptor α

RESULTS

Masculine sexual behavior

In both males and females expression of sexual behavior varied with genotype and drug treatment (p < 0.05 at least for each comparison). As shown in Figure2, the majority of wild-type males mounted, performed pelvic thrusts, and intromitted regardless of drug treatment. In addition, nearly all wild-type males ejaculated whereas none of the vehicle-treated ERαKO males intromitted or ejaculated. All the ERαKO males given apomorphine mounted, thrusted, and intromitted with stimulus females (Fig. 2). Fifty percent of the ERαKOs treated with APO ejaculated.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

The percentage of wild-type (WT) and estrogen receptor α knock-out (ERαKO) mice treated with either vehicle (V) or apomorphine (APO) showing mounting and thrusting behavior toward a receptive female. *Significantly lower than the other groups p < 0.05.

Similar results were noted in females (Fig. 2). There was a significant difference in the frequency of females that mounted other females and thrusted during their mounts, and the frequency varied with genotype and drug treatment (p < 0.05 at least). None of the ERαKO females treated with vehicle mounted or displayed thrusts. In the APO-treated ERαKO group, 70% of the females mounted and thrusted. This rate was comparable to that noted in wild-type females (Fig. 2).

Behavior of males that engaged in sexual activity was examined. No differences between numbers of mounts, mounts with thrusts, or mounts with intromissions or the latencies to perform these behaviors were noted (Table 1). This finding shows that wild-type males were not adversely affected by APO treatment and that ERαKO males treated with APO did not differ in the display of their sexual behavior as compared with wild-type.

View this table:

• View inline
• View popup

Table 1.

Data from male and female wild-type and ERαKO mice tested for masculine sexual behavior

Females that displayed mount bouts and thrusts were also basically similar in all measures, regardless of genotype and drug treatment (Table 1). The only exception was in total number of mounts displayed (F_(2,19) = 5.32; p < 0.02). ERαKO females treated with APO mounted significantly more than wild-type females that received either vehicle or APO treatment (p < 0.05).

Social preference

The amount of time males spent sniffing the anesthetized stimulus animals varied with genotype and treatment. Time spent sniffing the stimulus female varied with genotype (F_(1,39) = 17.7; p < 0.0003), drug treatment (F_(1,39) = 4.4; p < 0.05), and there was a significant interaction between the factors (F_(1,39) = 15.5; p < 0.0005). The ERαKO vehicle-treated males were responsible for these effects; they spent less than one-third the amount of time sniffing females as compared to males in the other three groups (p < 0.05; Fig.3). A similar pattern was seen for time spent sniffing the stimulus male (main effect of genotype and an interaction; F_(1,39) = 5.95;p < 0.02 for both). The difference between times spent sniffing a female versus a male was similar as well (main effect of genotype and an interaction; F_(1,39) = 11.5, 9.9 respectively; p < 0.004 at least). None of the other measures were significant; these included time spent in the compartment containing the male versus the female and the number of visits to each compartment.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

The mean amount of time in seconds (+SEM) male wild-type (WT) and estrogen receptor α knock-out (ERαKO) mice treated with either vehicle (V) or apomorphine (APO) spent engaged in chemoinvestigation of an anesthetized estrogen-treated female or an anesthetized gonad-intact male. *Significantly less time spent investigating the male versus the female, p< 0.05. **ERαKO vehicle-treatment animals spent significantly less total time (p < 0.05) engaged in chemoinvestigation than mice in the other three treatment groups.

Females did not exhibit social preferences (Fig.4). There was a trend for ERαKO females that received vehicle to spend more time sniffing the stimulus females than the subjects in the other three groups (F_(1,38) = 3.39; p = 0.074).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

The mean amount of time in seconds (+SEM) female wild-type (WT) and estrogen receptor α knock-out (ERαKO) mice treated with either vehicle (V) or apomorphine (APO) spent engaged in chemoinvestigation of an anesthetized estrogen-treated female or an anesthetized gonad-intact male.

DISCUSSION

The present results replicate our past findings that gonadectomized, T-treated male and female ERαKO mice fail to exhibit normal masculine sexual behavior as compared with similarly treated wild-type littermates (Wersinger et al., 1997). However, here we show that normal masculine behavior can be elicited in adulthood if T is supplemented by APO. A similar result was obtained for chemoinvestigatory behavior in males. We have shown that ERαKO males fail to preferentially investigate anesthetized E₂-treated stimulus females (Wersinger and Rissman, 2000). In the present study the same effect was noted, but APO treatment corrected this deficiency in ERαKO males. Chemoinvestigatory behavior is displayed by wild-type and ERαKO females, but they do not have social preferences. Moreover, unlike masculine sexual behavior, female-directed chemoinvestigation was not activated by T alone or T supplemented with APO in females. Taken together our results show that masculine sexual behavior is not particularly sexually dimorphic in wild-type mice. Aspects of male copulatory behavior that do not depend on a penis can be activated in females by elevated levels of T. Moreover, this male-typical copulatory performance is not dependent on the ERα in mice of either sex. In contrast, female-directed chemoinvestigation is sexually dimorphic in mice, and increasing levels of T did not facilitate display of this behavior in females. Although APO can activate this behavior in male ERαKOs, drug treatment had no effect on preference behavior in female mice of either genotype.

The HPLC data show that neural content of several catecholamines and their metabolites is largely similar between wild-type and ERαKO mice. However, for masculine sex behavior in rats it is critical that DA is not only present, but is released into the extracellular environment where it can bind its postsynaptic receptors (Hull et al., 1995, 1997). The question of DA availability in synapses in the ERαKO brain still needs to be addressed. We found sex differences in catecholamine levels in many regions (Table 2). Wild-type females, despite having lower levels of catecholamines than males, expressed masculine sexual behavior, demonstrating that neural catecholamine content is sufficient in females for the expression of these behaviors. All mice in our study received equivalent T treatment, thus, differences in steroid levels do not explain the sex differences in catecholamine content. These sex differences could be caused by differential sensitivity to adult T. Regardless of the nature of the sex difference, it is clear that its development does not rely on ERα. The striatum was the only region in which we detected an effect of genotype on catecholamine content. The striatum has been implicated in male sexual behavior. In male rats, extracellular dopamine increased in the striatum after copulation (Damsma et al., 1992). Thus, it is possible that ERαKO males, like castrated rats (Hull et al., 1995) accumulate, but do not release DA in the brain. This inability to release DA may contribute to the failure of ERαKOs to display masculine sexual behaviors.

Many lines of evidence show that dopamine is involved in male sexual behavior. Sexually experienced, long-term castrated rats can display copulatory behavior, including ejaculation in some cases, after treatment with APO (Scaletta and Hull, 1990). More reliable sexual behavior is seen when castrated male rats are treated with subthreshold doses of T and APO is provided (Hull et al., 1997). Dopamine-deficient knock-out mice require both T and dopamine to display sexual behavior (Szczypka et al., 1998). In addition, DA antagonists impair copulatory behavior (Pehek et al., 1988) in rats. Hull et al. (1997) have proposed a pathway in which T upregulates nitric oxide synthase (NOS) in the MPOA, NOS in turn enhances DA release. Immunoreactivity for NOS neurons in the MPOA is enhanced in male rats exposed to females (Dominguez and Hull, 1999). Castration leads to fewer NOS-IR cells in the MPOA and bed nucleus of the stria terminalis (Du and Hull, 1999). Pilot data collected in our lab show that ERαKO males have fewer NOS-ir cells in the MPOA than wild-type males (J. Perkins and E. Rissman, unpublished observations). These data suggest that T regulates NOS via actions on ERα. Data collected on endothelial NO production supports this hypothesis (Shaul, 1999). Moreover, basal release of endothelium-derived NO is significantly lower in aorta tissue from ERαKO as compared to wild-type male mice (Rubanyi et al., 1997). Thus, ERα may act during development and/or adulthood as a modulator of NOS activity in brain.

Another link between dopamine, steroid receptors, and sexual behavior is the progesterone receptor (PR). Interestingly estrogen can induce PR (mRNA and protein) in the female ERαKO brain (Shughrue et al., 1997;Moffatt et al., 1998). In the caudal ventromedial hypothalamus, the numbers of estrogen-induced PR-IR cells are only 50% lower than that seen in wild-type females (Moffatt et al., 1998). In female rats and mice DA can stimulate sexual behavior by activating the PR in a ligand-independent manner (Mani et al., 1994, 1996). Although generally associated with female sexual behavior, several lines of evidence have shown that the PR may be involved in male sexual behavior. For example, on their first behavior test, male PR knock-out mice display reduced levels of masculine sexual behavior, compared with wild-type males (Phelps et al., 1998). If similar mechanisms are in place in males, it is possible that APO may act on PR in the MPOA. Perhaps in the ERαKO and wild-type mouse, APO activates unoccupied PR and this stimulates masculine sexual behavior, given the correct hormone and testing conditions.

The involvement of dopamine in differentiation of sex differences has been examined in two contexts. Several researchers have treated pregnant rats, or their pups, with DA agonists or antagonists and tested the offspring for masculine sexual behavior (Hull et al., 1984; Gonzales and Leret, 1992). Dopamine agonists and antagonists are both able to demasculinize males (Hull et al., 1984; Gonzales and Leret, 1992). Thus, the interpretation of these data has to be that some optimal level of DA is required during development, either too much or too little can demasculinize genetic males. Reisert and Pilgrim (1991) have extensively studied sex differences in dopaminergic cellsin vitro . In mouse and rat, fetal dopaminergic neurons from midbrain or hypothalamus are removed before gonad differentiation. After several days in culture sex differences develop, either in the absence of steroids, or in the presence of equivalent titers of steroid (Beyer et al., 1991). Thus, a possibility for the effects of APO in ERαKO mice is that dopamine acts on a pathway that is not influenced by the lack of ERα during development.

Dopamine can stimulate copulatory behavior in male and female mice that lack the ERα. Thus, ERα is not required for neural development of masculine sexual behavior circuits in mice. This idea is a radical departure from the dogma, however, the organizational/activational hypothesis does not explain all sexual dimorphic behaviors (Arnold, 1996). Moreover, although strain differences exist, mice in general do not have as pronounced sexual dimorphisms in brain as do other rodents (Brown et al., 1999). Yet, social preference, as measured by chemoinvestigation of females, is more sexually dimorphic in mice than is masculine copulatory behavior. The accessory olfactory system is sexually dimorphic in rats (Guillamon and Segovia, 1997). Exposure to steroid hormones controls the development of this dimorphism, which can be reversed by steroids during the critical period (Collado et al., 1998). The neural projection pathway from the AOB includes the MA, bed nucleus of the stria terminalis, and the hypothalamus. One terminal region, the anterioventral periventricular region is sexually dimorphic, and this dimorphism is nearly reversed in male ERαKO brains (Simerly et al., 1997). In ongoing studies we are examining sex differences in neural pathways that underlie perception of chemosensory cues in mice.

Our data compliment those collected by others using the ERαKO model. Estrogenic responses can be elicited in these animals under certain conditions (Das et al., 1997; Shughrue et al., 1997; Moffatt et al., 1998; Singh et al., 2000). For example, estradiol can phosphorylate extracellular signal-regulated kinase 1 in brains of ERαKO females (Singh et al., 2000). It is possible that an estrogen-responsive protein other than ER mediates these estrogenic effects. Alternatively, the disrupted ERα gene is transcribed to form a truncated, presumable inactive, protein (Couse et al., 1995). Our finding elaborates on data collected by others in showing that the ERα deficit can be overridden by a nonestrogenic mechanism.