The biologically active estrogen, estradiol, has important roles in adult brain physiology and sexual behavior. A single gene, Cyp19a1, encodes aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol in the testis and brain of male mice. Estradiol formation was shown to regulate sexual activity in various species, but the relative contributions to sexual behavior of estrogen arising in the brain versus gonads remained unclear. To determine the role of brain aromatase in regulating male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse. A newly generated whole-body total aromatase knockout mouse of the same genetic background served as a positive control. Here we demonstrate that local aromatase expression and estrogen production in the brain is partially required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited decreased sexual activity in the presence of strikingly elevated circulating testosterone. In castrated adult bArKO mice, administration of testosterone only partially restored sexual behavior; full sexual behavior, however, was achieved only when both estradiol and testosterone were administered together. Thus, aromatase in the brain is, in part, necessary for testosterone-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating testosterone of testicular origin. Our findings suggest testosterone activates male sexual behavior in part via conversion to estradiol in the brain. These studies provide foundational evidence that sexual behavior may be modified through inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators.

This data collection details the following supplemental figures and movies associated with the manuscript, determining the role of aromatase and estrogen in male sexual behavior.

Supplemental Figure 1. Fertility of bArKO and tArKO mice. Average litter size of bArKO (A) or tArKO male mice (B). The average number of pups per litter was calculated when bArKO or control (heterozygous) males were mated with age-matched WT females or tArKO or control (heterozygous) males were mated with age-matched WT females for 4 months. 2-tailed Student’s t test, *P < 0.05, n=5 per group.

Supplemental Figure 2. Body weight of bArKO and tArKO mice. Body weight  was assessed weekly from 3 to 48 weeks of age in (A) bArKO and (B) tArKO mice. Age-matched heterozygous male mice were used as controls. 2-tailed Student’s t test, *P < 0.05, n=7-8 for controls, n=8 for bArKO mice, n=7 for tArKO mice.

Supplemental Figure 3. Tissue testosterone (T) and androstenedione (A4) levels in bArKO mice. Brain levels of T (A) and A4 (B) and testis levels of T (C) and A4 (D) were measured by LC-MS2 assay. 2-tailed Student’s t test, n=9-12 for heterozygous controls, n=6 for bArKO mice. Mouse tissues were collected from 8- to 26-week-old mice (A-D).

Supplemental Figure 4. Tissue androgen levels in tArKO mice. Brain levels of testosterone (T) (A) and androstenedione (A4) (B) and testis levels of T (C) and A4 (D) were measured by LC-MS2 assay. 2-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparison test, *P < 0.05, **P < 0.01, ****P < 0.0001, n=21 for WT, n=25 for heterozygous controls (Het), and n=16 for tArKO mice (A and B); n=8-9 for WT, n=6-7 for heterozygous controls (Het), and n=6 for tArKO mice (C and D). Mouse tissues were collected from 8- to 26-week-old mice (A-D).

Movie S1. Representative sexual behavior movie for a male bArKO mouse (black). The white mouse is an unfamiliar, hormonally primed, and sexually experienced female mouse.

Movie S2. Representative sexual behavior movie for a male heterozygous mouse (Control, black) served as a control for bArKO mice. The white mouse is an unfamiliar, hormonally primed, and sexually experienced female mouse.

Movie S3. Representative sexual behavior movie for a male tArKO mouse (black). The white mouse is an unfamiliar, hormonally primed, and sexually experienced female mouse.

Movie S4. Representative sexual behavior movie for a male heterozygous mouse (Het, black) served as a control for tArKO mice. The white mouse is an unfamiliar, hormonally primed, and sexually experienced female mouse.

Movie S5. Representative sexual behavior movie for a male WT mouse (black) served as a control for tArKO mice. The white mouse is an unfamiliar, hormonally primed, and sexually experienced female mouse.