Louisiana State University LSU Digital Commons Faculty Publications Department of Biological Sciences 12-1-2010 Reproductive status regulates expression of sex steroid and GnRH receptors in the olfactory bulb Karen P. Maruska Stanford University Russell D. Fernald Stanford University Follow this and additional works at: https://digitalcommons.lsu.edu/biosci_pubs Recommended Citation Maruska, K., & Fernald, R. (2010). Reproductive status regulates expression of sex steroid and GnRH receptors in the olfactory bulb. Behavioural Brain Research, 213 (2), 208-217. https://doi.org/10.1016/j.bbr.2010.04.058 This Article is brought to you for free and open access by the Department of Biological Sciences at LSU Digital Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of LSU Digital Commons. For more information, please contact ir@lsu.edu. Reproductive Status Regulates Expression of Sex Steroid andGnRH Receptors in the Olfactory BulbKaren P. Maruska and Russell D. FernaldBiology Department Stanford University 371 Serra Mall Stanford, CA. 94305 Karen P. Maruska:maruska@stanford.edu Russell D. Fernald: rfernald@stanford.edu Phone: 650-723-0881AbstractNeuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integratean animal's internal physiological state with incoming external cues, and can have profound effectson the processing of behaviorally-relevant information, particularly from the olfactory system. WhileGnRH and steroid receptors are present in olfactory processing regions across vertebrates, little isknown about whether their expression levels change with internal physiological state or externalsocial cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα,ARβ), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapiaburtoni. We asked whether these receptor levels changed with reproductive condition in females, orwith social status, which regulates reproductive capacity in males. Our results reveal that mRNAlevels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb varywith sex, social status in males, and reproductive condition in females, which highlights the potentialimportance of changing receptor levels in fine-tuning the olfactory system during the reproductivecycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levelsin males, but negatively correlated in females, suggesting different regulatory control between sexes.These results provide support for the hypothesis that the first-order olfactory relay station is asubstrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levelscould be an important mechanism for regulating reproductive, social, and seasonal plasticity inolfactory perception observed across vertebrates.Keywordsandrogen receptor; aromatase; Astatotilapia burtoni; estrogen receptor; gonadotropin-releasinghormone; mRNA; olfaction; teleost1. IntroductionAnimals must integrate signals from their external and internal environments to makeappropriate and context-dependent behavioral decisions. One key player in this process, theolfactory system, is important for vertebrate behavior, since many taxa use chemicalcommunication in their courtship and dominance displays [1-4]. In many fish species, for© 2010 Elsevier B.V. All rights reserved.Correspondence to: Karen P. Maruska.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptBehav Brain Res. Author manuscript; available in PMC 2011 December 1.Published in final edited form as:Behav Brain Res. 2010 December 1; 213(2): 208–217. doi:10.1016/j.bbr.2010.04.058.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptexample, males determine female reproductive state through compounds released by gravid orpre-ovulatory individuals [5-7], male urine can be used as a form of sexual signaling to denotesocial rank [3,8-10], and chemical cues are used in mother-young social bonds [11,12]. Howmight the internal physiological state of an individual influence its perception of these chemicalcues? Steroid hormones and neuropeptides are known to fine-tune sensory responsiveness tobehaviorally relevant cues [4,13-15], providing a potential link between internal state andexternal signals, but the mechanisms and site of action for such modulators in the olfactorysystem are not well understood.Previous studies in fishes and other vertebrates show that steroids and the modulatoryneuropeptide gonadotropin-releasing hormone (GnRH) can influence olfactory processing,and that these effects often vary between sexes or across reproductive seasons [13,14,16-21].For example, more olfactory receptor neurons responded to GnRH application during thebreeding season compared to the non-breeding season in the mudpuppy Necturus maculosus[17,22], olfactory responsiveness differed between sex hormone-treated male and femalegoldfish Carassius auratus [18], and steroid-related response differences were reported inseveral mammal species [19]. For any modulator to influence the olfactory system, however,cognate receptors must be present in the tissues implicated, so quantification of receptor levelscan be used as a proxy for physiological action. One potential mechanism to explain changesin olfactory responsiveness is variations in steroid and GnRH receptor abundance withinprocessing centers such as the olfactory bulb. However, little is known about how receptorlevels change with reproductive state or social status in the olfactory system of any vertebrate.Here, we tested for changes in receptor mRNA levels of key modulators in the first-orderolfactory processing center of the brain, the olfactory bulb, using the highly social Africancichlid fish Astatotilapia burtoni to test the hypothesis that receptor levels vary withreproductive and social state.Cichlid fishes use multiple sensory cues (e.g., visual, olfactory, auditory, mechanosensory) tocoordinate their complex social behaviors, and show great diversity in reproductive andparental care strategies, which makes them excellent models for discovering how hormonesinfluence sensory function. The African cichlid A. burtoni is endemic to shallow shore poolsof Lake Tanganyika and lives in a lek-like social system where males exist in one of twophenotypes: 1) dominant territorial males (~10-30% of population) that are brightly colored,aggressively defend a spawning territory, and actively court and spawn with females; and 2)subordinate non-territorial males that are drab-colored, frequently chased by dominant males,and do not court females [23]. Males can rapidly and reversibly switch between dominant andsubordinate phenotypes depending on the composition of the social environment. Importantly,this social transformation in males causes a suite of behavioral and physiological changes alongthe reproductive axis [24]. Females do not have a similarly organized social system, buttypically school with subordinate males and enter the territories of dominant males only to eatand spawn. After spawning, females rear the developing young in their mouths (mouthbrooding) for ~2 weeks before releasing them, and then physiologically recover (~25-30 days)before spawning again. While visual cues are important for social behaviors in this species[23,25,26], olfactory cues may also provide crucial information on sex, reproductive condition,and dominance status. Previous studies in A. burtoni show that olfactory signals are used forperception of social information [25,27], and that the olfactory system is responsive to putativepheromone compounds [28-30]. In the closely related tilapia Oreochromis mossambicus,chemical communication is also used by both males and females for reproductive anddominance behaviors [6,31]. These studies provide support for the importance of olfactory-mediated behaviors in A. burtoni, but whether the olfactory system might be influenced by theanimals’ internal physiological state remains unknown.Maruska and Fernald Page 2Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptWe do know from previous studies that GnRH receptors and sex steroid receptors are foundwithin the olfactory bulb of several teleosts including A. burtoni [Munchrath and Hofmann,pers. comm. [32-36], and thus the olfactory bulb could be an important substrate forneuropeptide and steroid-mediated olfaction. Here we asked whether social status and/orreproductive state influenced mRNA levels of GnRH receptors, sex steroid receptors, andaromatase in the central olfactory system of male and female A. burtoni, and whether thesechanges were correlated with circulating steroid levels. Our results show that mRNA levels ofmultiple sex steroid receptor subtypes, GnRH receptor subtypes, and aromatase in the olfactorybulb of a cichlid fish vary with sex, social status in males, and reproductive condition in females.These results provide support for the hypothesis that the first-order olfactory relay station is asubstrate for both GnRH and sex steroid action, and that changing receptor expression mayprovide a mechanism to influence context-dependent olfactory responsiveness.2. Methods2.1. AnimalsLaboratory-bred adult male and female Astatotilapia burtoni, derived from wild-caught stockin Lake Tanganyika, Africa, were maintained in aquaria under environmental conditions thatmimic their natural equatorial habitat (28°C; pH 8.0; 12h light: 12h dark with full spectrumillumination; constant aeration), and fed cichlid pellets and flakes (AquaDine, Healdsburg, CA,USA) each morning. Aquaria contained gravel-covered bottoms with terra cotta pots cut inhalf to serve as spawning territories. All experimental procedures were approved by theStanford Administrative Panel for Laboratory Animal Care.Stable dominant and subordinate males were established by initially placing two previouslydominant territorial males from separate community tanks together in an aquarium with a singleterra cotta pot territory and 4 females. In this situation, social interactions between the twomales result in one male quickly becoming dominant over the other. The dominant male thendefends the territory and uses it to court and spawn with females, while the subordinate malebecomes drab-colored, is frequently chased by the newly dominant individual, and becomesreproductively suppressed. Fish were observed daily to verify that the dominant andsubordinate males were stable phenotypes and maintained their social status for 4-5 weeks, atime sufficient to ensure reproductive suppression in the subordinate animals [37].Female A. burtoni breed year round and provide sole parental care to developing young.Females have three distinct reproductive phases, which were selected for analysis: 1) Mouthbrooding females had mouths filled with large full-term embryos that they had been broodingfor 14 days. Mouth brooding females generally do not eat while carrying developing young;2) Gravid females had visibly swollen abdomens, numerous large “ready-to-spawn” eggs(some eggs were readily released from the ovary upon dissection in all individuals indicatingthey were at or near ovulation), and a correspondingly high gonadosomatic index (GSI), and;3) Recovering females (neither gravid nor mouth brooding) were created by releasing full-termfry from mouth brooders to initiate ovarian recrudescence, and then returning them to theircommunity tanks to recover for 12 days (~equivalent to half of the average ovarian cycle periodof 25-30 days) so that they were all approximately in the same state of ovarian growth at thetime of sampling.Fish used in this experiment were those used to compare gene expression in the inner ear aspart of a separate study [38]. Gonadosomatic index and plasma steroid concentration data wereused here only to test for correlations between receptor mRNA levels in the olfactory bulb andGSI and circulating steroid levels.Maruska and Fernald Page 3Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2.2. Tissue preparationAll fish used in this study were sacrificed at the same time of day (9:30-10:30 am) to controlfor any potential diurnal changes in gene expression and were size-matched to account for anydifferences due to body size (SL = 59.4 ± 4.5 mm SD). Fish were captured from their tanks,anesthetized in ice-cold tank water, and standard length (SL) and total body mass (BM)measured. Immediately before sacrifice by rapid cervical transection, blood samples (50-100μl) were collected from the caudal vein by caudal severance with heparinized 100 μl capillarytubes within 2 min of capture. Blood was centrifuged for 10 min at 8000 rpm, and the plasmawas removed and stored at -80°C until assayed.The paired olfactory bulbs are the first relay station in the olfactory system that link odorantdetection to the brain. In fishes, they receive input from sensory neurons within the olfactoryrosette positioned on the snout, and then convey information to higher brain centers. We choseto examine the olfactory bulb because it processes olfactory information prior to integrationwith other senses or motor systems, and previous studies suggested that many steroidmodulatory effects occur here or in higher brain centers rather than at the peripheral olfactoryepithelium [18,28,39-41]. We also knew from previous studies in A. burtoni that GnRHreceptors and steroid receptors were located primarily within the internal cellular layer of theolfactory bulb. Both olfactory bulbs, which included a portion of the olfactory nerves on thedistal end but none of the rostral telencephalon on the proximal end, were transected from thebrain at the olfactory tracts (connection between the olfactory bulbs and rostral telencephalon)(Fig. 1), flash frozen, and stored at -80°C until analysis. Testes and ovaries were also removedand weighed to calculate the gonadosomatic index [GSI = (gonad mass/body mass) × 100].Olfactory bulb tissue was homogenized and RNA extracted following standard protocols(RNeasy Micro kit, Qiagen) as previously reported [38]. RNA was treated with DNase (RNase-free DNase set, Qiagen) during the isolation procedure according to kit instructions to removecontaminating genomic DNA. RNA concentration and purity was estimated fromspectrophotometric absorbance (260 nm and 280 nm) for all samples. Approximately 0.15 μgof total RNA was reverse transcribed to cDNA (iScript cDNA synthesis kit, Bio-Rad) andsamples were diluted 1:10 prior to use as a template for quantitative RT-PCR reactions.2.3. Quantitative Reverse Transcription-PCR (qRT-PCR)Astatotilapia burtoni express two androgen receptor (ARα, ARβ), three estrogen receptor(ERα, ERβa, ERβb [32,42], and two GnRH receptor (GnRH-R1, GnRH-R2) genes [33,43,44]. Quantitative RT-PCR was used to measure mRNA expression of these seven differentreceptor subtypes, plus the aromatase enzyme CYP19a (converts T into E2) from the olfactorybulb of both males and females using previously described protocols [38]. Gene-specificprimers, including those for two reference genes, 18s rRNA and glyceraldehyde 3-phosphodehydrogenase (G3PDH), were commercially synthesized (Invitrogen) and identicalto those used in previous studies [38,42,45,46]. Each primer pair produced a single melt curvepeak in the presence of cDNA template, and showed no amplification when water was used asa template, or when reverse transcriptase was omitted from the cDNA synthesis reaction(negative controls). Quantitative RT-PCR was performed on an iCycler (MyiQ, Bio-Rad,Hercules, CA) and the reaction progress in 30 μl volumes was monitored by fluorescencedetection at 490 nm during each annealing step. Reaction parameters were 3 min at 95°Cfollowed by 45 cycles of 95°C, 60°C, and 72°C for 30 s each, and followed by a melting curveanalysis over the temperature range of 95°C to 50°C (decrease by 0.5°C increments each cycle).Amplification occurred prior to cycle 32 for all genes and all samples (mean CT values for all8 target genes ranged from 23.11 – 31.65). All reactions were performed in duplicate andseveral reaction products per gene were verified by DNA sequencing (Sequetech, MountainView, CA).Maruska and Fernald Page 4Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFluorescence thresholds for each sample were automatically measured (MyiQ software, Bio-Rad) and then Real-Time PCR Miner [46] was used to calculate reaction efficiencies and cyclethresholds from the background subtracted fluorescence readings of individual wells duringthe reaction. This curve-fitting real-time PCR algorithm objectively calculates reactionefficiency and the fractional cycle number at threshold (CT) of the amplification curve formore accurate computation of mRNA levels. By using the kinetics of individual reactions,estimates of efficiency and CT are independent of the specific equipment used to perform PCRreactions and data can be more reliably compared across plates. The relative amount of targetgene mRNA was then normalized to the geometric mean of two housekeeping genes (18s andG3PDH) that were also measured in each sample with the following equation: relative targetmRNA levels = [1/(1+Etarget)^CTtarget] / [1/(1+Egeomean)^CTgeomean] × 100, where E is thereaction efficiency and CT is the average cycle threshold of the duplicates [42,45,47].Normalization to multiple reference genes, rather than a single gene, provides a more accuratequantification of mRNA levels [48,49]. Mean CT values for 18s and G3PDH did not differbetween male phenotypes (18s, p = 0.091; G3PDH, p = 0.192) or among female groups (18s,p = 0.082; G3PDH, p = 0.061), nor did they differ between the sexes (18s, p = 0.958; G3PDH,p = 0.689), demonstrating they are appropriate reference genes for this study.2.4. Steroid assaysPlasma testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) were measuredwith commercially available reagents (Enzyme ImmunoAssay kits; Cayman Chemical, Inc.).Full details on the extraction protocol, steroid assays, and validation of EIA kits can be foundin [38]. Briefly, for each assay, a 5 μl sample of plasma from each subject was extracted threetimes using 200 μl of ethyl ether and evaporated under a fume hood prior to re-constitution inassay buffer (1:40 dilution; extraction efficiencies 87-89%). EIA kit protocols were then strictlyfollowed, plates were read at 405 nm using a microplate reader (UVmax Microplate Reader,Molecular Devices), and steroid concentrations determined based on standard curves. Allsamples were assayed in duplicate, and intra-assay coefficients of variation were: T (6.2%,10.3%); 11-KT (4.9%, 3.1%); E2 (6.0%, 9.1%), while inter-assay coefficients of variation were:T (8.4%); 11-KT (4.2%); E2 (6.2%).2.5. StatisticsData sets that were normally distributed with equal variances were analyzed with Student's t-tests or one-way analysis of variance (ANOVA) with post-hoc Tukey's tests for multiplecomparisons. Data that did not meet the assumptions of parametric statistics were comparedwith Mann-Whitney rank sum tests or Kruskal-Wallis tests (KW) with post-hoc Dunn's tests.For consistency, however, all data are plotted as mean ± standard errors (SE) with appropriatestatistical test values reported in the text. To test for sex differences in mRNA levels for eachreceptor subtype, data were also combined within each sex and then compared with Mann-Whitney tests. Correlations were assessed with Pearson product moment or Spearman ranktests. Our results are presented and interpreted without the use of conservative corrections formultiple comparisons such as the Bonferroni correction. While the use of Bonferroni andrelated procedures may reduce Type I errors, they also reduce statistical power and increasethe chance of Type II error, especially in cases of smaller sample sizes (i.e., n < 30) [50]. Wetherefore chose to report observed effect size (e.g., r values) along with exact p-values to allowreader evaluation of biological importance, rather than utilize the overly conservativeBonferroni correction. However, Bonferroni corrected p-values are also indicated in thecorrelation tables for reference (Bonferroni corrected p = 0.05/n, where n is the number ofhypotheses tested on a set of data). Statistical comparisons were performed with Sigma Plot11.0 (Systat Software, Inc., San Jose, CA.).Maruska and Fernald Page 5Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript3. Results3.1. GSI and circulating steroid levelsGSI and circulating steroid levels for these animals were previously reported as part of aseparate study [38] and those values are summarized here in Table 1. GSI differed among allthree reproductive phases in females (KW, H = 28.10, p < 0.001, Dunn's test, p < 0.05), andwas two-fold greater in dominant males compared to subordinate males (Student's t-test, t =-6.50, p < 0.001). Serum E2, T, and 11-KT levels differed among all three female reproductivestages (ANOVA or KW, all p < 0.001). In males, dominant individuals had higher serum E2,T, and 11-KT levels compared to subordinate individuals (Mann-Whitney tests, all p < 0.001).3.2. GnRH receptor mRNA expression in the olfactory bulbLevels of GnRH-R1 mRNA were approximately 3-fold higher in subordinate as compared todominant males (Student's t-test, t = 3.724, p = 0.001), but there was no difference in GnRH-R2 mRNA levels between social states (Student's t-test, t = -0.845, p = 0.408) (Fig. 2A). Levelsof GnRH-R1 were also positively correlated with GnRH-R2 in subordinate males (r = 0.85,p = 0.001), but not dominant males (r = 0.56, p = 0.077). In females, GnRH-R1 mRNA levelswere higher in both brooding and gravid individuals compared to recovering individuals(ANOVA, F = 4.472, p = 0.015) (Fig. 2B). Similar to males, there was no difference in GnRH-R2 mRNA levels across female reproductive states (KW, H = 2.043, p = 0.360). Levels ofGnRH-R1 were positively correlated with GnRH-R2 levels in brooding females (r = 0.88, p <0.001), but not gravid (r = -0.32, p = 0.334) or recovering (r = 0.14, p = 0.657) females.Olfactory bulb mRNA levels of GnRH-R1 were also higher than GnRH-R2 in both sexes(Mann-Whitney rank sum tests, males: U = 70.0, p < 0.001; females: U = 130.0, p < 0.001).3.3. Sex steroid receptor mRNA expression in the olfactory bulbAndrogen receptors (Student's t-tests, ARα: t = -3.395, p = 0.003; ARβ: t = 2.271, p = 0.036),estrogen receptors (ERα: t = -2.260, p = 0.035; ERβa: t = 2.438, p = 0.024; ERβb: t = -2.050,p = 0.054), and aromatase (t = -2.209, p = 0.039) mRNA levels were all higher in dominantcompared to subordinate males (Fig. 3). Gravid females had lower mRNA levels of androgenreceptors (KW, ARα: H = 9.10, p = 0.010; ARβ: H = 12.437, p = 0.002, Dunn's tests, p < 0.05)and estrogen receptors (ERα: H = 17.132, p < 0.001; ERβa: H = 10.864, p = 0.004; ERβb: H= 18.588, p < 0.001) compared to both brooding and recovering females (Fig. 4). AromatasemRNA expression differed among all three reproductive states where brooding individuals hadthe highest levels and gravid females the lowest (H = 13.201, p = 0.001).3.4. Sex differences in receptor mRNA expression in the olfactory bulbMales had 2-3 fold higher mRNA levels of both GnRH receptor subtypes compared to females(Table 2). In contrast, with the exception of ERβa, females had higher mRNA levels of allsteroid receptor subtypes and aromatase compared to males (Table 2).3.5. Correlations between receptor mRNA levels, GSI and circulating steroid levelsLevels of GnRH-R1 mRNA in males were negatively correlated with E2 and T, but not 11-KT, and were positively correlated only with T in females (Table 3). GnRH-R2 mRNA levelswere not correlated with any circulating steroid levels in males or females (all p > 0.10). Therewere also no correlations between GnRH receptor subtypes and circulating steroid levels whenmale and female individuals were examined separately by reproductive state (all p > 0.10).In males, all steroid receptor subtypes and aromatase were positively correlated with circulatinglevels of both T and 11-KT (Table 3). In contrast to the androgens, circulating E2 levels wereonly positively correlated with ARα in males. When dominant and subordinate males wereMaruska and Fernald Page 6Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptexamined separately, there were no correlations between mRNA levels and any plasma steroidlevels in subordinate males (all p > 0.05), while in dominant males there were positivecorrelations between some mRNA levels and both T (ARα, ARβ, aromatase) and 11-KT(ARα, ARβ, ERα, ERβa, ERβb, aromatase), but not E2 (p > 0.05).There were fewer relationships between circulating steroid levels and mRNA levels in theolfactory bulb of females, but in contrast to data from males, all correlations were negative(Table 3). There was a negative correlation between mRNA levels of ERα and aromatase andcirculating levels of E2, T, and 11-KT. There was also a negative correlation between ERβband both circulating E2 and 11-KT, but not T. When females within each reproductive statewere examined separately, there were no correlations between mRNA levels and anycirculating steroids in gravid or recovering animals (all p > 0.05), and a negative correlationonly between ERβa and T in brooding individuals (p = 0.034).Correlations between receptor mRNA levels in the olfactory bulb and GSI showed norelationships between GSI and any receptor subtype in all males together (all r < 0.40, p >0.05) (Table 4), or when dominant and subordinate animals were examined alone (all p > 0.05).In contrast, female GSI was negatively correlated with all steroid receptor subtypes, butpositively correlated with GnRH-R1 mRNA levels (Table 4), which may be due in part to the10-fold differences in female GSI among reproductive states compared to the 2-fold GSIdifference in males. When examined within each female reproductive state, there were nocorrelations between GSI and mRNA levels in the olfactory bulb (all p > 0.05).4. DiscussionOur results show that mRNA levels of multiple sex steroid and GnRH receptor subtypes in theolfactory bulb of a cichlid fish vary with sex, social status in males, and reproductive state infemales. To our knowledge, this is the first quantification of mRNA levels of GnRH receptors,sex steroid receptors, and aromatase specifically in the olfactory bulb in any non-mammalianvertebrate. While previous studies in many vertebrates showed that both GnRH and steroidscan influence olfactory processing, and that these effects often vary with sex and reproductivecondition, our results show that changes in receptor mRNA levels within the olfactory bulbmay be one potential mechanism to explain this chemosensory plasticity.4.1. GnRH receptorsGnRH-R1 mRNA levels in the olfactory bulb of A. burtoni differed with sex, social status inmales, and reproductive state in females, while GnRH-R2 mRNA levels only differed betweensexes with higher levels found in males. These subtype-specific changes in receptor expressionassociated with reproductive capacity further support the hypothesis that GnRH-R1 and GnRH-R2 serve different functions, even within the same tissue [33]. Similar to other vertebrates[14,51], varicose GnRH-immunoreactive (-ir) fibers are abundant in the olfactory bulb of A.burtoni and likely originate from GnRH3 neurons in the terminal nerve ganglion, althoughprojections from midbrain GnRH2 and possibly even preoptic area GnRH1 cells cannot beentirely ruled out. A previous study in A. burtoni used in situ hybridization to show that GnRH-R2 mRNA was widely distributed throughout the brain including the olfactory bulbs, butGnRH-R1 mRNA was concentrated in the pituitary, hypothalamus and preoptic area with lowerexpression in the telencephalon and olfactory bulbs [33]. Since both GnRH receptor subtypeshave similar affinities for all GnRH ligands found in A. burtoni (GnRH1, GnRH2, GnRH3)[43,44], variations in expression levels of receptor subtypes may reflect functionalspecialization. Since brain levels of the extra-hypothalamic GnRH3 and GnRH2 do not varybetween dominant and subordinate males, or among females in different reproductive states[37], any changes in GnRH action in the olfactory bulb may result from either the variationsMaruska and Fernald Page 7Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptin receptor levels reported here, or possibly via alterations in GnRH release from varicosities,membrane receptor abundance, or other post-transcriptional mechanisms.The olfactory bulbs in teleosts receive dense GnRH-ir projections and are thought to be primetargets for neuromodulation, but the physiological effects of GnRH at this processing levelremain unexamined in any vertebrate [14]. In goldfish, however, GnRH content in the olfactorybulb of males was increased after exposure to pre-ovulatory females, suggesting a role forGnRH in olfactory-mediated reproduction [52]. In several teleost fishes, including A.burtoni, GnRH receptors are localized to the internal granule cellular layer of the olfactorybulb [33,53,54] and in mammals GnRH-R1 is also expressed in mitral cells [55]. Our dataprovide the first quantification of multiple GnRH receptor subtypes in the olfactory bulb of avertebrate, and suggest that the hypothesized modulatory effects of GnRH on olfactory bulbresponsiveness could be regulated by changes in GnRH-R1 expression. Moreover, thedifferential regulation of the GnRH receptor subtypes supports the hypothesis that these tworeceptors, which arose from a gene duplication event, may be in the process ofsubfunctionalization [33].Previous work suggested that the function of GnRH in the olfactory bulb may be closelyassociated with physiological condition and reproductive status of the fish [56,57]. Our resultsthat show differences in GnRH-R1 mRNA levels in the olfactory bulb associated withreproductive state in both males and females provides further support for this hypothesis, asdoes a study in the bullfrog (bf) that demonstrated differential expression of multiple splicevariants of the bfGnRHR-3 transcript in the olfactory lobe between hibernation and pre-breeding seasons [58]. The exact functional significance of GnRH action in the fish olfactorybulb remains unknown, but based on a suite of morphological, physiological and behavioralstudies across vertebrates it is hypothesized to enhance detection of reproductive-related odors,enhance general odorant contrast, or modulate olfactory-mediated context-dependentbehavioral responses (i.e., feeding versus reproductive) [14].One unexpected finding in our study was the higher GnRH-R1 mRNA levels found in theolfactory bulb of subordinate compared to dominant males, especially since subordinate maleshave a significantly suppressed reproductive axis from the brain to the testis and do not holdspawning territories [24]. While it is unknown whether or not GnRH has any effect on olfactorybulb responsiveness in A. burtoni, we can hypothesize about what these changes in GnRHreceptor levels might mean to the fish based on what we do know about these distinct malephenotypes. One explanation is that higher GnRH-R1 levels in the olfactory bulb helps toenhance detection of non-reproductive odorant cues, such as food or predators. Subordinatemales do not maintain a feeding territory, but often put more energy into growth so that theirchances of acquiring and holding a territory in the near future are improved [59]. An alternativehypothesis is that elevated GnRH-R1 levels in subordinate males could improve theirperception of olfactory cues released by females and/or males during spawning, which wouldallow them to better localize spawning pairs for sneak fertilizations. However, while sneakingattempts by subordinate males have been observed in aquaria (personal observations), this doesnot appear to be a common strategy. It is also possible that GnRH release within the olfactorybulb has inhibitory effects on olfactory processing that are mediated by GnRH-R1 expressionon GABA (γ-aminobutyric acid)-containing granule cells in the internal cellular layer.Inhibitory effects of GnRH are also observed in midbrain central gray neurons in rats [60],bullfrog sympathetic neurons [61], and olfactory neurons in the mudpuppy [16,17]. Thefunctional relationships between GnRH receptor expression and olfactory perception have yetto be described in any taxa, however, and future work is needed to test what role receptors playin GnRH-mediated olfactory modulation.Maruska and Fernald Page 8Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4.2. Steroid receptors in malesDominant male A. burtoni had higher mRNA levels of both AR subtypes in the olfactory bulbcompared to subordinate males, and AR mRNA levels were positively correlated withcirculating androgen levels. These data suggest that olfactory bulbs in dominant reproductivelyactive males could be more sensitive to sex steroids, which may be an adaptation that facilitatesolfactory discrimination of conspecifics during territorial and reproductive behaviors. In fishes,gravid females often release pheromones that can influence the behavior and reproductivereadiness of males [6,7], and chemical cues are used by males to signal social rank [3,9,31].Previous work in A. burtoni also indicates that males can discriminate sex and breeding stateof conspecifics based on olfactory cues [25]. In the round goby Neogobius melanostomus, maleolfactory response thresholds to putative pheromones varied with reproductive stage of the fish[62], and in the cyprinid fish Puntius schwanenfeldi, androgen implants increased themagnitude and sensitivity of electro-olfactogram (EOG) responses to pheromones [13]. Whilethese increases in sensitivity were measured at the level of the olfactory epithelium, suggestingdirect hormone effects at the periphery, the authors also acknowledge that the response couldbe mediated by steroid action in the olfactory bulb and conveyed via efferent projections tothe epithelium. Another study showed that neither gender nor reproductive condition had aninfluence on peripheral EOG responses to female pre-ovulatory pheromones, suggesting thatthe steroid influence on olfactory-mediated behavioral responsiveness seen in male goldfishoccurs at central processing sites [63]. Androgen treatment in several cyprinid species alsoshowed that there were marked sex differences in EOG responses to prostaglandins, but not tosteroids [15], while in A. burtoni there is evidence for sex differences in response to someconjugated steroids [28]. These studies suggest that sexual dimorphisms may be compound-specific or differ among species. Another relevant study, however, showed that there areseasonal changes in the number and position of crypt cells (type of olfactory sensory neuronthought to respond to sex pheromones) in the olfactory epithelium of C. auratus [64],suggesting morphological changes in sensory neuron-olfactory bulb wiring could alsocontribute to changes in olfactory perception. In A. burtoni, the physical and social environmentis dynamic such that an individual male might only maintain a spawning territory and reproducefor a few weeks before being usurped [23,59]. It would be advantageous, therefore, fordominant males to enhance the detection of any sensory signals that would increasereproductive fitness during their territory tenure. Further support of the importance of steroid-mediated olfactory processing is provided by previous findings of ARs within other olfactoryprocessing regions in the brain of A. burtoni (e.g., central nucleus of the dorsal telencephalon,ventral nucleus of the ventral telencephalon) [32], as well as the higher mRNA levels of ARsfound in the anterior brain of dominant compared to subordinate males [42].Similar to AR expression, dominant male A. burtoni also had higher mRNA levels of all ERsand aromatase in the olfactory bulb compared to subordinate males. However, there were norelationships between ER mRNA levels and circulating E2 levels in males, but there was apositive correlation between ERs, aromatase, and circulating androgens. This suggests thatcirculating T may be converted to E2 within the olfactory bulb and then acts on ERs, which issupported by the abundant aromatase expression seen in the olfactory bulb of other teleosts[36,65-67]. However, it is important to note that we only measured CYP19a aromatase hereand future work is needed to test whether the predominant brain aromatase form (CYP19b)shows a similar pattern. Estradiol was shown to enhance olfactory perception at multipleprocessing levels including the olfactory bulbs in male mammals [19], and E2 also increasedolfactory bulb sensitivity to NaCl in goldfish, where the effects were much greater in malescompared to females [18]. E2 can also reduce levels of inhibitory GABA and GABA receptorsin the olfactory bulb of some mammals to facilitate the responses of mitral cells to conspecificsignals [19], but similar experiments have not yet been performed in fishes. Regardless of theMaruska and Fernald Page 9Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptmechanisms, the elevated levels of ERs in the olfactory bulb of dominant male A. burtonisuggest E2 may also be an important modulator of olfactory processing in males.4.3. Steroid receptors in femalesAnother surprising finding of our study was that reproductive gravid female A. burtoni hadlower mRNA levels of ARs and ERs in the olfactory bulb compared to both mouth broodingand recovering females. Females may benefit from greater olfactory sensitivity when they areready to spawn in order to facilitate detection of any chemicals released by dominant courtingmales. We therefore predicted that gravid females would have higher steroid receptor levelscompared to the other groups, suggesting greater steroid sensitivity of the olfactory bulbs.However, this hypothesis was not supported by our results. It is possible that the changes insteroid receptor mRNA levels in females may simply be a homeostatic mechanism to maintainconstant steroid sensitivity of the olfactory bulbs as the circulating hormone levels fluctuateacross the reproductive cycle. Interestingly, in the brain (remainder of brain without olfactorybulbs) of the same females used in this study, mRNA levels of ARs and ERs were higher ingravid females compared to recovering and mouth brooding individuals [38]. This indicatesthat steroid receptor levels are regulated by diverse mechanisms in different regions of thebrain, and that changes in receptors could also contribute to functional specialization.In many fishes, female perception of male odors is also important for reproductive success[1]. For example, reproductive female round gobies [68] and tilapia [9,69] are behaviorallyand physiologically sensitive to compounds released into the water from reproductive males.As noted above for males, the olfactory responsiveness of females often depends onreproductive state, a phenomenon also true for mammals which may be regulated byneuromodulators [19]. In addition to a role in reproduction, chemical cues may also beimportant in brooding females for recognition of their young. In another maternal mouthbrooding cichlid, Cichlasoma nigrofasciatum, mothers were shown to prefer water from theirown young [12,70], suggesting that larval odors and olfactory perception play a role in mother-young parental associations. Olfaction is also critical for maternal behaviors in many mammals,and steroids are thought to play a role in the fine-adjustment of mother-infant interactions[2]. The higher mRNA levels of all steroid receptors in the olfactory bulb of female A.burtoni compared to males suggests steroid modulation is also important for female olfactoryprocessing, but the context may vary across the reproductive cycle.Levels of AR and ER mRNA in the olfactory bulb of female A. burtoni were also negativelycorrelated with GSI, which suggests ligand down-regulation of steroid receptors in theolfactory bulb of females rather than the up-regulation seen in males. Few studies haveexamined reproductive-related changes in steroid receptor expression specifically in theolfactory bulbs of females, so it is unclear whether similar variations exist in other taxa. Infemale rats, age-dependent differences in AR and ER expression were found in the olfactorybulbs [71-73], but there were no changes in ERs within the olfactory bulb across differentstages of pregnancy [74]. Nevertheless, the changes in AR, ER, and aromatase mRNA levelsin the olfactory bulb associated with reproductive condition in female A. burtoni suggest thatsteroids play some important role in olfactory processing or maintenance during reproductionor parental care.4.4. Sex differences in receptor mRNA levelsThe tight correlations between plasma hormone levels and steroid receptor mRNA levels inthe olfactory bulb of males, but not females, raises the possibility that modulation of olfactorycues by steroids is more important for males than for females. It is also possible, however, thatthe correlations between circulating steroid levels and receptor mRNA levels in the olfactorybulb of males result from the greater difference in plasma steroid levels between maleMaruska and Fernald Page 10Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptphenotypes compared to that of females of different reproductive states (e.g., ~10-20 fold inmales; ~2-11 fold in females). A more finely-tuned male olfactory system, however, was alsosuggested in cyprinids due to the crucial importance of male recognition of female ovulatorystatus, a cue that might hold less relevance for females [75]. Dominant male A. burtoni mayalso need a keen olfactory sense for both successful establishment of dominance and forreproduction that might require enhanced odorant contrast detection or discrimination abilities.Comparatively, since females do not hold territories or have a strict dominance hierarchy, theirolfactory discrimination may be most important during reproductive and brooding activities.This idea is supported by studies in rats that show odor preference in males is more susceptibleto internal hormonal state than it is in females [76]. In the tilapia Oreochromis mossambicus,which has a similar social system to A. burtoni, females have high olfactory sensitivity tosubstances released in male urine [69], and males increase the frequency of urination as partof their courtship display, especially in the presence of pre-ovulatory females [6,31]. Male A.burtoni can also distinguish female reproductive state from olfactory cues [25], and there issome evidence for higher EOG responses to certain conjugated steroids in males compared tofemales [28]. Collectively, these studies show that pre-ovulatory females release compoundsthat can be used by males to discern reproductive status, and that dominant males also sendchemical signals to females via urine release. Thus it is possible that context-dependentolfactory communication is important for both sexes in A. burtoni. There are examples in theliterature of sex steroids both enhancing and attenuating olfactory sensitivity, so future studiesare needed to determine how changes in steroid receptor levels might influence olfactory acuityin this and other fishes.In summary, our results show that mRNA levels of multiple sex steroid receptors, GnRHreceptor subtypes, and aromatase in the olfactory bulb of a cichlid fish vary with sex, socialstatus, and reproductive state. Future studies are needed to directly test how changes in GnRHand steroid receptor expression might influence olfactory processing, whether changes inreceptor mRNA expression are localized to specific olfactory bulb cell types and whether theyare reflected in functional protein abundance, and what effects these modulators have on theperception of dominance and reproductive-related olfactory information. Our results indicatethat the olfactory bulb is likely a substrate for both GnRH and sex steroid-mediatedneuromodulation, and suggest that changes in modulatory receptor levels could be onemechanism to regulate the plasticity in olfactory responsiveness observed across vertebrates.AcknowledgmentsWe thank Brian Grone, Julie Desjardins, Russ Carpenter, and two anonymous reviewers for their insightful commentsthat improved the manuscript. This research was funded by National Institutes of Health (NIH) NRSA F32NS061431to KPM and NIH NS 034950 to RDF.References1. Derby CD, Sorensen PW. 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Abbreviations: Ce, cerebellum; Dl-d,dorsal division of lateral zone of the dorsal telencephalon; Dl-v1, ventral division of the lateralzone of the dorsal telencephalon – subdivision 1; Dm-1, medial part of the dorsal telencephalon– subdivision 1; Hyp, hypothalamus; M, medulla; nI, cranial nerve I or olfactory nerve; nII,cranial nerve II or optic nerve; OB, olfactory bulb; T, tectum; Tel, telencephalon. Scale bars:1 mm (A), 200 μm (B). Modified from [77] (A) and [78] (B).Maruska and Fernald Page 16Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 2.GnRH receptor mRNA levels in the olfactory bulb of male and female Astatotilapia burtoni.A) GnRH-R1 mRNA levels were three-fold higher in subordinate compared to dominant males,but GnRH-R2 mRNA levels did not differ between social states. B) Brooding and gravidfemales had higher GnRH-R1 mRNA levels compared to recovering individuals, but there wasno difference in GnRH-R2 levels among reproductive states. Data are plotted as relative mRNAlevels (mean ± SE) referenced to the geometric mean of two housekeeping genes (18s andG3PDH). Bars with different letters represent significant differences (p < 0.05) and samplesizes are indicated within each bar.Maruska and Fernald Page 17Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 3.Steroid receptor and aromatase mRNA levels in the olfactory bulb of male A. burtoni.Dominant males had higher mRNA levels of all sex steroid receptors (ARα, ARβ, ERα,ERβa, ERβb) and aromatase compared to subordinate males. Data are plotted as relative mRNAlevels (mean ± SE) referenced to the geometric mean of two housekeeping genes (18s andG3PDH). Bars with different letters represent significant differences (p < 0.05) and samplesizes are indicated within each bar.Maruska and Fernald Page 18Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 4.Steroid receptor and aromatase mRNA levels in the olfactory bulb of female A. burtoni. Levelsof all androgen receptors (ARα, ARβ) and estrogen receptors (ERα, ERβa, ERβb) were lowerin gravid females compared to both brooding and recovering individuals, while aromatasemRNA levels differed among all three reproductive states. Data are plotted as relative mRNAlevels (mean ± SE) referenced to the geometric mean of two housekeeping genes (18s andG3PDH). Bars with different letters represent significant differences (p < 0.05) and samplesizes are indicated within each bar.Maruska and Fernald Page 19Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptMaruska and Fernald Page 20Table 1Gonadosomatic index (GSI) and plasma steroid concentrations (ng ml-1) in male and female Astatotilapia burtoni.NGSIE2T11-KTMales Dominant111.03 ± 0.07115.2 ± 13.7108.9 ± 17.94.65 ± 0.8 Subordinate110.43 ± 0.065.2 ± 2.44.7 ± 1.50.47 ± 0.1Females Brooding120.86 ± 0.123.7 ± 0.91.5 ± 0.30.23 ± 0.01 Recovering122.24 ± 0.3020.7 ± 3.13.8 ± 0.60.30 ± 0.03 Gravid118.72 ± 0.7543.6 ± 9.113.4 ± 3.20.40 ± 0.03Data are expressed as mean ± SE. E2, estradiol; T, testosterone; 11-KT, 11-ketotestosterone. N = number of animals. These values are a summary of data previously reported as part of a separate study on thesame individuals [38].Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptMaruska and Fernald Page 21Table 2Sex comparisons of GnRH receptors, steroid receptors, and aromatase mRNA levels in the olfactory bulb of Astatotilapia burtoni.Receptor subtype U statistic p SummaryGnRH-R1 122.0 < 0.001 M > FGnRH-R2 18.0 < 0.001 M > FARα 17.0 < 0.001 M < FARβ 88.0 < 0.001 M < FERα 51.0 < 0.001 M < FERβa 275.0 0.073 M = FERβb 75.0 < 0.001 M < FAromatase 129.0 < 0.001 M < FFemale data are combined brooding, recovering and gravid individuals; male data are combined subordinate and dominant individuals. U statisticsand p-values are from Mann-Whitney Rank Sum tests and bold values indicate differences at p < 0.05 (Bonferroni correction p < 0.006).Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptMaruska and Fernald Page 22Table 3Correlations between GnRH receptors, steroid receptors, and aromatase mRNA levels in the olfactory bulb and circulating steroid concentrations in Astatotilapia burtoni.FemalesE2T11-KTrprprpGnRH-R10.180.2930.350.0420.320.062GnRH-R2-0.240.170-0.230.182-0.260.139ARα-0.170.341-0.190.277-0.190.264ARβ-0.320.062-0.290.087-0.330.052ERα-0.390.020-0.330.054-0.380.025ERβa-0.270.112-0.240.164-0.220.200ERβb-0.350.039-0.310.073-0.350.041Aromatase-0.420.012-0.330.054-0.380.025MalesE2T11-KTrprprpGnRH-R1-0.540.011-0.450.039-0.390.081GnRH-R20.260.2640.330.1390.300.192ARα0.490.0250.85<0.0010.77<0.001ARβ0.360.1090.68<0.0010.71<0.001ERα0.300.1830.620.0030.70<0.001ERβa0.320.1530.68<0.0010.72<0.001ERβb0.260.2500.640.0020.70<0.001Aromatase0.260.2550.75<0.0010.70<0.001E 2, estradiol; T, testosterone; 11-KT, 11-ketotestosterone. Correlation coefficients (r) and p-values are from Pearson Product moment tests. P-values in bold indicate significant relationships at p < 0.05 (withinsex Bonferroni correction p < 0.002).Behav Brain Res. Author manuscript; available in PMC 2011 December 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptMaruska and Fernald Page 23Table 4Correlations between mRNA levels in the olfactory bulb and gonadosomatic index for male and female Astatotilapia burtoni.Receptor subtype Females Malesr p r pGnRH-R1 0.38 0.026 -0.39 0.076GnRH-R2 -0.28 0.104 0.18 0.430ARα -0.37 0.030 0.39 0.072ARβ -0.45 0.006 0.34 0.124ERα -0.47 0.004 0.28 0.207ERβa -0.34 0.045 0.34 0.106ERβb -0.47 0.005 0.38 0.080Aromatase -0.43 0.010 0.27 0.219Correlation coefficients (r) and p-values are from Pearson Product moment tests. P-values in bold indicate significant relationships at p < 0.05 (withinsex Bonferroni correction p < 0.006).Behav Brain Res. Author manuscript; available in PMC 2011 December 1.

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