Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Brain and circulating steroids in an electric fish: Relevance for non-breeding aggression

  • Lucia Zubizarreta ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

    ‡ LZ and CJ are co-first authors on this work.

    Affiliations Laboratorio de Neurofisiología Celular y Sináptica, Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, Departamento de Neurofisiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay

    ⨯
  • Cecilia Jalabert ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    ‡ LZ and CJ are co-first authors on this work.

    Affiliations Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada

    ⨯
  • Ana C. Silva,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing

    Affiliation Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay

    ⨯
  • Kiran K. Soma ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing – review & editing

    lquintana@iibce.edu.uy (LQ); ksoma@psych.ubc.ca (KKS)

    Affiliations Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada, Department of Psychology, The University of British Columbia, Vancouver, British Columbia, Canada

    ⨯
  • Laura Quintana

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    lquintana@iibce.edu.uy (LQ); ksoma@psych.ubc.ca (KKS)

    Affiliation Departamento de Neurofisiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay

    ⨯

Abstract

Steroids play a crucial role in modulating brain and behavior. While traditionally it is thought that the brain is a target of sex steroids produced in endocrine glands (e.g. gonads), the brain itself produces steroids, known as neurosteroids. Neurosteroids can be produced in regions involved in the regulation of social behaviors and may act locally to regulate social behaviors, such as reproduction and aggression. Our model species, the weakly electric fish Gymnotus omarorum, displays non-breeding aggression in both sexes. This is a valuable natural behavior to understand neuroendocrine mechanisms that differ from those underlying breeding aggression. In the non-breeding season, circulating sex steroid levels are low, which facilitates the study of neurosteroids. Here, for the first time in a teleost fish, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify a panel of 8 steroids in both plasma and brain to characterize steroid profiles in wild non-breeding adult males and females. We show that: 1) systemic steroid levels in the non-breeding season are similar in both sexes, although only males have detectable circulating 11-ketotestosterone, 2) brain steroid levels are sexually dimorphic, as females display higher levels of androstenedione, testosterone and estrone, and only males had detectable 11-ketotestosterone, 3) systemic androgens such as androstenedione and testosterone in the non-breeding season are potential precursors for neuroestrogen synthesis, and 4) estrogens, which play a key role in non-breeding aggression, are detectable in the brain (but not the plasma) in both sexes. These data are consistent with previous studies of G. omarorum that show non-breeding aggression is dependent on estrogen signaling, as has also been shown in bird and mammal models. Overall, our results provide a foundation for understanding the role of neurosteroids, the interplay between central and peripheral steroids and potential sex differences in the regulation of social behaviors.

Citation: Zubizarreta L, Jalabert C, Silva AC, Soma KK, Quintana L (2023) Brain and circulating steroids in an electric fish: Relevance for non-breeding aggression. PLoS ONE 18(10): e0289461. https://doi.org/10.1371/journal.pone.0289461

Editor: Irfan Ahmad Bhat, University of Iceland, ICELAND

Received: July 18, 2023; Accepted: September 23, 2023; Published: October 10, 2023

Copyright: © 2023 Zubizarreta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data are available from OSF at: https://doi.org/10.17605/OSF.IO/W8ZMJ.

Funding: Agencia de Investigacion e Inovacion (POS_NAC_2014_1_102353) to LZ Comision academica de postgrado UdelaR (42458413) to LZ Emerging Leaders in the Americas program graduate, Global Affairs Canada, Canadian Bureau for international education (2018-2019) to LZ Agencia Nacional de Investigación e Innovación (POS_EXT_2016_1_134441) to CJ Zoology Graduate Fellowship, University of British Columbia (6444) to CJ Canadian Institutes of Health Research (133606) to KKS Canadian Institutes of Health Research (426405) to KKS Canada Foundation for Innovation Grant (32631) to KKS Agencia de Investigacion e Inovacion (FCE 136381) To LQ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Steroids are potent signaling molecules that modulate the brain and behavior. Historically, the brain has been considered a target for steroids produced in peripheral endocrine glands. However, the identification of steroidogenic enzymes in the brain reveals that the brain can also produce steroids locally (reviewed in [1, 2]). Neurally synthesized steroids, ‘neurosteroids’, play a wide variety of roles, such as the maintenance of synaptic transmission and connectivity in the hippocampus (reviewed in [3]), the regulation of neuroinflammatory responses (reviewed in [4]), neuroprotective processes [5], and pain [6]. In addition, in both birds and mammals, neuroestrogens in particular are important in the regulation of social behaviors, including reproduction [7–9] and aggression [10–12].

Several studies have quantified steroid levels in specific brain regions involved in natural behaviors. For example, in zebra finches, brain microdialysis coupled with immunoassay techniques have shown that conspecific song produces a transient increase of androgens in the auditory cortex, with no effect on circulating androgens. These neuroandrogens, which can be aromatized into neuroestrogens, influence song processing and sensorimotor integration [13]. In the year-round territorial song sparrow, Melospiza melodia, 10 steroids in 10 microdissected regions of the social behavior network were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study showed seasonal patterns in different regions of the social behavior network that differ greatly from those in the blood [14]. The use of ultrasensitive LC-MS/MS assays that can measure multiple steroids at low analyte levels can further our understanding of local steroidogenesis, the precursors involved, and the balance between local and systemic steroid signaling [14–16].

Sex steroids are key regulators of aggression, an adaptive behavior that is displayed across species in different contexts. Although aggression is mostly studied in reproductive contexts, many species display this behavior outside of the breeding season, when circulating levels of sex steroids are often very low. These species offer an opportunity to study the neuromodulation of aggression independently from gonadal steroid secretion [11, 17–20]. For example, song sparrows maintain high male-male territorial aggression during the non-breeding season, when neuroestrogens are key in sustaining this behavior [11, 21–27], similar to some rodents [10, 28]. These neuroendocrine mechanisms supporting aggression may be a common strategy across vertebrates [29, 30].

Males and females of the weakly electric fish Gymnotus omarorum [31] display high levels of aggression in the non-breeding season [32–34]. In this species, castration does not affect aggressive behavior in non-breeding males. However, the inhibition of aromatase reduces non-breeding aggression in both males and females, indicating that extra-gonadal estrogens are key regulators of non-breeding aggression [20, 33]. Moreover, a transcriptomic analysis of the forebrain shows that dominant non-breeding male G. omarorum have a steroidogenic pathway directed towards estrogen synthesis, whereas subordinate males have a pathway directed towards the production of non-aromatizable androgens [35]. These results support the hypothesis that brain-derived estrogens play an important role in the regulation of non-breeding aggression. This estrogenic effect may be direct or via other neuromodulators, such as arginine vasotocin and serotonin, which affect non-breeding aggression in this species [36–41]. However, no attempts have been made so far to measure blood and brain levels of sex steroids to reveal whether there is neural synthesis of key steroids and whether there are sex differences.

Here, for the first time in a teleost fish, we used LC-MS/MS to quantify a panel of 8 steroids in both plasma and brain of wild non-breeding male and female G. omarorum. We measured progesterone, cortisol, dehydroepiandrosterone (DHEA), androstenedione (AE), testosterone (T), 11-ketotestosterone (11-KT), estrone (E1), and 17β-estradiol (E2). Moreover, we examined sex differences in plasma and forebrain steroid levels. Finally, we evaluated the possibility of the brain as a potential source of estrogens and other sex steroids.

Materials and methods

Field procedures

Free-living adult male (n = 11) and female (n = 11) G. omarorum were captured in the non-breeding season (June 2018) in Laguna de los Cisnes, Maldonado, Uruguay (34° 48´ S, 55° 180`W). Sample collection was carried out during daytime, which corresponds to the resting phase of the animals. The capture method consisted of locating the individuals with a detector (without disturbing them) and then using a rigid net to quickly lift the vegetation and the fish. Individuals were anesthetized immediately after capture by immersion in a fast-acting eugenol solution (1.2 mg/l). Blood was immediately extracted from the caudal vein with a heparinized syringe, and body length was measured. Subjects were rapidly decapitated, and the brain was removed from the skull, quickly frozen in powdered dry ice, and stored on dry ice until arrival at the laboratory. To prevent a stress response from affecting basal steroid levels, there was a maximum of 3 min between capture and decapitation [42–44]. The time between decapitation and brain freezing was always less than 90 sec. Blood was kept on wet ice until centrifugation in the laboratory (approximately 5 h). The gonads were then removed and stored on ice. Once in the laboratory, gonad and body weights were determined. To calculate the gonadosomatic index, the weight of the gonads was added to the body weight, and the index was calculated as follows: [gonad weight / body weight] x 100. The blood was centrifuged (14,000g for 10 min) and plasma was collected and stored at -80 °C, along with the brains. Additional subjects (n = 14) were used for method development.

All research procedures complied with ASAP/ABS Guidelines for the Use of Animals in Research and were approved by the Institutional Ethical Committee (Comisión de Ética en el Uso de Animales, Instituto Clemente Estable, MEC, CEUA-IIBCE: 001/02/2018).

Brain dissection

In each individual, we obtained a section containing the forebrain and the midbrain. To do so, dissection was carried out on a metal plate surrounded by dry ice, under a magnifying glass. Frozen brains were mounted in tissue-tek on a glass petri dish by their dorsal surface, exposing the ventral surface. We performed a coronal section with a heated cryostat blade immediately caudal to the inferior lobe (following [37]). Then, from the rostral section we removed the cerebellum, optic tectum, and pituitary gland (which do not have a major role in social behaviors). Each resulting sample was weighed, placed in 2 ml polypropylene vials (Sarstedt AG and Co.), and stored at -80°C until processing.

Steroid extraction

Extraction was performed using liquid-liquid extraction followed by solid phase extraction (based on [45]). Steroids were extracted from 25 μL plasma and ~50 mg brain tissue. Samples were placed in 2-mL polypropylene vials (Sarstedt AG & Co., Nümbrecht, Germany) containing five zirconium ceramic oxide beads (1.4-mm diameter, Fisher Scientific). Then 50 μL of the deuterated internal standards (progesterone-d9, cortisol-d4, DHEA-d6, T-d5, E2-d4; C/D/N Isotopes Inc., Pointe-Claire, Canada) in 50% HPLC-grade methanol were added to each sample, standards, and water blanks (except double blanks) to track recovery and matrix interference for each sample. The T-d5 internal standard was used for AE, T, and 11-KT; the E2-d4 internal standard was used for E1 and E2; and progesterone-d9, cortisol-d4, and DHEA-d6 were used for their respective analytes. Then, 1 mL of GC-grade ethyl acetate was added to each vial, and samples were homogenized using a bead mill homogenizer at 4 m/s for 30 s (Omni International Inc., Kennesaw, GA). Samples were then centrifuged at 16,100g for 5 min, and 1 mL of supernatant was transferred to a borosilicate glass culture tube pre-cleaned with HPLC-grade methanol (VWR International). Then 1 mL of ethyl acetate was added to the remaining sample (to maximize extraction efficiency), homogenized, and centrifuged as before, and again 1 mL of supernatant was collected and combined with the initial ethyl acetate. Then 0.5 mL of Milli-Q water was added, and samples were vortexed and centrifuged at 3200g for 2 min. The water was removed and discarded, and the ethyl acetate was dried in a vacuum centrifuge at 60°C for 45 min (ThermoElectron SPD111V). The pellets were reconstituted with HPLC-grade methanol and then subjected to solid phase extraction [45]. Plasma, standards, and blanks were reconstituted in 0.5 mL of methanol, whereas brain samples were reconstituted in 1 mL of methanol and only 0.5 mL was used to avoid matrix effects (internal standards corrected for this sample reduction). Columns (C18, Agilent, Santa Clara, CA; catalog no. 12113045) were previously conditioned with 3 mL HPLC-grade hexane and then 3 mL HPLC-grade acetone and equilibrated with 3 mL HPLC-grade methanol. Extracts were then loaded onto the column (0.5 mL per sample), eluted with 2 mL HPLC-grade methanol, and eluates were collected. Samples were vacuum dried as above. Dried pellets were reconstituted with 55 μL of 25% HPLC-grade methanol in MilliQ water, transferred to 0.6 mL polypropylene microcentrifuge tubes (Fisher Scientific), and centrifuged at 16,100g for 2min. Then 50 μL of supernatant were transferred to a glass LC vial insert (Agilent, Santa Clara, CA) and stored overnight at -20°C until injection.

Steroid analysis by LC-MS/MS

Steroids were quantified using a Sciex QTRAP 6500 UHPLC-MS/MS system as previously described [45]. Samples were transferred into a refrigerated autoinjector (15°C). Then, 45 μL of resuspended sample were injected into a Nexera X2 UHPLC system (Shimadzu Corp., Kyoto, Japan), passed through a KrudKatcher ULTRA HPLC In-Line Filter (Phenomenex, Torrance, CA) followed by a Poroshell 120 HPH C18 guard column (2.1 mm) and separated on a Poroshell 120 HPH C18 column (2.1 x 50 mm; 2.7 μm; at 40°C) using 0.1 mM ammonium fluoride in MilliQ water as mobile phase A (MPA) and HPLC-grade methanol as mobile phase B (MPB). The flow rate was 0.4 mL/min. During loading, MPB was at 10% for 0.5 min, from 0.6 to 4 min the gradient profile was at 42% MPB, which was ramped to 60% MPB until 9.4 min. From 9.4 to 9.5 min the gradient was 60–70% MPB, which was ramped to 98% MPB until 11.9 min and finally a column wash from 11.9 to 13.4 min at 98% MPB. The MPB was then returned to starting conditions of 10% MPB for 1 min. Total run time was 14.9 min. The needle was rinsed externally before and after each sample injection with 100% isopropanol.

We used 2 multiple reaction monitoring transitions for each steroid and 1 multiple reaction monitoring transition for each deuterated internal standard (Table 1). Steroid concentrations were acquired on a Sciex 6500 QTRAP triple quadrupole tandem mass spectrometer (Sciex LLC, Framingham, MA) in positive electrospray ionization mode for all steroids except E1 and E2, which were acquired in negative electrospray ionization mode.

thumbnail
Download:
Table 1. Scheduled multiple reaction monitoring for LC-MS/MS.

https://doi.org/10.1371/journal.pone.0289461.t001

Calibration curves were made from certified reference standards (Cerilliant Co., Round Rock, TX) prepared in 50% HPLC-grade methanol. The calibration curve range was 0.2 to 1000 pg/tube for T and E1; 0.4 to 1000 pg/tube for AE and 11-KT; 0.8 to 1000 pg/tube for progesterone and E2; 2 to 1000 pg/tube for cortisol, and 20 to 10,000 pg/tube for DHEA (Table 2). Lower limit of quantification was calculated as the lowest standard on the calibration curve divided by the amount of sample (25 μL for plasma and 25 mg for brain) (Table 2). All blanks and double blanks were below the lowest standard on the calibration curves.

thumbnail
Download:
Table 2. Lower limit of quantification in different matrices.

https://doi.org/10.1371/journal.pone.0289461.t002

Matrix effects were assessed by extracting steroids from samples of different amounts (5, 15, 25, 50 μL for plasma; and 2, 10, 20 mg for brain, n = 3 per sample amount). For each sample, we measured the internal standard peak areas and compared them to those in neat solution. Recovery was assessed using plasma and brain pools and comparing unspiked samples with samples spiked with a known amount of steroid (n = 5 replicates per sample type). Matrix effects can cause either ion enhancement or, more commonly, ion suppression. When sample cleanup was not sufficient and matrix effects were higher than 50% (indicated by the internal standard) the sample was removed from the analysis. Accuracy was assessed by measuring quality controls with a known concentration in neat solution. Precision was evaluated by comparing replicates of quality controls within runs (intra-assay variation) and between runs (inter-assay variation). A total of 5 quality control replicates were run in each assay.

Statistical analysis

A value was considered non-detectable if it was below the lowest standard on the calibration curve. When the percentage of detectable samples was ≥ 20% of the total values in a group, the non-detectable values were estimated via quantile regression imputation of left-censored missing data using MetImp web tool [14, 16, 46–49]. When the percentage of detectable samples in a group was < 20% of the total values in the group, the non-detectable values were set to 0 to perform statistical analysis.

We were interested in comparing brain and blood concentrations of steroids, and the use of plasma overestimates steroid concentrations in the blood [50–52]. Therefore, we estimated blood steroid concentrations from plasma steroid concentrations. For this, we measured the hematocrit in adult male and female non-breeding G. omarorum (n = 3). Plasma volume corresponds to 56% of blood volume, and therefore steroid levels in plasma were multiplied by 0.56 to estimate steroid levels in whole blood.

Statistics were conducted using GraphPad Prism version 9.02 (GraphPad Software, La Jolla, CA, USA). Data were analyzed using parametric statistics. When necessary, data were log transformed prior to analysis. In cases where we had to compare a group with all non-detectable values to a group with detectable values, we added 1 to all values of both groups, data were log transformed, and then a parametric test was performed. Comparisons between groups were made by t-tests: unpaired t-tests for sex differences in plasma and brain, and paired t-tests to compare between blood and brain levels (paired variables in the same fish). Correlations between steroid levels were examined using Spearman’s rho correlations with Benjamini-Hochberg correction for multiple comparisons (the corrected p values are reported). Significance criterion was set at p ≤ 0.05 for all analyses. Graphs show the mean ± standard error of the mean (SEM) and are presented using the non-transformed data.

Results

LC-MS/MS assay development and validation

We developed a specific and sensitive method to quantify a panel of eight steroids by LC-MS/MS in both plasma and brain of G. omarorum. Analytes had distinct retention times and multiple reaction monitoring transitions, which provide specificity (Table 1). Matrix effects, calculated by comparing internal standard peak areas of samples with those in neat solution (n = 3/sample type), were similar for all steroids and within an acceptable range for 25 μL of plasma and 25 mg of brain tissue (Table 3). Recovery was assessed by subtracting unspiked sample pools from spiked sample pools and dividing by the amount of steroid added (n = 5/sample type, Table 4). Recovery was within an acceptable range for most steroids in both plasma and brain tissue. Recovery for progesterone was high (Table 4), suggesting that our assay overestimates progesterone levels; nonetheless, progesterone was non-detectable in plasma and brain tissue (see below). The assay demonstrated high accuracy and precision, with quality control measurements within the acceptable limits (Table 5). As expected, assay blanks did not contain detectable analyte peaks, and “double blanks” did not contain detectable analyte or internal standards peaks.

thumbnail
Download:
Table 3. Matrix effects in biological samples.

https://doi.org/10.1371/journal.pone.0289461.t003

thumbnail
Download:
Table 4. Recovery in biological samples.

https://doi.org/10.1371/journal.pone.0289461.t004

thumbnail
Download:
Table 5. Assay accuracy and precision.

https://doi.org/10.1371/journal.pone.0289461.t005

Systemic steroid levels

As shown in Fig 1A, non-breeding males had 4 detectable steroids in plasma: AE (0.14 ± 0.03 ng/ml), T (0.12 ± 0.02 ng/ml), 11-KT (0.07± 0.02 ng/ml), and cortisol (3.70 ± 0.95 ng/ml). Similarly, females had detectable levels of AE (0.15 ± 0.04 ng/ml), T (0.15 ± 0.04 ng/ml), and cortisol (5.2 ± 2.1 ng/ml), but did not have detectable 11-KT levels in plasma. Circulating levels of steroids were not different between sexes (AE: t = 0.11, p = 0.90; T: t = 0.08, p = 0.93; and cortisol: t = 0.45, p = 0.66; Fig 1A) except for 11-KT (t = 4.3, p = 0.0005).

thumbnail
Download:
Fig 1. Steroid profiles of male and female G. omarorum in the non-breeding season.

Bar graphs show concentrations in plasma (A) and forebrain (B) expressed as mean ± SEM, n = 11 per group. Abbreviations: AE, androstenedione; T, testosterone; 11-KT, 11-ketotestosterone; E1, estrone; nd, non-detectable. * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001.

https://doi.org/10.1371/journal.pone.0289461.g001

Neither females nor males had detectable progesterone, DHEA, E2 and E1 in plasma.

Brain steroid levels

Forebrain steroid profiling in non-breeding males and females showed sex differences in both androgens and estrogens (Fig 1B). Males had detectable forebrain levels of AE (0.03 ± 0.004 ng/g), T (0.06 ± 0.002 ng/g), 11-KT (0.04± 0.007 ng/g), E1 (0.005 ± 0.0005 ng/g), and cortisol (2.54 ± 0.3 ng/g). Similarly, females had detectable forebrain AE (0.05 ± 0.006 ng/g), T (0.01 ± 0.001 ng/g), E1 (0.014 ± 0.004 ng/g), and cortisol (3.83 ± 1.6 ng/g) (Fig 1B), but not detectable 11-KT. Interestingly, females had higher forebrain levels of AE (t = 2.152, p = 0.045), T (t = 2.246, p = 0.038), and E1 (t = 2.805, p = 0.01) than males. In contrast, forebrain 11-KT were higher in males than females (t = 5.3, p < 0.0001) as 11-KT was only consistently detectable in males (Fig 1B). Cortisol levels did not show sex differences (t = 0.243, p = 0.81).

Neither females nor males had detectable forebrain levels of progesterone, DHEA, or E2.

Comparison between circulating and brain steroid levels

We compared blood and brain steroid levels for each subject by paired comparisons (Fig 2). Interestingly, both sexes showed higher levels of estrogens in the brain than in blood, whereas androgens showed the opposite pattern. In this regard, E1 was detectable in forebrain samples but not in plasma samples (males: t = 9.19; p < 0.0001; females: t = 3.35; p = 0.007; Fig 2). On the other hand, AE and T levels were higher in the blood than in the forebrain in both sexes (males: AE, t = 3.05; p = 0.02; females: t = 2.15; p = 0.07; males: T, t = 13.35; p < 0.0001; females: T, t = 8.83; p < 0.0001; Fig 2). The analysis for 11-KT in males showed that blood and forebrain levels were not significantly different (t = 0.86; p = 0.42 Fig 2).

thumbnail
Download:
Fig 2. Comparison of circulating and brain steroid levels in male and female G. omarorum in the non-breeding season.

Blood and forebrain steroid concentrations were compared using paired t-tests. Lines connect values for the same fish. Abbreviations: AE, androstenedione; T, testosterone; 11-KT, 11-ketotestosterone; E1, estrone. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.

https://doi.org/10.1371/journal.pone.0289461.g002

Correlations between circulating and brain steroid levels

We assessed the relationships between circulating and brain steroid levels using correlation matrices (Spearman’s with Benjamini-Hochberg correction) (Fig 3). In males, the three circulating androgens showed positive correlations (AE and T in plasma, r = 0.76, p = 0.013; AE and 11-KT in plasma, r = 0.67, p = 0.008; and T and 11-KT in plasma, r = 0.76, p = 0.012).

thumbnail
Download:
Fig 3. Correlation matrices for steroid levels in plasma and forebrain.

Analyses were performed in males (A) and females (B). All values are expressed in Spearman’s rho and corrected for multiple comparisons using the Benjamini-Hochberg method. Abbreviations: AE, androstenedione; T, testosterone; 11-KT, 11-ketotestosterone; E1, estrone. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

https://doi.org/10.1371/journal.pone.0289461.g003

In females, circulating AE and T showed a positive correlation (r = 0.97, p < 0.0001). In addition, brain AE and T also showed a positive correlation (r = 0.82, p = 0.001). Brain E1 levels showed positive correlations with two circulating androgens (brain E1 and plasma AE, r = 0.75, p = 0.05; brain E1 and plasma T, r = 0.77, p = 0.02), and a positive correlation with brain AE (r = 0.79, p = 0.006).

Discussion

This study is the first report of steroid profiling in teleost fish brain using mass spectrometry. A panel of 8 steroids was validated for forebrain and plasma samples in male and female G. omarorum, a seasonal breeder in which both sexes display aggression in the non-breeding season. Here we show that: i) systemic steroids in the non-breeding season are similar in both sexes, although only males have circulating 11-KT, ii) brain steroid levels are sexually dimorphic, as females have higher levels of AE, T and E1, and only males have 11-KT, iii) systemic androgens such as AE and T in the non-breeding season are potential precursors for neuroestrogen synthesis, iv) estrogens, which play a key role in non-breeding aggression, are detectable in the brain (but not the circulation) in both sexes. Taken together, these data provide fundamental insights into steroid regulation of behavior during the non-breeding season.

Steroid measurement by mass spectrometry

The protocol developed in this study allowed us to describe brain and systemic steroid profiles in a teleost for the first time. The method combines liquid-liquid extraction with solid phase extraction to remove interference caused by the matrices, which is particularly challenging in lipid-rich brain samples. The LC-MS/MS assay has high specificity and high sensitivity (detection limits were generally 0.2 to 0.8 pg per sample). This is especially advantageous when quantifying steroids in non-breeding samples, as it allows for accurate quantification of low levels of analytes. In contrast, immunoassays often overestimate analyte levels (due to antibody cross-reactivity), particularly when analyte concentrations are low [53, 54]. Mass spectrometry also enables the measurement of steroids that are not commonly measured by immunoassays (e.g. AE and E1) and simultaneous quantification of multiple steroids in a sample. This allows a more comprehensive endocrine profile and a deeper understanding of systemic and local steroid levels in individual subjects.

Plasma and brain steroid profiles and sex differences

There are few reports of brain steroid levels in non-breeding wild animals. Resting, undisturbed non-breeding male and female G. omarorum had detectable circulating and forebrain levels of AE, T, and cortisol, while only males had detectable circulating and forebrain 11-KT. In contrast, E2, DHEA, and progesterone were not detectable in plasma or forebrain. In addition, E1 was detectable in both sexes exclusively in forebrain samples. The thorough steroid profiling achieved in this study is extremely valuable in the framework of understanding neural steroid synthesis and the hormonal mechanisms underlying social behaviors.

Circulating and forebrain cortisol levels in G. omarorum were at least an order of magnitude higher than the other steroids (Fig 1A and 1B). Systemic glucocorticoid levels were comparable to those of non-breeding birds [14] and other teleost fish [55], although much lower than those of non-breeding hamsters [56]. In non-breeding G. omarorum, cortisol levels did not differ between the sexes (Fig 1A), as reported in other teleosts (reviewed in [55]). Cortisol levels were similar between blood and brain (Fig 1) suggesting that brain cortisol comes from a peripheral source.

Several studies have examined the relationship between progesterone and aggression [15, 57–61]. Progesterone can be converted to brain androgens and estrogens in teleosts [62], and this process might be important for non-breeding aggression in song sparrows [14]. For these reasons, we were especially interested in the measurement of progesterone in G. omarorum. The lack of circulating and brain progesterone observed in this study could be due to several reasons: i) progesterone might be rapidly converted to metabolites, which are biologically active in other teleosts [63] but not measured here, ii) progesterone might transiently increase during aggressive interactions.

Androgens were detectable both in circulation as well as in brain tissue. Circulating 11-KT levels of non-breeding males were similar to those measured in this species by ELISA [20] and to levels of other teleosts in the non-breeding season [64, 65]. The only previous report of brain androgens in fish is in the bidirectional hermaphrodite Lythrypnus dalli, in which the authors explored the changes in 11-KT and T that accompany sex reversal, parental care, and aggression [66–68]. In L. dalli, brain T and 11-KT were present in both males and females [68], most probably related to their particular sexual physiology. In non-breeding male song sparrows, basal levels of AE and T measured by LC-MS/MS were non-detectable [14].

Overall, systemic steroid levels were similar between males and females, except for 11-KT, a typical male androgen [69] (Fig 1A). This lack of dimorphism was expected for non-breeding subjects, as G. omarorum is a monomorphic species that shows no sex differences in body size, body condition, basal electric organ discharge [31], spatial distribution in the field, or territory size [70]. Moreover, non-breeding territorial behavior shows no sex differences in dynamics, contest duration, outcome, or communication signals [32, 33].

Surprisingly, in spite of the lack of sex differences in physiology and behavior, forebrain steroid levels showed sex differences (Fig 1B). Females had higher levels of AE, T and E1 than males (Fig 1B) while males had higher forebrain 11-KT than females (11-KT was non-detectable in females). This may be explained by: i) a sex difference in the expression of steroid binding globulins, leading to differences in brain uptake and/or retention of steroids [71, 72], or ii) sex differences in brain steroidogenic enzymes that synthesize or metabolize steroids [73–75]. Although brain sex differences are usually related to sexually biased behavior, they can also compensate for basal physiological sex differences and ultimately produce a monomorphic behavioral output [76]. In the breeding season there is evidence of sexually differentiated behavior in G. omarorum which may be associated with anatomical or functional brain differences. In G. omarorum males and females have different territory size determinants, suggesting different energetic requirements and associated foraging behavior [70]. Furthermore, males of the genus Gymnotus are reported to show paternal care [77, 78]. Seasonal changes in the brain may compensate for these differences, ultimately leading to monomorphic behaviors in the non-breeding season.

Neuroestrogens and the mechanisms underlying non-breeding aggression

The presence of steroidogenic enzymes to produce steroids in the brain, even de novo, has been demonstrated in teleosts [62, 79–81]. However, when measuring enzyme activity, enzyme substrates and co-factors are often used in saturating concentrations. This may give us limited information about the brain as a steroid source in natural processes, because the post-translational modifications of enzymes, concentration of precursors, and local metabolism are dynamic factors that influence local steroid levels.

An important role for circulating steroid hormones during the non-breeding season is to serve as precursors that reach the brain and are locally converted into signaling molecules that are key in certain neurobiological processes [27, 82, 83]. In that sense, the quantification of circulating DHEA was of particular interest as it is an inactive androgen precursor that has been linked with the maintenance of non-breeding aggression in birds and mammals [29, 74, 82, 84]. However, circulating and brain DHEA were non-detectable in G. omarorum during the non-breeding season, indicating that DHEA is absent or present at very low concentrations, similar to a recent report in song sparrows [14].

Brain-synthesized sex steroids are key in the regulation of social behavior. In zebra finch, quantification of brain steroids in the auditory cortex shows local synthesis of androgens and estrogens in response to social stimuli [13, 85]. In the fish, dusky gregories, androgen receptor antagonism in the non-breeding season reduces aggression in males but not females [64]. In song sparrows, non-breeding aggression is not affected by androgen receptor antagonism [86] or castration [27]. However, aromatase inhibition reduces aggression, indicating that the conversion of androgens into estrogens is crucial for promoting non-breeding aggression [11]. Similarly, in G. omarorum non-breeding aggression also depends on extragonadal synthesis of estrogens [20, 33]. In the present study, the data suggest that androgens, either from the circulation or from the brain, may be acting as precursors for neural synthesis of estrogens. Our results unequivocally show neuroestrogen synthesis in non-breeding female and male G. omarorum. In particular, E1 was detected in all forebrain samples in both sexes, while its plasma levels were non-detectable (Figs 1 and 2). The correlations between hormonal precursors and their products provide insight into the pathways of steroid neurosynthesis. Our data showed sex differences in matrix correlations most probably reflecting different synthetic pathways. The direct precursor of E1 is AE, which is detectable in plasma and brain, although in much higher levels in circulation (Fig 2). In females, there is a positive correlation between forebrain AE and E1, as well as between circulating AE and brain E1 (Fig 3B). This suggests that peripheral AE is taken up by the brain and converted to E1. Both AE and T are substrates of aromatase, and direct precursors to estrogens (E1 and E2 respectively). Nevertheless, brain AE concentrations are almost five-fold higher than those of T, and thus may be a stronger competitor for aromatase, accounting for the detection of E1 but not E2. The neurosynthesis of estrogens is consistent with the observation that in G. omarorum the dynamics and outcome of aggressive behavior depends on non-gonadal estrogen [20, 33, 87] and that there is differential expression of forebrain aromatase related to dominance and subordination [35]. Further, in G. omarorum the effects of estrogens on aggression are rapid and act in a timeframe that is incompatible with the classic genomic (nuclear initiated) mechanism of action [20, 33, 88, 89]. Brain synthesized estrogens act via non-genomic mechanisms by binding to membrane-associated receptors and affecting intracellular signaling cascades [90, 91]. In all, we propose that in G. omarorum neuroestrogens may promote non-breeding aggression by non-genomic mechanisms.

Conclusions

Plasma and forebrain steroid profiles were characterized for the first time in non-breeding males and females of a teleost fish. Our results demonstrate brain production of an estrogen in both sexes, most probably derived from circulating androgens. Taken together, these data provide fundamental insights into the regulation of non-breeding aggression and common neuroendocrine strategies across species.

Acknowledgments

We thank Dr. Chunqi Ma for her assistance with LC-MS/MS protocol development, Sofia Gray for assistance with the statistical analysis, Adriana Migliaro, Guillermo Valiño, and Rossana Perrone for help with fieldwork.

References

  1. 1. Compagnone NA, Mellon SH. Neurosteroids: Biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol. 2000;21(1):1–56. pmid:10662535
  2. 2. Mellon SH, Griffin LD. Neurosteroids: biochemistry and clinical significance. TRENDSin Endocrinol Metab. 2002;13(1). pmid:11750861
  3. 3. Fester L, Rune GM. Sex neurosteroids: Hormones made by the brain for the brain [Internet]. Neurosci Lett [Internet]. Elsevier B.V.; 2021;753(December 2020):135849. Available from: pmid:33775739
  4. 4. Yilmaz C, Karali K, Fodelianaki G, Gravanis A, Chavakis T, Charalampopoulos I, et al. Neurosteroids as regulators of neuroinflammation. Front Neuroendocrinol. 2019;55:100788. pmid:31513776
  5. 5. Arevalo MA, Azcoitia I, Garcia-Segura LM. The neuroprotective actions of oestradiol and oestrogen receptors. Nat Rev Neurosci. Nature Publishing Group; 2015;16(1):17–29. pmid:25423896
  6. 6. Amandusson Å, Blomqvist A. Estrogenic influences in pain processing [Internet]. Front Neuroendocrinol [Internet]. Elsevier Inc.; 2013;34:329–49. Available from: http://dx.doi.org/10.1016/j.yfrne.2013.06.001 pmid:23817054
  7. 7. Cornil CA, Court L. Neuroestrogens in the control of sexual behavior: Past, present, and future [Internet]. Curr Opin Endocr Metab Res [Internet]. Elsevier Ltd; 2022;24:100334. Available from:
  8. 8. Court L, Balthazart J, Ball GF, Cornil CA. Effect of chronic intracerebroventricular administration of an aromatase inhibitor on the expression of socio-sexual behaviors in male Japanese quail [Internet]. Behav Brain Res [Internet]. Elsevier B.V.; 2021;410(April):113315. Available from: pmid:33901434
  9. 9. Brooks DC, Coon JSV, Ercan CM, Xu X, Dong H, Levine JE, et al. Brain aromatase and the regulation of sexual activity in male mice. Endocrinol (United States). 2020;161(10):1–15. pmid:32910181
  10. 10. Trainor BC, Finy M Sima, Nelson RJ. Rapid effects of estradiol on male aggression depend on photoperiod in reproductively non-responsive mice [Internet]. Horm Behav [Internet]. 2008 Jan;53(1):192–9. Available from: http://www.sciencedirect.com/science/article/pii/S0018506X07002334 pmid:17976598
  11. 11. Soma KK, Tramontin AD, Wingfield JC. Oestrogen regulates male aggression in the non-breeding season. Proc R Soc B Biol Sci. 2000;267(1448):1089–96. pmid:10885513
  12. 12. Heimovics SA, Merritt JR, Jalabert C, Ma C, Maney DL, Soma KK. Rapid effects of 17β-estradiol on aggressive behavior in songbirds: Environmental and genetic influences. Horm Behav. 2018;104:41–51.
  13. 13. de Bournonville C, McGrath A, Remage-Healey L. Testosterone synthesis in the female songbird brain [Internet]. Horm Behav [Internet]. Elsevier; 2020;121(June 2019):104716. Available from: pmid:32061616
  14. 14. Jalabert C, Ma C, Soma KK. Profiling of systemic and brain steroids in male songbirds: Seasonal changes in neurosteroids. J Neuroendocrinol. 2021;33(1):1–16. pmid:33314446
  15. 15. Munley KM, Trinidad JC, Deyoe JE, Adaniya CH, Nowakowski AM, Ren CC, et al. Melatonin-dependent changes in neurosteroids are associated with increased aggression in a seasonally breeding rodent. J Neuroendocrinol. 2021;33(3):1–18.
  16. 16. Jalabert C, Shock MA, Ma C, Bootsma TJ, Liu MQ, Soma KK. Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS. eNeuro. 2022;9(4):1–16. pmid:35788106
  17. 17. Logan CA, Wingfield JC. Autumnal territorial aggression is independent of plasma testosterone in mockingbirds [Internet]. Horm Behav [Internet]. 1990 Dec;24(4):568–81. Available from: http://www.sciencedirect.com/science/article/pii/0018506X9090042V pmid:2286368
  18. 18. Jasnow AM, Huhman KL, Bartness TJ, Demas GE. Short-Day Increases in Aggression Are Inversely Related to Circulating Testosterone Concentrations in Male Siberian Hamsters (Phodopus sungorus) [Internet]. Horm Behav [Internet]. 2000 Sep;38(2):102–10. Available from: http://www.sciencedirect.com/science/article/pii/S0018506X00916040 pmid:10964524
  19. 19. Hau M, Stoddard ST, Soma KK. Territorial aggression and hormones during the non-breeding season in a tropical bird. Horm Behav. 2004;45(1):40–9. pmid:14733890
  20. 20. Jalabert C, Quintana L, Pessina P, Silva A. Extra-gonadal steroids modulate non-breeding territorial aggression in weakly electric fish [Internet]. Horm Behav [Internet]. Elsevier Inc.; 2015;72:60–7. Available from: http://dx.doi.org/10.1016/j.yhbeh.2015.05.003 pmid:25989595
  21. 21. Soma KK, Schlinger BA, Wingfield JC, Saldanha CJ. Brain aromatase, 5α-reductase, and 5β-reductase change seasonally in wild male song sparrows: Relationship to aggressive and sexual behavior [Internet]. J Neurobiol [Internet]. Wiley Subscription Services, Inc., A Wiley Company; 2003 Sep 5;56(3):209–21. Available from: http://dx.doi.org/10.1002/neu.10225
  22. 22. Soma KK, Sullivan K, Wingfield JC. Combined Aromatase Inhibitor and Antiandrogen Treatment Decreases Territorial Aggression in a Wild Songbird during the Nonbreeding Season [Internet]. Gen Comp Endocrinol [Internet]. 1999 Sep;115(3):442–53. Available from: http://www.sciencedirect.com/science/article/pii/S0016648099973343 pmid:10480996
  23. 23. Wacker DW, Wingfield JC, Davis JE, Meddle SL. Seasonal Changes in Aromatase and Androgen Receptor, but not Estrogen Receptor mRNA Expression in the Brain of the Free-Living Male Song Sparrow, Melospiza melodia morphna. J Comp Neurol. 2010;518(18):3819–35. pmid:20653036
  24. 24. Soma KK, Sullivan KA, Tramontin AD, Saldanha CJ, Schlinger BA, Wingfield JC. Acute and chronic effects of an aromatase inhibitor on territorial aggression in breeding and nonbreeding male song sparrows. J Comp Physiol—A Sensory, Neural, Behav Physiol. 2000;186(7–8):759–69. pmid:11016791
  25. 25. Heimovics SA, Ferris JK, Soma KK. Non-invasive administration of 17β-estradiol rapidly increases aggressive behavior in non-breeding, but not breeding, male song sparrows [Internet]. Horm Behav [Internet]. Elsevier B.V.; 2015;69:31–8. Available from: http://dx.doi.org/10.1016/j.yhbeh.2014.11.012
  26. 26. Wingfield JC, Hahn TP. Testosterone and territorial behaviour in sedentary and migratory sparrows [Internet]. Anim Behav [Internet]. 1994 Jan;47(1):77–89. Available from: http://www.sciencedirect.com/science/article/pii/S0003347284710098
  27. 27. Wingfield JC. Regulation of territorial behavior in the sedentary song sparrow, Melospiza melodia morphna. Hormones and Behavior. 1994 p. 1–15. pmid:8034278
  28. 28. Trainor BC, Lin S, Finy MS, Rowland MR, Nelson RJ. Photoperiod reverses the effects of estrogens on male aggression via genomic and nongenomic pathways [Internet]. Proc Natl Acad Sci [Internet]. 2007 Jun 5;104(23):9840–5. Available from: http://www.pnas.org/content/104/23/9840.abstract pmid:17525148
  29. 29. Soma KK, Rendon NM, Boonstra R, Albers HE, Demas GE. DHEA effects on brain and behavior: Insights from comparative studies of aggression. Journal of Steroid Biochemistry and Molecular Biology. 2015. pmid:24928552
  30. 30. Quintana L, Jalabert C, Fokidis HB, Soma KK, Zubizarreta L. Neuroendocrine Mechanisms Underlying Non-breeding Aggression: Common Strategies Between Birds and Fish. Front Neural Circuits. 2021;15(July). pmid:34393727
  31. 31. Richer-de-Forges MM, Crampton WGR, Albert JS. A New Species of Gymnotus (Gymnotiformes, Gymnotidae) from Uruguay: Description of a Model Species in Neurophysiological Research [Internet]. Copeia [Internet]. 2009 Sep [cited 2011 Apr 27];3:538–44. Available from: http://www.bioone.org/doi/abs/10.1643/CI-07-103
  32. 32. Batista G, Zubizarreta L, Perrone R, Silva A. Non-sex-biased dominance in a sexually monomorphic electric fish: Fight structure and submissive electric signalling [Internet]. Ethology [Internet]. Blackwell Publishing Ltd; 2012 Apr 1;118(4):398–410. Available from: http://dx.doi.org/10.1111/j.1439-0310.2012.02022.x
  33. 33. Zubizarreta L, Silva AC, Quintana L. The estrogenic pathway modulates non-breeding female aggression in a teleost fish [Internet]. Physiol Behav [Internet]. Elsevier; 2020;220(March):112883. Available from: pmid:32199998
  34. 34. Silva AC, Zubizarreta L, Quintana L. A Teleost Fish Model to Understand Hormonal Mechanisms of Non-breeding Territorial Behavior. Front Endocrinol (Lausanne). 2020;11:468. pmid:32793118
  35. 35. Eastman G, Valiño G, Radío S, Young RL, Quintana L, Zakon HH, et al. Brain transcriptomics of agonistic behaviour in the weakly electric fish Gymnotus omarorum, a wild teleost model of non-breeding aggression. Sci Rep. 2020;10(1):1–11.
  36. 36. Perrone R, Silva AC. Status-dependent vasotocin modulation of dominance and subordination in the weakly electric fish Gymnotus omarorum. Front Behav Neurosci. 2018;12(January). pmid:29403366
  37. 37. Pouso P, Radmilovich M, Silva A. An immunohistochemical study on the distribution of vasotocin neurons in the brain of two weakly electric fish, Gymnotus omarorum and Brachyhypopomus gauderio [Internet]. Tissue Cell [Internet]. Elsevier Ltd; 2017;49(2):257–69. Available from: http://dx.doi.org/10.1016/j.tice.2017.02.003
  38. 38. Silva AC, Perrone R, Zubizarreta L, Batista G, Stoddard PK. Neuromodulation of the agonistic behavior in two species of weakly electric fish that display different types of aggression [Internet]. J Exp Biol [Internet]. 2013 Jul 1;216(13):2412–20. Available from: http://jeb.biologists.org/content/216/13/2412.abstract pmid:23761466
  39. 39. Zubizarreta L, Perrone R, Stoddard PK, Costa G, Silva AC. Differential serotonergic modulation of two types of aggression in weakly electric fish [Internet]. Front Behav Neurosci [Internet]. Frontiers Media S.A.; 2012 Nov 19;6:77. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500767/ pmid:23181014
  40. 40. Zubizarreta L, Stoddard PK, Silva A. Aggression levels affect social interaction in the non-breeding territorial aggression of the weakly electric fish, gymnotus omarorum [Internet]. Ethology [Internet]. 2014 Jan 1;121(1):8–16. Available from: http://dx.doi.org/10.1111/eth.12299
  41. 41. Borde M, Quintana L, Comas V, Silva A. Hormone-mediated modulation of the electromotor CPG in pulse-type weakly electric fish. Commonalities and differences across species. Dev Neurobiol. 2020;80(1–2):70–80. pmid:31955508
  42. 42. Dunlap KD, Smith GT, Yekta A. Temperature Dependence of Electrocommunication Signals and Their Underlying Neural Rhythms in the Weakly Electric Fish, Apteronotus leptorhynchus [Internet]. Brain Behav Evol [Internet]. 2000 Jul 7;55(3):152–62. Available from: pmid:10899709
  43. 43. Fox HE, White SA, Kao MHF, Fernald RD. Stress and dominance in a social fish. J Neurosci. Soc Neuroscience; 1997;17(16):6463–9.
  44. 44. Salazar VL, Stoddard PK. Sex differences in energetic costs explain sexual dimorphism in the circadian rhythm modulation of the electrocommunication signal of the gymnotiform fish Brachyhypopomus pinnicaudatus. J Exp Biol. 2008;211(6):1012–20. pmid:18310126
  45. 45. Tobiansky DJ, Korol AM, Ma C, Hamden JE, Jalabert C, Tomm RJ, et al. Testosterone and corticosterone in the mesocorticolimbic system of male rats: Effects of gonadectomy and caloric restriction. Endocrinology. 2018;159(1):450–64. pmid:29069423
  46. 46. Tobiansky DJ, Kachkovski G V, Enos RT, Schmidt KL, Murphy EA, Soma KK. Sucrose consumption alters steroid and dopamine signalling in the female rat brain. J Endocrinol. 2020;245(2):231–46. pmid:32112695
  47. 47. Tobiansky DJ, Kachkovski G V, Enos RT, Schmidt KL, Murphy EA, Floresco SB, et al. Maternal sucrose consumption alters behaviour and steroids in adult rat offspring. J Endocrinol. 2021;251(3):161–80. pmid:34582358
  48. 48. Wei R, Wang J, Su M, Jia E, Chen S, Chen T, et al. Missing Value Imputation Approach for Mass Spectrometry-based Metabolomics Data. Sci Rep. Springer US; 2018;8(1):1–10. pmid:29330539
  49. 49. Wei R, Wang J, Jia E, Chen T, Ni Y, Jia W. GSimp: A Gibbs sampler based left-censored missing value imputation approach for metabolomics studies. PLoS Comput Biol. 2018;14(1):e1005973. pmid:29385130
  50. 50. Taves MD, Ma C, Heimovics SA, Saldanha CJ, Soma KK. Measurement of steroid concentrations in brain tissue: Methodological considerations. Front Endocrinol (Lausanne). 2011;2:39. pmid:22654806
  51. 51. Taves MD, Schmidt KL, Ruhr IM, Kapusta K, Prior NH, Soma KK. Steroid concentrations in plasma, whole blood and brain: Effects of saline perfusion to remove blood contamination from brain. PLoS One. 2010;5(12):e15727. pmid:21206751
  52. 52. Taves MD, Plumb AW, Sandkam BA, Ma C, Van Der Gugten JG, Holmes DT, et al. Steroid profiling reveals widespread local regulation of glucocorticoid levels during mouse development. Endocrinology. 2015;156(2):511–22. pmid:25406014
  53. 53. Grebe SKG, Singh RJ. LC-MS/MS in the clinical laboratory—Where to from here? Clin Biochem Rev. 2011;32(1):5. pmid:21451775
  54. 54. Wudy SA, Schuler G, Sánchez-guijo A, Hartmann MF. The art of measuring steroids Principles and practice of current hormonal steroid analysis. J Steroid Biochem Mol Biol. Elsevier; 2018;179:88–103. pmid:28962971
  55. 55. Milla S, Wang N, Mandiki SNM, Kestemont P. Corticosteroids: Friends or foes of teleost fish reproduction? [Internet]. Comp Biochem Physiol—A [Internet]. Elsevier Inc.; 2009;153:242–51. Available from: http://dx.doi.org/10.1016/j.cbpa.2009.02.027 pmid:19254778
  56. 56. Scotti MAL, Rendon NM, Greives TJ, Romeo RD, Demas GE. Short-day aggression is independent of changes in cortisol or glucocorticoid receptors in male Siberian hamsters (Phodopus sungorus). J Exp Zool Part A Ecol Genet Physiol. 2015;323(5):331–42. pmid:25802186
  57. 57. Davis ES, Marler CA. The progesterone challenge: Steroid hormone changes following a simulated territorial intrusion in female Peromyscus californicus. Horm Behav. 2003;44(3):185–98. pmid:14609541
  58. 58. Kohlert JG, Meisel RL. Inhibition of aggression by progesterone and its metabolites in female Syrian hamsters. Aggress Behav. 2001;27(5):372–81.
  59. 59. Albert DJ, Jonik RH, Walsh ML. Interaction of estradiol, testosterone, and progesterone in the modulation of hormone-dependent aggression in the female rat. Physiol Behav. 1992;52(4):773–9. pmid:1409952
  60. 60. Goymann W, Wittenzellner A, Schwabl I, Makomba M. Progesterone modulates aggression in sex-role reversed female African black coucals. Proc R Soc B Biol Sci. 2008;275:1053–60. pmid:18252672
  61. 61. O’Connell LA, Ding JH, Hofmann HA. Sex differences and similarities in the neuroendocrine regulation of social behavior in an African cichlid fish [Internet]. Horm Behav [Internet]. Elsevier Inc.; 2013;64(3):468–76. Available from: http://dx.doi.org/10.1016/j.yhbeh.2013.07.003 pmid:23899762
  62. 62. Diotel N, Do Rego JL, Anglade I, Vaillant C, Pellegrini E, Vaudry H, et al. The brain of teleost fish, a source, and a target of sexual steroids. Front Neurosci. 2011;5:1–15.
  63. 63. Kime DE. Classical and non-clssical reproductive steroids in fish. Rev Fish Biol Fish. 1993;3:160–80.
  64. 64. Vullioud P, Bshary R, Ros AFH. Intra- and interspecific aggression do not modulate androgen levels in dusky gregories, yet male aggression is reduced by an androgen blocker [Internet]. Horm Behav [Internet]. 2013 Aug;64(3):430–8. Available from: http://www.sciencedirect.com/science/article/pii/S0018506X13001360 pmid:23838629
  65. 65. Vitousek MN, Johnson MA, Donald JW, Francis CD, Fuxjager MJ, Goymann W, et al. Data Descriptor: HormoneBase, a population-level database of steroid hormone levels across vertebrates. Sci Data. 2018;5:1–7.
  66. 66. Lorenzi V, Earley RL, Grober MS. Differential Responses of Brain, Gonad and Muscle Steroid Levels to Changes in Social Status and Sex in a Sequential and Bidirectional Hermaphroditic Fish. PLoS One. 2012;7(12).
  67. 67. Pradhan DS, Connor KR, Pritchett EM, Grober MS. Contextual modulation of androgen effects on agonistic interactions [Internet]. Horm Behav [Internet]. Elsevier Inc.; 2014;65(1):47–56. Available from: http://dx.doi.org/10.1016/j.yhbeh.2013.11.006 pmid:24315925
  68. 68. White KJ, Rivas MG, Pradhan DS. Sex differences in aggressive intensities and brain steroids during status resolution in a sex changing fish, Lythrypnus dalli [Internet]. Horm Behav [Internet]. Elsevier Inc.; 2023;153:105373. Available from: pmid:37182511
  69. 69. Borg B. Androgens in teleost fishes [Internet]. Comp Biochem Physiol Part C Comp [Internet]. 1994 Nov;109(3):219–45. Available from: http://www.sciencedirect.com/science/article/pii/074284139400063G
  70. 70. Zubizarreta L, Quintana L, Hernández D, De Mello FT, Meerhoff M, Honjii RM, et al. Seasonal and social factors associated with spacing in a wild territorial electric fish. PLoS One. 2020;15(6):1–19.
  71. 71. Bobe J, Guiguen Y, Fostier A. Diversity and biological significance of sex hormone-binding globulin in fish, an evolutionary perspective. Mol Cell Endocrinol. 2010;316:66–78. pmid:19772892
  72. 72. Marivin E, Yano A, Guérin A, Nguyen TV, Fostier A, Bobe J, et al. Sex hormone-binding globulins characterization and gonadal gene expression during sex differentiation in the rainbow trout, Oncorhynchus mykiss. Mol Reprod Dev. 2014;81(8):757–65. pmid:24889418
  73. 73. Santillo A, Falvo S, Di Fiore MM, Chieffi Baccari G. Seasonal changes and sexual dimorphism in gene expression of StAR protein, steroidogenic enzymes and sex hormone receptors in the frog brain [Internet]. Gen Comp Endocrinol [Internet]. Elsevier Inc.; 2017;246:226–32. Available from: http://dx.doi.org/10.1016/j.ygcen.2016.12.012 pmid:28027903
  74. 74. Munley KM, Han Y, Lansing MX, Demas GE. Winter madness: Melatonin as a neuroendocrine regulator of seasonal aggression. J Exp Zool Part A Ecol Integr Physiol. 2022;337:873–89. pmid:35451566
  75. 75. Wartenberg P, Farkas I, Csillag V, Colledge WH, Hrabovszky E, Boehm U. Sexually dimorphic neurosteroid synthesis regulates neuronal activity in the murine brain. J Neurosci. 2021;41(44):9177–91. pmid:34561233
  76. 76. De Vries GJ. Minireview: Sex Differences in Adult and Developing Brains: Compensation, Compensation, Compensation. Endocrinology. 2004;145(3):1063–8. pmid:14670982
  77. 77. Crampton AWGR, Hopkins CD. Nesting and Paternal Care in the Weakly Electric Fish Gymnotus (Gymnotiformes: Gymnotidae) with Descriptions of Larval and Adult Electric Organ Discharges of Two Species [Internet]. Am Soc Ichthyol Herpetol [Internet]. 2005;1:48–60. Available from:
  78. 78. Kirschbaum F, Schugardt C. Reproductive strategies and developmental aspects in mormyrid and gymnotiform fishes. J Physiol. Elsevier; 2002;96(5–6):557–66. pmid:14692503
  79. 79. Andersson E, Borg B, Lambert JGD. Aromatase activity in brain and pituitary of immature and mature atlantic salmon (Salmo salar L.) parr. Gen Comp Endocrinol. 1988;72(3):394–401. pmid:3240849
  80. 80. Schulz R, Blum V. Extragonadal 17β-hydroxysteroid dehydrogenase activity in rainbow trout. Gen Comp Endocrinol. 1991;82(2):197–205.
  81. 81. Pasmanik M, Callard G V. Aromatase and 5α-reductase in the teleost brain, spinal cord, and pituitary gland. Gen Comp Endocrinol. 1985;60(2):244–51.
  82. 82. Soma KK, Scotti M-AL, Newman AEM, Charlier TD, Demas GE. Novel mechanisms for neuroendocrine regulation of aggression [Internet]. Front Neuroendocrinol [Internet]. 2008 Oct;29(4):476–89. Available from: http://www.sciencedirect.com/science/article/pii/S0091302207000751 pmid:18280561
  83. 83. Wingfield JC, Goymann W, Jalabert C, Soma KK. Concepts derived from the Challenge Hypothesis. Horm Behav. 2019;115:104550. pmid:31265826
  84. 84. Jalabert C, Munley KM, Demas GE, Soma KK. Aggressive Behavior [Internet]. second. Encyclopedia of Reproduction. Academic Press; 2018 [cited 2018 Sep 13] p. 242–7. https://www.sciencedirect.com/science/article/pii/B9780128012383645919?via%3Dihub
  85. 85. Remage-Healey L, Maidment NT, Schlinger BA. Forebrain steroid levels fluctuate rapidly during social interactions. Nat Neurosci. 2008;11(11):1327–34. pmid:18820691
  86. 86. Sperry T, Wacker DW, Wingfield JC. The role of androgen receptors in regulating territorial aggression in male song sparrows. Horm Behav. 2010;57(1):86–95. pmid:19799905
  87. 87. Quintana L, Zubizarreta L, Jalabert C, Batista G, Perrone R, Silva A. Building the case for a novel teleost model of non-breeding aggression and its neuroendocrine control [Internet]. J Physiol Paris [Internet]. Elsevier Ltd; 2016;110(3):224–32. Available from: http://dx.doi.org/10.1016/j.jphysparis.2016.11.009 pmid:27915075
  88. 88. Micevych P, Dominguez R. Membrane estradiol signaling in the brain [Internet]. Front Neuroendocrinol [Internet]. Elsevier Inc.; 2009;30(3):315–27. Available from: http://dx.doi.org/10.1016/j.yfrne.2009.04.011 pmid:19416735
  89. 89. Merritt JR, Davis MT, Jalabert C, Libecap TJ, Williams DR, Soma KK, et al. Rapid effects of estradiol on aggression depend on genotype in a species with an estrogen receptor polymorphism [Internet]. Horm Behav [Internet]. 2018 Feb [cited 2018 Aug 13];98:210–8. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0018506X17303501 pmid:29277700
  90. 90. Balthazart J, Ball GF. Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci. 2006;29(5):241–9. pmid:16580076
  91. 91. Saldanha CJ, Remage-Healey L, Schlinger BA. Synaptocrine signaling: Steroid synthesis and action at the synapse. Endocr Rev. 2011;32(4):532–49. pmid:21622487