Sexual responses of the male rat medial preoptic area and medial amygdala to estrogen II: Site specific effects of selective estrogenic drugs

Abstract

In the medial preoptic area (MPO) and medial amygdala (MEA), estradiol (E₂) aromatized from testosterone (T) may act via either estrogen receptor (ER) α or ERβ to mediate mating in male rats. We tested the hypothesis that, in the MPO, ERα exclusively mediates sexual responses to E₂ by monitoring mating in four groups of castrated male rats administered dihydrotestosterone (DHT) subcutaneously and MPO implants delivering either: cholesterol, E₂, propyl pyrazole triol (PPT, ERα-agonist) or diarylpropionitrile (DPN, ER β-agonist); a fifth group of intact males served as DPN toxicity control, receiving DPN MPO implants. In a follow-up study, either 1-methyl-4-phenyl pyridinium (MPP, ERα-antagonist) or blank MPO cannulae were implanted in castrated male rats receiving T subcutaneously, whereas intact MPP toxicity controls received MPP MEA implants. PPT or E₂ MPO implants maintained mating, but cholesterol or DPN MPO implants did not. Moreover, MPP MPO implants interfered with T reinstatement of mating suggesting that, in the MPO, ERα is necessary and sufficient for mating in androgen-maintained male rats and ERβ is not sufficient. Because it is unknown which ER subtype(s) mediate sexual responses of the MEA to E₂, we examined mating following MEA implants of cholesterol, E₂, PPT or DPN in four groups of castrated male rats administered DHT subcutaneously. E₂ MEA implants maintained mounting but mating was significantly decreased in groups receiving PPT, DPN or cholesterol MEA implants suggesting that, unlike the MPO where ERα alone is essential, sexual responses of the MEA to E₂ require simultaneous interactions among multiple ER subtypes.

Introduction

Male rat mating behavior is dependent on testosterone (T), which is enzymatically converted in the brain into estradiol (E₂) by aromatase (Naftolin et al., 1975) and into the non-aromatizable androgen, dihydrotestosterone (DHT) by 5α reductase (Martini, 1982, Massa et al., 1972). These metabolites, together with T, act on steroid sensitive neurons, including those containing estrogen receptors (ER) in the medial preoptic area (MPO) (Vagell and McGinnis, 1997) and medial amygdala (MEA) (Huddleston et al., 2003, Huddleston et al., 2006) of the male rat brain to promote mating behavior. Thus, mating declines after castration and is reinstated by exogenous T (Christensen and Clemens, 1975, Davidson, 1966a, Davidson, 1966b, Davidson, 1969). Furthermore, castrated male rats mate normally (to ejaculation) after being treated with a combination of E₂ and DHT, given in physiological dose ranges, and their mating behavior is comparable to that of rats treated with physiological levels of T (Baum and Vreeburg, 1973, Davidson, 1966a, Davidson, 1966b, Davidson, 1969, Larsson et al., 1973). When administered separately, however, neither E₂ (Baum and Vreeburg, 1973, Davidson, 1969, Södersten, 1973) nor DHT (Feder, 1971, Feder et al., 1974, McGinnis and Dreifuss, 1989) fully maintains mating behavior, indicating that estrogens and androgens both are necessary for the normal expression of male rat sexual behavior. Fadrozole, a non-steroidal aromatase inhibitor (Lipton et al., 1990, Vagell and McGinnis, 1997) that blocks formation of E₂ from T, administered either systemically (Bonsall et al., 1992) or intracerebrally into the MPO (Clancy et al., 1995) or MEA (Huddleston et al., 2006) attenuates male rat mating behavior, an effect that can be partially reversed via administration of E₂ (Bonsall et al., 1992). Mating is also maintained by implants of E₂ into the MPO (Clancy et al., 2000) or MEA (Huddleston et al., 2003) of gonadally intact male rats receiving fadrozole subcutaneously (s.c.) that blocks E₂ formation elsewhere throughout the body and brain. Collectively, these studies suggest that estrogen is required for the expression of male rat mating behavior and specifically that estrogenic metabolites of T act on estrogen sensitive neurons in both the MPO (Clancy et al., 1995, Clancy et al., 2000) and the MEA (Huddleston et al., 2003, Huddleston et al., 2006).

Studies of mating-induced expression of Fos immunoreactivity (ir) show that Fos-ir occurs in MPO and MEA cells that contain receptors for gonadal steroids, including those that express ER-ir (Baum and Everitt, 1992, Coolen et al., 1996, Gréco et al., 1996, Gréco et al., 1998, Gréco et al., 2003). Steroid sensitive cells of the MPO and MEA are critical for expression of male rat mating behavior (Huddleston et al., 2007b, Simerly and Swanson, 1986).

Multiple subtypes of ER exist (Gréco et al., 2003, Kuiper et al., 1996), some of which may be located within the cell membranes of E₂ responsive neurons (Evinger and Levin, 2005, Thomas et al., 2005). The second type of ER to be discovered was termed ERβ and the original ER is now known as ERα (Kuiper et al., 1996). ERα and ERβ are both expressed in steroid sensitive neurons of the rat MPO and MEA (Abraham et al., 2004, Gréco et al., 2003, Nomura et al., 2003, Shughrue and Merchanthaler, 2001, Shughrue et al., 1997). ER-Knock-Out (ERKO) experiments in male mice have shown that the deactivation of ERα significantly reduces mating behavior, while βERKO does not greatly decrease mating behavior (Ogawa et al., 1997, Ogawa et al., 1998, Ogawa et al., 2000, Rissman et al., 1997). However, the combined knock-out, of both ERα and ERβ completely eliminates all sexual behaviors of the male mice (Ogawa et al., 2000). This suggests that ERα may play a dominant role, and ERβ a minor role, in the expression of male sexual behavior (Ogawa et al., 1997, Ogawa et al., 1998, Ogawa et al., 2000, Rissman et al., 1997, Scordalakes et al., 2002). In light of this, it is noteworthy that infusion of an ERα antisense oligodeoxynucleotide (AS-ODN) to the MPO significantly decreased male rat mating behavior (Paisley et al., in press). In contrast, ERα AS-ODN infusion of the MEA locally inhibited ERα expression in the MEA, but there was no significant decrease in the mating behavior of male rats (Paisley et al., in press). These findings suggest two hypotheses: (i) in the MPO, ERα mediates sexual responses of male rats to E₂ and (ii) the MEA responds differently to E₂ than the MPO in that a different ER pathway, not ERα but perhaps ERβ, mediates the sexual response of the male rat MEA to E₂.

Recently developed, highly selective estrogenic drugs that modulate ERα and/or ERβ activity may serve as powerful tools that might aid in identifying the behaviorally relevant ER isoform(s) in the male rat MPO and MEA that mediate sexual responses to E₂ (Harrington et al., 2003, Hillisch et al., 2004). Therefore, we used selective estrogenic drugs implanted into the MPO to test the first hypothesis that, in the MPO, ERα mediates sexual responses of male rats to E₂. In one experiment, four groups of male rats were castrated and administered DHT s.c. and then received bilateral MPO implants carrying one of the following drugs: i) cholesterol (negative control), ii) E₂ (positive control), iii) propyl pyrazole triol (PPT, an ERα agonist; Stauffer et al., 2000) or iv) diarylpropionitrile (DPN, an ERβ agonist; Meyers et al., 2001). A fifth group was used as a DPN toxicity control that consisted of gonadally intact male rats that also received bilateral MPO DPN implants. In a second experiment, two groups of male rats were castrated, tested until mating behavior declined, and then implanted with T s.c. and bilateral MPO implants delivering either no drugs (blank) or the drug 1-methyl-4-phenyl pyridinium (MPP, an ERα antagonist; Zhou et al., 2009). In addition, a group of gonadally intact male rats received MPP bilateral MEA implants and served as an MPP toxicity control.

We also tested, using selective estrogenic drugs, the second hypothesis that the MEA responds differently to E₂ than the MPO in that a different ER pathway, not ERα but perhaps ERβ, mediates the sexual response of the male rat MEA to E₂ by comparing the sexual behavior of castrated male rats before and after the drug implantation into the MEA. Four groups of castrated, DHT s.c. maintained male rats received bilateral MEA implants containing one of the following drugs: (i) cholesterol (negative control), (ii) E₂ (positive control), (iii) PPT (ERα agonist; Stauffer et al., 2000) or (iv) DPN (ERβ agonist; Meyers et al., 2001).