cyp19a1b-deficient males exhibit severely impaired male-typical mating and aggressive behaviors.

(A and B) Levels of E2, testosterone, and 11KT in the brain of adult cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− males (A) and females (B) (n = 3 per genotype and sex). (C) Set-up for testing the mating behavior of cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− males. (D) Latency of cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− males (n = 18 per genotype) to initiate each mating act toward the stimulus female. (E) Set-up for testing mating behavior using an esr2b-deficient female as the stimulus. (F) Latency of cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− males (n = 16, 15, and 17, respectively) to initiate each mating act toward the esr2b-deficient female. (G) Number of each mating act performed. (H) Latency of cyp19a1b+/+ and cyp19a1b−/− males and cyp19a1b+/+ females (n = 15 each) to receive courtship displays from the esr2b-deficient female. (I) Set-up for testing aggressive behavior among grouped males. (J) Total number of each aggressive act observed among cyp19a1b+/+, cyp19a1b+/−, or cyp19a1b−/− males in the tank (n = 6, 7, and 5, respectively). Statistical differences were assessed by Bonferroni’s or Dunn’s post hoc test (A, B, G, and J) and Gehan-Breslow-Wilcoxon test with Bonferroni’s correction (D, F, and H). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Neuroestrogens facilitate male-typical behaviors probably by stimulating brain AR expression.

(A) Set-up for testing the mating behavior of E2-treated cyp19a1b+/+ and cyp19a1b−/− males. (B) Latency of cyp19a1b+/+ males (n = 12) to initiate courtship displays toward the stimulus female before (day 0) and after (day 4) E2 treatment. (C) Number of courtship displays performed by cyp19a1b+/+ males. (D) Latency of cyp19a1b−/− males (n = 12) to initiate courtship displays before (day 0) and after (day 4) E2 treatment. (E) Number of courtship displays performed by cyp19a1b−/− males. (F) Total area of ara expression signal in each brain nucleus of cyp19a1b+/+ (n = 6 except for pPPp, where n = 5) and cyp19a1b−/− (n = 7) males. The data are displayed in two graphs for visual clarity. (G) Representative images of ara expression in the PPa, pPPp, and NVT. (H) Total area of arb expression signal in each brain nucleus of cyp19a1b+/+ (n = 6) and cyp19a1b−/− (n = 7 except for NVT, where n = 6) males. The data are displayed in two graphs for visual clarity. (I) Representative images of arb expression in the PMp, aPPp, and NPT. (J) Total area of ara expression signal in the PPa, pPPp, and NVT of cyp19a1b+/+ and cyp19a1b−/− males treated with vehicle alone or E2 (n = 5 per group except for NVT of E2-treated cyp19a1b+/+ males, where n = 4; and PPa of vehicle-treated cyp19a1b+/+, E2-treated cyp19a1b+/+, and vehicle-treated cyp19a1b−/− males, where n = 3). (K) Total area of arb expression signal in the PMp, aPPp, and NPT of cyp19a1b+/+ and cyp19a1b−/− males treated with vehicle alone or E2 (n = 5 per group except for NPT of vehicle-treated cyp19a1b+/+ males, where n = 4). Scale bars represent 50 μm. For abbreviations of brain nuclei, see Table S1. Statistical differences were assessed by Gehan-Breslow-Wilcoxon test (B and D) and unpaired t test, with Welch’s correction where appropriate (C, E, F, H, J, and K). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

cyp19a1b deficiency impairs behaviorally relevant signaling pathways downstream of ARs.

(A) Total area of vt expression signal in the PMp/PPa/PMm/PMg, SC/aNVT, and pNVT of cyp19a1b+/+ and cyp19a1b−/− males and females (n = 4 and 5 per genotype for males and females, respectively). (B) Representative images of vt expression in the pNVT. (C) Total area of gal expression signal in the aPMp/PPa, pPMp, PPp, and NAT/NVT/NRL of cyp19a1b+/+ and cyp19a1b−/− males and females (n = 4 except for cyp19a1b+/+ males, where n = 3). (D) Representative images of gal expression in the pPMp. Scale bars represent 50 μm. For abbreviations of brain nuclei, see Table S1. Statistical differences were assessed by unpaired t test, with Welch’s correction where appropriate (A and C). Error bars represent SEM. **P < 0.01.

Estrogens directly stimulate the transcription of ARs through ESRs.

(A and B) Schematic of ara (A) and arb (B) loci showing the location of the canonical bipartite ERE-like sequences. Bent arrows mark the transcription initiation sites. Nucleotides of the ERE-like sequences are denoted by capital letters, and those identical to the consensus ERE (AGGTCAnnnTGACCT) are gray-shaded. (C) Ability of E2 to directly activate ara transcription. Cultured cells were transfected with a luciferase reporter construct containing a genomic fragment upstream of exon 3 of ara, together with an Esr1, Esr2a, or Esr2b expression construct. The cells were stimulated with different concentrations of E2 and luciferase activity was measured. (D) Effect of mutations in the ERE-like sequences on the E2-induced activation of ara transcription. Cultured cells were transfected with a wild-type luciferase construct or a construct carrying a mutation in the ERE-like sequence at position +1699 (mut+1699) or +2050 (mut+2050), together with an Esr1, Esr2a, or Esr2b expression construct. The cells were stimulated with or without E2, and luciferase activity was measured. (E) Ability of E2 to directly activate arb transcription. The assay described in C was performed with a luciferase construct containing a genomic fragment upstream of the first methionine codon of arb. (F) Effect of mutations in the ERE-like sequences on the E2-induced activation of arb transcription. The assay described in D was performed with luciferase constructs each carrying a mutation in the ERE-like sequence at position –1272 (mut-1272), –2180 (mut-2180), or –3327 (mut-3327). Values are expressed as a fold change relative to a control without E2 stimulation (C and E) or a control using the wild-type construct without E2 stimulation (D and F). Statistical differences were assessed by Dunnett’s post hoc test (C and E) and unpaired t test with Bonferroni–Dunn correction (D and F). Error bars represent SEM. **P < 0.01; ***P < 0.001.

Neuroestrogens stimulate ara and arb expression in behaviorally relevant brain regions primarily through Esr2a and Esr1, respectively.

(A) Total area of arb expression signal in each brain nucleus of esr1+/+ and esr1−/− males (n = 5 per genotype). The data are displayed in two graphs for visual clarity. (B) Representative images of arb expression in the PMp and aPPp. (C) Total area of ara expression signal in each brain nucleus of esr2a+/+ and esr2a−/− males (Δ8 line; n = 5 per genotype except for NPT of esr2a+/+ males and Vs/Vp and PMp of esr2a−/− males, where n = 4). The data are displayed in two graphs for visual clarity. (D) Representative images of ara expression in the PPa, pPPp, and NVT. (E) Set-up for testing the mating behavior of esr1+/+ and esr1−/− males using an esr2b-deficient female as the stimulus. (F) Latency of esr1+/+ and esr1−/− males (n = 24 and 31, respectively) to initiate each mating act toward the stimulus female. (G) Number of each mating act performed. (H) Set-up for testing the mating behavior of esr2a+/+ and esr2a−/− males using an esr2b-deficient female as the stimulus. (I) Latency of esr2a+/+ and esr2a−/− males (Δ8 line; n = 23 and 22, respectively) to initiate each mating act toward the stimulus female. (J) Number of each mating act performed. (K and L) Total number of each aggressive act observed among esr1+/+ or esr1−/− males (n = 6 per genotype) (K) and among esr2a+/+ or esr2a−/− males (Δ8 line; n = 8 and 7, respectively) (L) in the tank. Scale bars represent 50 μm. For abbreviations of brain nuclei, see Table S1. Statistical differences were assessed by unpaired t test, with Welch’s correction where appropriate (A, C, G, J, K, and L) and Gehan-Breslow-Wilcoxon test (F and I). Error bars represent SEM. *P < 0.05, **P < 0.01.

cyp19a1b-deficient females are less receptive to males and instead court other females.

(A) Set-up for testing the mating behavior of cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− females. (B) Latency of cyp19a1b+/+, cyp19a1b+/−, and cyp19a1b−/− females (n = 10 per genotype) to receive each mating act from the stimulus male. (C) Number of each mating act received. (D) Percentage of time cyp19a1b+/+ and cyp19a1b−/− females (n = 6 per genotype) spent at different distances from the stimulus male. (E) Set-up for testing the mating behavior of cyp19a1b+/+ and cyp19a1b−/− females toward a stimulus female. (F) Latency of cyp19a1b+/+ (n = 9) or cyp19a1b−/− (n = 10) females to initiate courtship displays toward the stimulus female. (G and H) Total area (G) and representative images (H) of npba expression signal in the Vs/Vp of cyp19a1b+/+ and cyp19a1b−/− males and females (n = 5 per genotype and sex). (I and J) Total area (I) and representative images (J) of npba expression signal in the PMm/PMg of cyp19a1b+/+ and cyp19a1b−/− males and females (n = 5 per genotype and sex). (K) Total area of esr2b expression signal in each brain nucleus of cyp19a1b+/+ or cyp19a1b−/− females (n = 5 per genotype). The data are displayed in two graphs for visual clarity. Scale bars represent 50 μm. For abbreviations of brain nuclei, see Table S1. Statistical differences were assessed by Gehan-Breslow-Wilcoxon test, with Bonferroni’s correction where appropriate (B and F), Bonferroni’s post hoc test (C), unpaired t test with Bonferroni–Dunn correction (D), and unpaired t test, with Welch’s correction where appropriate (G, I, and K). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.