Article Figures & Data
Figures
- Figure 1.
Representative punch locations in the song sparrow brain. A–F, Coronal sections (300 µm) presented from rostral to caudal. Punches (1 mm diameter) are represented by circles (approximately to scale). Abbreviations: POA, caudal preoptic area; LS, lateral septum; BNST, bed nucleus of the stria terminalis; CoA, anterior commissure; AH, anterior hypothalamus; VMH, ventromedial hypothalamus; OM, tractus occipito-mesencephalicus; NIII, nervus oculomotorius; VTA, ventral tegmental area; PC, posterior commissure; CG, central gray; NCM, caudomedial nidopallium; TnA, nucleus taeniae of the amygdala; Cb, cerebellum. Adapted from Jalabert et al. (2021).
- Figure 2.
Calibration curves of (A) androstenedione and (B) testosterone, with insets displaying the lowest standards on the curve. The area ratio is calculated by dividing an analyte peak area with its deuterated internal standard peak area in the same sample. Calibration curves ranged from 0.03 to 1,000 pg.
- Figure 3.
Effect of a 10 min STI on aggressive responses of wild adult male song sparrows during the breeding (green) and non-breeding (blue) seasons. Bar graphs represent (A) song and (B) flight latencies, for control and STI subjects. Values are expressed as the mean ± SEM. n = 10 for breeding group, n = 9 for non-breeding group. ****p ≤ 0.0001.
- Figure 4.
Effect of a 10 min STI on aggressive responses of wild adult male song sparrows during the breeding (green) and non-breeding (blue) seasons. Bar graphs represent (A) number of songs, (B) number of flights, and (C) time in 5 m, for control and STI subjects. Values are expressed as the mean ± SEM. n = 10 for breeding group, n = 9 for non-breeding group. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
- Figure 5.
Effect of a 10 min STI on circulating and brain steroid levels of wild adult male song sparrows during the breeding season. Bar graphs represent concentrations of (A) progesterone and (B) corticosterone for control and STI subjects. Values are expressed as the mean ± SEM. n = 10 per group. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.
- Figure 6.
Effect of a 10 min STI on circulating and brain androgen levels of wild adult male song sparrows during the breeding season. Bar graphs represent concentrations of (A) androstenedione, (B) testosterone, and (C) 5α-dihydrotestosterone for control and STI subjects. Values are expressed as the mean ± SEM. n = 10 per group.
- Figure 7.
Effect of a 10 min STI on circulating and brain estrogen levels of wild adult male song sparrows during the breeding season. Bar graphs represent concentrations of (A) estrone and (B) 17β-estradiol for control and STI subjects. Values are expressed as the mean ± SEM. n = 10 per group. nd, non-detectable.
- Figure 8.
Effect of a 10 min STI on circulating and brain steroid levels of wild adult male song sparrows during the non-breeding season. Bar graphs represent concentrations of (A) progesterone and (B) corticosterone, for control and STI subjects. Values are expressed as the mean ± SEM. n = 9 per group. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. nd, non-detectable.
- Figure 9.
Representative chromatograms for the two MRM transitions of androstenedione (left panel, quantifier m/z 287.2 → 109.1, qualifier m/z 287.2 → 97.2) and testosterone (right panel, quantifier m/z 289.0 → 97.0, qualifier m/z 289.0 → 109.1). Retention times for androstenedione (7.40 min) and testosterone (7.98 min) are indicated by the arrows. Samples were neat solution (in gray scale), (A, I) blank (no analyte) and (B, J) certified reference standard (0.1 pg), and wild male song sparrow blood and brain from breeding (green) and non-breeding (blue) seasons. C, Blood breeding control, (D) caudomedial nidopallium (NCM) breeding control, (E) blood non-breeding control, (F) NCM non-breeding control, (G) blood non-breeding STI (simulated territorial intrusion), (H) NCM non-breeding STI, (K) blood breeding control, (L) anterior hypothalamus (AH) breeding control, (M) blood non-breeding control, (N) AH non-breeding control, (O) blood non-breeding STI, (P) AH non-breeding STI. The ion ratio between quantifier and qualifier ions was similar between detectable samples and standards. For analyte quantification, a peak signal/noise ratio >10 was the criterion for detection. Blanks and non-breeding control samples were non-detectable for both analytes. Standards and breeding samples were detectable for both analytes. During the non-breeding season, both androstenedione and testosterone were detectable in brain samples but non-detectable in blood samples. The intensity was measured in counts per second (cps). n = 10 for breeding season and n = 9 for non-breeding season. The LLOQs were the lowest standard on the calibration curve in which the analyte peak had a signal/noise ratio >10.
- Figure 10.
Effect of a 10 min STI on circulating and brain androgen levels of wild adult male song sparrows during the non-breeding season. Bar graphs represent concentrations of (A) androstenedione and (B) testosterone for control and STI subjects. Values are expressed as the mean ± SEM. n = 9 per group. ***p ≤ 0.001, ****p ≤ 0.0001. nd, non-detectable.
Tables
- Table 1.
LC-MS/MS calibration curves and analyte retention times: linear regressions and lower limits of quantification (LLOQ)
Steroid r2 p LLOQ (pg) Retention time (min) Pregnenolone 0.9997 <0.0001 5 10.66 Progesterone 0.9983 <0.0001 0.05 10.35 Corticosterone 0.9999 <0.0001 0.05 5.96 Dehydroepiandrosterone 0.9945 <0.0001 1 8.57 Androstenedione 0.9998 <0.0001 0.03 7.21 Testosterone 0.9998 <0.0001 0.03 8.00 5α-DHT 0.9994 <0.0001 0.05 9.67 Estrone 0.9999 <0.0001 0.1 7.39 17α-estradiol 0.9991 <0.0001 0.2 8.08 17β-estradiol 0.9999 <0.0001 0.2 7.49 Estriol 0.9967 <0.0001 0.2 2.58 Note: Calibration curves contained 13 points. The LLOQs were the lowest standard on the calibration curve with a signal/noise ratio >10. Abbreviation: 5α-DHT, 5α-dihydrotestosterone.
Steroid Low QC High QC Precision % (intra-assay) Precision % (inter-assay) Accuracy % Precision % (intra-assay) Precision % (inter-assay) Accuracy % Pregnenolone n/a n/a n/a 4.1 7.5 92.9 Progesterone 3.4 4.5 85.4 2.1 6.7 86.4 Corticosterone 4.5 12.1 96.0 1.3 5.1 93.3 Dehydroepiandrosterone 11.4 12.2 99.3 2.3 5.6 98.9 Androstenedione 2.7 6.3 96.2 3.9 6.4 96.0 Testosterone 2.0 3.9 90.0 1.2 3.4 89.1 5α-DHT 2.1 11.3 71.6 2.2 6.7 73.5 Estrone 4.0 6.3 82.5 4.3 7.6 84.2 17α-estradiol 9.1 12.0 74.7 4.8 6.9 77.3 17β-estradiol 15.1 16.1 69.7 4.5 8.3 80.1 Estriol 3.2 5.0 84.5 3.9 8.9 90.3 Note: Low QCs (n = 15 total across five assays) contained 0.8 pg of each steroid except for dehydroepiandrosterone (8 pg). High QCs (n = 15 total across five assays) contained 50 pg of each steroid except for dehydroepiandrosterone (500 pg). Precision was measured by the coefficient of variation of replicates within assays (intra-assay) or among assays (inter-assay). Accuracy was assessed by the measured values of the QCs compared with their known values. Low QCs were below the lower limit of quantification for pregnenolone. n/a, not applicable. Abbreviation: 5α-DHT, 5α-dihydrotestosterone.
- Table 3.
Percent of samples with detectable androstenedione and testosterone in wild adult male song sparrows exposed to control or STI
Breeding Non-breeding Control STI Control STI Androstenedione Blood 100 100 0 0 Plasma 100 100 0 0 POA 80 70 0 11 AH 90 90 0 11 LS 60 70 11 11 BNST 70 50 22 55 VMH 90 90 33 33 VTA 70 80 22 11 CG 70 70 11 11 NCM 100 90 11 55 TnA 90 90 11 44 Cb 70 70 11 44 Testosterone Blood 100 100 0 0 Plasma 100 100 0 0 POA 100 100 11 55 AH 100 100 0 44 LS 100 100 44 33 BNST 100 100 44 44 VMH 100 100 11 11 VTA 100 100 11 11 CG 100 100 66 33 NCM 100 100 44 55 TnA 100 100 11 77 Cb 100 100 44 55 Note: n = 10 subjects in breeding season, n = 9 subjects in non-breeding season. Sample amounts were 5 µl for blood and 10 µl for plasma. The amount of brain tissue varied by region (0.98 mg for POA, AH, LS, BNST, VMH, VTA, and CG and 1.47 mg for NCM, TnA, and Cb). Abbreviations: POA, caudal preoptic area; AH, anterior hypothalamus; LS, lateral septum; BNST, bed nucleus of the stria terminalis; VMH, ventromedial hypothalamus; VTA, ventral tegmental area; CG, central gray; NCM, caudomedial nidopallium; TnA, nucleus taeniae of the amygdala; Cb, cerebellum.
