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. 2020 Oct 1;161(10):bqaa137.
doi: 10.1210/endocr/bqaa137.

Brain Aromatase and the Regulation of Sexual Activity in Male Mice

David C Brooks  1 , John S Coon V  1 , Cihangir M Ercan  1 , Xia Xu  2 , Hongxin Dong  3 , Jon E Levine  4 , Serdar E Bulun  1 , Hong Zhao  1
Affiliations

Brain Aromatase and the Regulation of Sexual Activity in Male Mice

David C Brooks et al. Endocrinology. .

Abstract

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 that arises in the brain versus from the 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.

Keywords: aromatase; brain; estrogen; sexual behavior; testosterone.

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Figures

Figure 1.
Figure 1.
Generation and characterization of bArKO and tArKO mice. (A) Schematic demonstrating generation of the floxed murine aromatase gene and its recombination after expression of recombinases. The upper diagram shows the targeting construct used to introduce loxP and FRT sites into the WT aromatase gene; restriction enzyme sites (Sca1 and EcoRV), the location of 5′ and 3′ probes, and the position of the primers (F1, F2, and R) for the PCR analysis are indicated. The middle diagram shows the targeting construct after homologous recombination. The aromatase allele together with the FRT-flanked neo cassette was flanked by LoxP sites (Aromfl/neo). The lower diagram indicates the floxed aromatase alleles after transient Flp recombinase expression with subsequent deletion of the neo cassette (Aromfl). Nestin-Cre recombinase expression with knockout of the flanked region of the aromatase gene (Aromko) in the brain generated bArKO mice, and Zp3-Cre recombinase expression with complete deletion of the flanked region of the aromatase gene (Aromdel) in whole body generated tArKO mice. I.f, a brain-specific exon 1. PII, a gonad-specific first exon. E2, exon 2. E3-E10, exon 3 to exon 10. (B) Southern blot analysis of different ES cell clones with the 5′ probe and 3′ probe after transfection with the LoxP/FRT flanked targeting construct. Clones #3 and #4 showed the expected bands of 18.9 kb (Aromwt allele) and 6.9 kb (Aromfl/neo allele) using the 5′ probe and the expected bands of 14.4 kb (Aromwt allele) and 8.2 kb (Aromfl/neo allele) using the 3′ probe, indicating that 1 copy of the WT aromatase gene was replaced by the targeting construct. (C) PCR analysis of DNA prepared from brain and testis of bArKO and tArKO mice. bArKO mice produced a 263-bp band (Aromko allele) in brain indicating a recombination event in brain and a 182-bp band (Aromfl allele) in testis indicating a loxP-flanked aromatase gene without recombination in testis. Aromatase heterozygous control mice contained an Aromwt allele (a 215-bp band) and an Aromko allele in brain, and an Aromwt allele and an Aromfl allele in testis. tArKO mice contained only the Aromdel allele (a 263-bp band) in brain and testis suggesting that recombination occurred in brain and testis. Aromatase heterozygous control mice (Het) contained an Aromwt allele and an Aromdel allele in brain and testis. WT mice only contained the Aromwt allele. (D) Real-time quantitative PCR demonstrating mRNA levels of aromatase-expressing tissues in control, bArKO, and tArKO mice. GAPDH mRNA levels served as loading controls. n = 5. E2 levels in the brain (E and G) and testis (F and H) of bArKO and tArKO mice were measured by the LC-MS/MS assay. 2-tailed Student t test or 1-way ANOVA with Tukey multiple comparison test, *P < 0.05, **P < 0.01. n = 9-12 for controls and n = 6 for bArKO mice; n = 21 for WT, n = 25 for Het, and n = 16 for tArKO mouse brain; n = 9 for WT, n = 6 for Het, and n = 6 for tArKO mouse testis.
Figure 2.
Figure 2.
Baseline sexual behavior of bArKO mice is decreased compared to controls. Sexual activity was measured in 12- to 14-week-old intact (noncastrated) bArKO, tArKO, and littermate control male mice over two 30-minute trials. The interactions were monitored and videotaped for the further analysis. (A) The number of events (mount or intromission), (B) time per event, (C) first event latency, and (D) total time for mounts or intromissions in bArKO mice. n = 7-8 for bArKO mice or heterozygous littermate controls. (E) The number of events (mount or intromission), (F) time per event, (G) first event latency, and (H) total time for mounts or intromissions in tArKO mice. n = 5 for tArKO mice, Het mice, or WT littermate controls. 2-tailed Student t test for bArKO mice, 1-way ANOVA with Tukey multiple comparison test for tArKO mice, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 3.
Figure 3.
Sexual behavior following hormone replacement in control and bArKO castrated mice. (A) Schematic diagram depicts the schedule of hormone replacement therapy and assays. (B) The number of mounts, (C) number of intromissions, (D) time per mount, (E) time per intromission, (F) first mount latency, (G) first intromission latency, (H) total time for mounts, and (I) total time for intromissions in castrated control and bArKO mice following each hormone treatment. n = 5 mice per group. Sexual activity was assessed in each mouse twice. Two-way ANOVA with Tukey multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 4.
Figure 4.
Serum hormone levels in bArKO and tArKO mice. Serum E2 (A and F), T (B and G), A4 (C and H), FSH (D and I), and LH (E and J) were measured in bArKO and tArKO mice, respectively. Serum levels from 8- to 26-week-old mice shown in panels A and F-H were measured by LC-MS/MS and serum levels from 12-week-old mice shown in panels B and C were measured by ELISA and in panels D, E, I, and J were measured by RIA. For bArKO mice, n = 13 for controls and n = 6 for bArKO mice in A, and n = 8-12 per group in B-E. For tArKO mice, n = 25 for Het controls and n = 16 for tArKO mice in F-G and n = 7-8 per group in I and J. Two-tailed Student t test, *P < 0.05, ***P < 0.001, ****P < 0.0001. Serum FSH (K) and LH (L) levels following steroid hormone replacement in castrated bArKO mice were measured by RIA. Castrated mice were treated with vehicle (Veh), E2, T, or T plus E2 (TE2) for 5 weeks. Mouse serum was collected from 14-week-old mice. One-way or 2-way ANOVA with Tukey multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 5 mice per group.
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