Aromatization Is Not Required for the Facilitation of Appetitive Sexual Behaviors in Ovariectomized Rats Treated With Estradiol and Testosterone

Abstract

Testosterone can be safely and effectively administered to estrogen-treated post-menopausal women experiencing hypoactive sexual desire. However, in the United States and Canada, although it is often administered off-label, testosterone co-administered with estradiol is not a federally approved treatment for sexual arousal/desire disorder, partly because its mechanism is poorly understood. One possible mechanism involves the aromatization of testosterone to estradiol. In an animal model, the administration of testosterone propionate (TP) given in combination with estradiol benzoate (EB) significantly increases sexually appetitive behaviors (i.e., solicitations and hops/darts) in ovariectomized (OVX) Long-Evans rats, compared to those treated with EB-alone. The goal of current study was to test whether blocking aromatization of testosterone to estradiol would disrupt the facilitation of sexual behaviors in OVX Long-Evans rats, and to determine group differences in Fos immunoreactivity within brain regions involved in sexual motivation and reward. Groups of sexually experienced OVX Long-Evans rats were treated with EB alone, EB+TP, or EB+TP and the aromatase inhibitor Fadrozole (EB+TP+FAD). Females treated with EB+TP+FAD displayed significantly more hops and darts, solicitations and lordosis magnitudes when compared to EB-alone females. Furthermore, TP, administered with or without FAD, induced the activation of Fos-immunoreactivity in brain areas implicated in sexual motivation and reward including the medial preoptic area, ventrolateral division of the ventromedial nucleus of the hypothalamus, the nucleus accumbens core, and the prefrontal cortex. These results suggest that aromatization may not be necessary for TP to enhance female sexual behavior and that EB+TP may act via androgenic pathways to increase the sensitivity of response to male-related cues, to induce female sexual desire.

Keywords: aromatase; estradiol; fadrozole; preclinical model; sexual desire; testosterone.

FIGURE 1

Experimental timeline. Females were ovariectomized 1 week after arrival into the colony, and given 1 week of recovery. All females were primed with estradiol benzoate (EB) 48 h before, and progesterone (P) 4 h prior to each of four sex-training sessions with males. After a 2 week hormone washout period females were randomly assigned to one of three experimental groups (n = 11/group): EB+Oil+Saline, EB+TP+Saline, or EB+TP+FAD (fadrozole). Estradiol benzoate (EB) was administered to experimental females by subcutaneous injection 48 h, and testosterone propionate (TP), or an equal volume of the oil control, 4 h before testing. FAD, or an equal volume of the vehicle control, was administered by subcutaneous injection at 8 a.m. and 8 p.m. every day for 3 days including the test day. For the experimental session, females were given 30 min to copulate with a sexually vigorous male.

FIGURE 2

Representative pictures of Fos-immunoreactive cells taken at 40X magnification in different hypothalamic and limbic structures in ovariectomized female rats treated with estradiol benzoate (EB) alone, or in combination with testosterone propionate (TP), or TP and the aromatase inhibitor fadrozole (FAD). Landmarks used to identify regions of interest include the claustrum (cl) and the forceps minor corpus callossum just superior to the cl for the IL; the ventral extent of the lateral ventricle and the anterior portion of the anterior commissure (aca) for the NAc; the 3rd ventricle, optic chiasm and continuous anterior commissure for the mPOA; the 3rd ventricle and arcuate nucleus (Arc) and the three distinct VMH subdivisions for VMH sections; the optic tract (opt), internal capsule (ic), and piriform cortex for MeA sections, and the dorsal 3rd ventricle, medial mammillary nuclei (ML and MM, lateral and medial, respectively) and the fasciculus retroflexus (fr) for the VTA. IL, Infralimbic prefrontal cortex; NAc, Nucleus Accumbens; mPOA, medial preoptic area; VMH, ventromedial hypothalamus; MeA, medial amygdala; VTA, ventral tegmental area.

FIGURE 3

Median frequency of hops/darts (A), solicitations (B), level changes (C), defensive behaviors (D), lordosis rating (E), and lordosis quotient (F) of ovariectomized Long-Evans rats (n = 11/group) treated with estradiol benzoate (EB) with or without testosterone propionate (TP) and the aromatase inhibitor fadrozole (FAD). Data were analyzed using Kruskall–Wallis to detect differences between groups, and significant effects were followed up using Mann–Whitney U, and p-values were adjusted using a Bonferroni correction. Boxes represent interquartile range, and whiskers each represent the top and bottom 25% of scores. ^o Outlier. +Extreme outlier. ^∗Different from EB-alone, p_adj < 0.05. ^#Tendency to differ from EB-alone, p < 0.05, or p_adj < 0.10. ^aTendency to differ from EB+TP, p < 0.05.

FIGURE 4

Median frequency of mounts (A), intromissions (B), and ejaculations (C) that males made toward ovariectomized Long-Evans rats (n = 11/group) treated with estradiol benzoate (EB) with or without testosterone propionate (TP) and the aromatase inhibitor fadrozole (FAD). Data were analyzed using Kruskall–Wallis to detect differences between groups, and significant effects were followed up using Mann–Whitney U, and p-values were adjusted using a Bonferroni correction. Boxes represent interquartile range, and whiskers each represent the top and bottom 25% of scores. ^o Outlier. ⁺Extreme outlier. ^∗Different from EB-alone, p_adj < 0.05. ^#Tendency to differ from EB-TP, p < 0.05; ^∗∗Different from EB-alone and EB+TP, both p_adj < 0.05.