BACKGROUND
the peripheral and central auditory system of vertebrates is sensitive to sex- and stress-related steroid hormones, which can have profound effects on how an animal perceives acoustic information and ultimately behaves during social interactions.
while steroids such as estradiol are known to influence hearing in many vertebrate taxa  <cit> , the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown.
in mammals, females often have "better" hearing  and later-onset of age-related hearing loss compared to males, as well as changes in hearing ability associated with the ovarian cycle and pregnancy  <cit> .
further, postmenopausal women who are on estrogen-based hormone replacement therapy  have better hearing than those who are not  <cit> , while progestin-based hrt can diminish hearing ability  <cit> .
these sex and female ovarian cycle variations in auditory ability are attributed to the protective effects of estrogen and may be partially related to estrogen receptor  expression in the cochlea.
this idea is supported by studies that demonstrate erβ knockout mice that have profound hearing impairment and hair cell loss  <cit> , and changes in erα abundance in the ear that are correlated with fluctuating estrogen levels during pregnancy in rats  <cit> .
a role for estrogen in hearing also extends to non-mammalian vertebrates.
in female midshipman fish , for example, estradiol shifts the tuning of primary afferent neurons that innervate the main hearing organ  possibly to better encode the higher frequency components of the male's advertisement call during the breeding season  <cit> .
moreover, erα and aromatase, an enzyme necessary for estrogen biosynthesis, are found in the inner ear, which suggests direct action of estradiol at the periphery  <cit> .
in contrast to these well-known effects of estradiol on hearing function  <cit> , relatively little is known about how androgens might influence hearing  or whether androgen receptors  are also expressed within the inner ear of vertebrates.
physical or emotional stress and the subsequent release of glucocorticoid hormones  can also regulate auditory sensitivity and inner ear homeostasis  <cit> .
moreover, glucocorticoids are used to treat several hearing disorders in humans such as hearing loss, tinnitus, meniere's disease and autoimmune disease because of their quick  therapeutic response in the inner ear  <cit> .
glucocorticoid  and mineralocorticoid receptors  that respond to both cortisol and aldosterone are found in the inner ear of several mammalian species   <cit> , but have not yet been described in other vertebrate taxa.
the conserved function of cortisol in the stress response system suggests a possible similar hypothalamic-pituitary-adrenal -hearing link in non-mammalian vertebrates.
for steroids to influence hearing at the periphery, steroid receptors must be present in the inner ear, but few studies have measured steroid receptor expression in the vertebrate ear.
sex and/or stress-related steroid receptors have been localized to the inner ear in one fish  <cit> , one bird  <cit> , and several mammals  <cit> , but little is known about how steroid receptor expression in the ear varies between the sexes within a species or across different reproductive states within a sex.
many vertebrates, especially teleost fishes, express multiple subtypes of a particular steroid receptor , and the distribution and abundance of these different forms within a tissue are known to be important determinants of how estrogen, androgen, and corticosteroid hormones function  <cit> .
our current understanding of the prevalence of multiple er, ar, and cr isoforms within the ear of a single vertebrate species, however, is incomplete.
indeed, only recently have investigators begun to study changes in receptor expression as a potential mechanism for auditory plasticity  <cit> .
to fully understand how steroid hormones might influence vertebrate inner ear function, we measured the expression patterns of all known ers, ars, and crs in a single species, along with circulating gonadal and stress hormone levels.
cichlid fishes use multiple sensory cues  to coordinate their complex social behaviors, and show great diversity in reproductive and parental care strategies, which makes them excellent models for analyzing how hormones influence sensory function.
we examined steroid receptor expression in the inner ear of the african cichlid fish astatotilapia burtoni, a species that is endemic to shallow shore pools of lake tanganyika.
a. burtoni live in a lek-like social system where males exist in one of two phenotypes: 1) dominant territorial males  that are brightly colored, aggressively defend a spawning territory, and actively court and spawn with females; and 2) subordinate non-territorial males that school with and resemble females in coloration, perform subordinate behaviors, and do not court females  <cit> .
males can rapidly and reversibly switch between dominant and subordinate appearance and behavior depending solely on the composition of the social environment.
importantly, this social transformation in males causes a suite of behavioral and physiological changes along the reproductive axis  <cit> .
females do not have a similarly organized social system, but typically school with subordinate males and enter the territories of dominant males only to eat and spawn.
after spawning, females rear the developing young in their mouths  for  <dig> weeks before releasing them, and then physiologically recover before spawning again.
while visual cues are critically important for social behaviors in this species, hydrodynamic and acoustic signals detected by the lateral line and inner ear are also likely essential for perception of social information.
territorial male a. burtoni produce courtship sounds, which are a series of pulses emitted during quivering behaviors in the presence of a female , as well as hydrodynamic sounds associated with body movements during specific behavioral acts  <cit> .
these a. burtoni courtship sounds are very similar to those described in other cichlids  <cit>  and may provide crucial information on reproductive condition, dominance status, fish size, or location of feeding and spawning territories that complement visual cues.
since auditory cues may be important for social behaviors in this species, the inner ear could be an important substrate for steroid-mediated auditory and social plasticity.
our goal for this study was to test whether steroid receptor mrna expression in the main hearing organ of a teleost fish, the saccule, varied between sexes or across reproductive and social states within a sex, and whether these changes were correlated with circulating steroid levels.
to our knowledge, this is the first comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any vertebrate.
our data provide support for the hypothesized conserved action of steroid hormones in the vertebrate inner ear.
RESULTS
gsi and circulating steroid levels
gsi differed among all three reproductive stages in females , where gravid females had mean values about four-fold greater than recovering females and ten-fold greater than brooding females .
gsi was also two-fold greater in dominant males compared to subordinate males  .
circulating cortisol levels did not differ significantly among females in different reproductive states  nor between male phenotypes  .
there was no correlation between serum cortisol levels and gsi for either males  or females .
serum e <dig> levels differed among all three female reproductive phases where gravid individuals had concentrations two-fold higher than recovering and over 10-fold higher than brooding individuals  .
in males, dominant individuals had serum e <dig> levels over 20-times higher than subordinate individuals .
gsi was also positively correlated with circulating e <dig> levels in both females  and males .
circulating t and 11-kt levels also differed among all three female reproductive phases in the same pattern as e <dig>  .
dominant males had approximately 10-fold higher serum t  and 11-kt  levels compared to subordinate males .
gsi was also positively correlated with serum t  and 11-kt  concentrations in both females and males.
androgen receptor mrna expression in the saccule
arα and arβ mrna levels differed among all three female reproductive phases where brooding individuals had the highest levels followed by recovering and then gravid animals  .
in contrast, there was no difference in either arα or arβ mrna levels of subordinate compared to dominant males  .
estrogen receptor and aromatase mrna expression in the saccule
in females, erα mrna levels differed among all three reproductive phases with brooding individuals showing the highest levels, followed by recovering and then gravid animals  .
in males, subordinates had greater levels of erα compared to dominant animals  .
erβa mrna levels were lower in gravid females compared to both brooding and recovering individuals  .
subordinate males also had two-fold higher erβa levels compared to dominant males  .
erβb mrna levels did not differ among reproductive phases in females , nor between social states in males  .
aromatase expression in the saccule of females differed among all three reproductive phases in an identical pattern to that of erα  .
in males, there was no difference in mrna levels of aromatase between subordinate and dominant phenotypes  .
corticosteroid receptor mrna expression in the saccule
gr <dig> mrna levels differed among the three female reproductive phases with the highest levels found in brooding females, followed by recovering and gravid individuals  .
gr1a and gr1b mrna levels were higher in recovering females compared to both brooding and gravid animals  .
mr levels were similar in brooding and recovering females, but both were greater than levels found in gravid females  .
in males, subordinate animals had higher levels of all four crs  compared to dominant animals  .
steroid receptor mrna expression in the female brain
the pattern of steroid receptor mrna levels in the female brain differed from that observed in the inner ear.
brain arα mrna levels were greater in gravid females compared to both brooding and recovering females  .
gravid females also showed higher levels of erα than both brooding and recovering females  .
in contrast to the saccule, there was no difference among females in aromatase expression in the brain  .
gr <dig> and mr mrna levels were higher in brooding females compared to both gravid and recovering females  .
sex differences in saccular steroid receptor mrna levels
on average, females had two to five-fold greater mrna levels of both ars compared to males  .
females also had higher levels of aromatase and erα, but not erβa or erβb, compared to males  .
in contrast, mrna levels of all four crs in the saccule were higher in males compared to females  .
female data were pooled from brooding, recovering and gravid individuals, while male data were pooled from subordinate and dominant individuals.
u statistics and p-values are from mann-whitney rank sum tests and bold values indicate differences at p <  <dig>
correlations between receptor mrna levels and circulating steroid levels and gsi
correlations between steroid receptor mrna levels in the saccule and circulating cs, e <dig>  t, and 11-kt levels are summarized in table  <dig>  and table  <dig> .
there was a positive correlation between serum cs levels and gr1a expression in the saccule of males, but no other relationships between any gr type and cs levels were found in either sex.
cs, cortisol; e <dig>  estradiol; t, testosterone; 11-kt, 11-ketotestosterone.
correlation coefficients  and p-values are from pearson product moment or spearman rank tests.
p-values in bold indicate significant relationships at p <  <dig>  .
cs, cortisol; e <dig>  estradiol; t, testosterone; 11-kt, 11-ketotestosterone.
correlation coefficients  and p-values are from pearson product moment or spearman rank tests.
p-values in bold indicate significant relationships at p <  <dig>  .
there was a negative correlation between serum e <dig> levels and both erα and erβa in the saccule of both males and females, but no relationship between e <dig> and erβb in either sex .
further, circulating e <dig> levels were also negatively correlated with aromatase expression in the saccule of females but not males.
there was a negative correlation between circulating androgen  levels and arα, arβ, erα, erβa and aromatase in the saccule of females .
in males, there was a negative correlation between circulating t levels and saccular expression of erβa and arα, but not arβ .
circulating t levels were also positively correlated with serum e <dig> levels in both females  and males .
in contrast to the saccule, receptor mrna levels in the female whole brain samples were not well correlated with circulating steroid levels, with the exception of a positive correlation between arα and t .
serum cs was not correlated with brain mrna levels of gr <dig>  or mr .
brain erα and aromatase mrna levels were also not correlated with either e <dig>  or t .
correlations between steroid receptor mrna levels in the saccule and gsi are summarized in table  <dig>  in females, gsi was negatively correlated with mrna levels of all sex-steroid receptor subtypes except erβb, as well as negatively correlated with the corticosteroid receptors gr <dig> and mr. in males, there was only a single negative correlation between gsi and gr1a.
correlation coefficients  and p-values are from pearson product moment or spearman rank tests.
p-values in bold indicate significant relationships at p <  <dig>  .
discussion
our data on mrna expression patterns of steroid receptors in the saccule of a. burtoni demonstrate that 1) multiple steroid receptor subtypes are expressed in the inner ear of a single fish species; and 2) steroid receptor mrna levels vary with sex, social status in males, and reproductive condition in females.
while previous studies in diverse taxa show the presence of some steroid receptors in both sensory and non-sensory tissues of the inner ear, their expression patterns are rarely quantified.
our study confirms expression of multiple ers, ars, crs, and aromatase in the inner ear of a single vertebrate species, and reveals a previously undescribed plasticity in receptor transcript abundance related to both internal reproductive state and the external social environment.
these data provide support for the hypothesis that, in addition to circulating steroid levels, auditory function and inner ear homeostasis could also be regulated by variations in receptor levels in the ear.
future studies are needed, however, to determine whether or not these changes reflect functional significance.
to our knowledge, this is the first study to demonstrate the presence of erβ, and multiple ars, grs and mr mrna in the inner ear of any non-mammalian vertebrate .
our data indicate that steroid receptor expression, and therefore steroid sensitivity, in peripheral auditory structures may be a conserved feature among vertebrates.
however, a whole-mount in situ hybridization study in zebrafish danio rerio did not detect er mrna within the inner ear, but did show abundant er expression within the morphologically and functionally similar lateral line neuromasts  <cit> , which also raises the possibility of species-specific differences even within broad taxonomic groups.
unique distribution profiles of multiple steroid receptor isoforms in the same species is common among vertebrates and suggests sub-functionalization  <cit> .
in teleost fishes, little is known about ligand-induced activation profiles of receptor subtypes in a single species, but there is evidence for both differential activation of ars, crs, and ers by the same ligand, as well as activation of a single receptor subtype by multiple ligands  <cit> .
this pattern of multiple interactions highlights the potential complexity of steroid-mediated transduction pathways, and given the profound organizational and activational effects of steroids in vertebrates, implicates the auditory system as an important target for all classes of steroid hormones.
corticosteroid receptor mrna expression
the corticosteroid signaling system in teleost fishes is complex, and in a. burtoni, all four types of crs are activated by both cortisol and aldosterone   <cit> .
expression levels of each cr subtype also differ among body tissues, which is indicative of functional specialization  <cit> .
here we show further differential expression of each cr subtype within the inner ear that is related to reproductive state in females and social status in males.
however, there was little evidence that changes in cr expression in the ear are regulated by circulating cortisol levels, with the exception of the positive correlation between gr1a and serum cs levels in males.
it is possible that cr expression is regulated by local glucocorticoid levels within the fluid of the inner ear that differs from circulating cortisol measures, or that crs are regulated independently of their ligands.
alternatively, crs may be regulated by compounds in the corticosteroid biosynthetic pathway other than cs such as 11-deoxycorticosterone or some yet unidentified aldosterone-like hormone.
glucocorticoids can both up-regulate and down-regulate synthesis of specific proteins within cochlear tissue of the rat  <cit> , which highlights the importance of examining cr subtype distribution, abundance, and transduction pathways.
in mammals, serum mineralocorticoid and glucocorticoid levels are correlated with na+, k+-atpase activity  in the cochlea  <cit> , but the hypothesis that na+, k+-atpase levels are regulated by gr or mr expression could not be verified  <cit> .
nevertheless, corticosteroids and cr expression are likely important for maintenance of ion balance and metabolic state of the inner ear across vertebrates, but the mechanisms remain to be elucidated.
estrogen receptor and aromatase mrna expression
saccular levels of erα, erβa and aromatase were lower in female a. burtoni undergoing ovarian recrudescence in preparation for spawning compared to mouth brooding females that put little investment into egg growth until the parental care period is over.
however, it is important to note that changes in the cyp19b aromatase form may be different from the cyp19a form measured here, and requires future investigation.
serum e <dig> levels in females were also negatively correlated with erα, erβa, and aromatase mrna levels in the saccule, indicating down-regulation of e2-sensitive pathways in the inner ear during ovarian recrudescence associated with high e <dig> production.
high circulating estrogen levels during pregnancy are also associated with a down-regulation of ers in the inner ear of mother rats  <cit> .
unfortunately, the relationship between steroid receptor expression in the ear, circulating hormone levels, and hearing ability within a single species are largely unknown so it is difficult to comment on whether high estrogens and low er expression translate into changes in hearing.
for example, in the midshipman fish, gravid females alter their auditory frequency tuning so they can better hear the male's advertisement call during the breeding season, a phenomenon mimicked by exogenous treatment with e <dig> or t  <cit> .
while these females have high circulating e <dig> levels just prior to the spawning period   <cit> , and they do express erα and cyp19b aromatase in the saccule  <cit> , it is not known whether this seasonal plasticity is regulated by changes in er expression in the ear.
in mice however, aromatase knockouts  show decreased erβ staining in the cochlea, and greater threshold shifts in response to acoustic trauma compared to wild-type individuals, but no difference in normal hearing thresholds  <cit> .
this indicates a very specific protective effect of erβ in response to trauma, which may also be species-specific.
in a. burtoni, females can reproduce and brood young year-round and changes in inner ear steroid receptor expression are coupled to gonadal state and serum hormone levels, which suggests some potential yet unknown consequence on saccular maintenance or function.
in contrast to erα and erβa, erβb mrna levels did not differ among females of different reproductive states or between males of different social status.
erβb mrna levels in the ear were also not correlated with circulating e <dig> levels in either sex, which suggests this subtype probably does not serve as a substrate for feedback regulation from estrogens.
in zebrafish, direct e <dig> exposure resulted in strong stimulation of erα expression, but reduced erβ <dig>  and had little effect on erβ <dig>  mrna in the liver  <cit> , indicating that er subtypes are differentially regulated by the same ligand and likely serve distinct physiological functions.
in mammals, the erβ subtype is important for maintaining hearing capabilities by preventing age-related hearing loss, and providing protection from acoustic trauma via a mechanism that involves brain-derived neurotrophic factor   <cit>  and possibly activation of intermediate filaments  <cit> .
the absence of status- and reproductive-related variations in erβb in a. burtoni suggests that this subtype may be important for regular maintenance of auditory function or homeostasis in non-mammalian vertebrates as well.
androgen receptor mrna expression
androgen receptors in the saccule of female a. burtoni differed among reproductive states, and females had two to three-fold greater ar mrna levels than males.
ar mrna expression in the inner ear has only been demonstrated in one other vertebrate, the midshipman fish  <cit> , so it is difficult to evaluate whether these reproductive- and sex-related variations seen in a. burtoni are present in other taxa.
ar expression in the ear, however, was negatively correlated with circulating androgens in females suggesting down-regulation of ars by high androgen levels produced during oocyte growth and maturation.
this ligand-receptor regulatory mechanism may also explain the elevated ar levels in the ear of females that have lower androgen levels compared to males.
in contrast to the well-known effects of estrogens on auditory function, relatively little is known about how androgens may influence hearing or inner ear homeostasis.
prenatal exposure to high levels of androgens are hypothesized to masculinize the cochlea and contribute to sex-differences in click-induced otoacoustic emissions in mammals  <cit> , but since ars have not yet been found in the cochlea, it is not known whether these organizational effects are mediated directly by ars or possibly via ers after aromatization.
in the midshipman fish, testosterone causes a shift in frequency tuning of saccular afferents that is similar to estradiol treatment  <cit> , and coupled with the recent finding of ar in the ear of that species  <cit>  suggests direct androgen influence at the periphery.
our discovery of multiple ars in the adult vertebrate inner ear and their plastic expression further supports the hypothesis that androgens can also have direct activational effects on the ear, but the mechanisms and impact on auditory function require further study.
comparison of steroid receptor mrna expression between the saccule and brain
the patterns of steroid receptor gene expression in the inner ear of a. burtoni differed from that observed in whole brain samples from the same female individuals.
for example, gravid females with high plasma androgen levels had the lowest levels of ar in the saccule, but had the highest ar levels in the brain.
this pattern was also true for males where sex-steroid receptor levels in the ear were higher in subordinate compared to dominant animals, but levels in sub-regions of the brain were higher in dominant compared to subordinate males  <cit> .
further, there were fewer correlations between circulating steroid levels and receptor mrna levels in the brain than there were in the inner ear.
these data raise the possibility that steroid receptors in the brain and inner ear are regulated by different mechanisms.
for example, receptor levels in the ear may be more influenced by peripheral steroid levels, while the brain may depend less on circulating hormones and more on local steroid production within specific brain regions to regulate receptors   <cit> .
it is also important to note that steroid receptors are found in many different brain nuclei and measures of receptor expression in whole brains, or large heterogeneous brain regions, may be very different from localized changes in specific processing centers.
functional consequences
in the cichlid fish a. burtoni, social status regulates reproductive opportunity and fertility.
subordinate males have a suppressed brain-pituitary-gonad axis with small gnrh <dig> neurons, low levels of gonadotropin hormones, reduced testicular size, and low circulating levels of sex steroids compared to dominant reproductively active males  <cit> .
here we provide evidence that the peripheral auditory system is also a substrate for social regulation of gene expression.
levels of erα, erβa, and all four crs were higher in the inner ear of subordinate compared to dominant males.
however, the negative correlations between circulating sex-steroid levels and receptor expression in the ear indicates that it may not be social status per se that regulates this plasticity, but rather, transformation of the reproductive axis causes variations in serum hormone concentrations that in turn regulate receptor levels.
while this mechanism may explain the pattern of higher er levels in the saccule of subordinate males, it does not rationalize the fact that levels of both ars are equivalent among male phenotypes despite subordinate animals having much lower serum androgen concentrations.
thus it is possible that social cues do have direct effects on ar expression in the inner ear of males that is independent of ligand-mediated control from the general circulation.
these data also suggest that the regulatory mechanisms of receptor expression in the ear may differ between the sexes such that receptor expression in females is influenced by internal physiological state, while in males, external social cues coupled with yet unknown pathways may play a larger role.
subordinate a. burtoni males also have higher cr expression in the saccule, a difference which for subtypes other than gr1a is not explained by either gsi or circulating cs levels.
social suppression alone may modulate inner ear cr expression directly, or act via mechanisms independent of the brain-pituitary-gonad or hypothalamic-pituitary-interrenal axis.
in previous studies, serum cs levels often do not differ between subordinate and dominant a. burtoni males when examined at ≤  <dig> weeks in an established community, but subordinate males do have much higher cs levels after longer time periods   <cit> .
thus it is possible that circulating cs levels would better correlate with cr expression at later time points.
examination of steroid receptor levels and serum steroid levels of males transitioning between subordinate and dominant states, or manipulation of plasma steroid levels independent of social status, will help resolve whether social status or reproductive state-related hormone concentrations regulate receptor expression in the ear.
estradiol appears to improve hearing ability in all vertebrate models studied to date  <cit> , particularly in females.
in a. burtoni, gravid females have high circulating e <dig> levels, and were previously shown to prefer dominant over subordinate males, as well as smaller more active males over less active ones  <cit> .
in cichlids, the peak frequency of courtship sounds is related to fish body size  <cit> , thus providing females with an honest signal for mate choice decisions.
for example, more active small males would produce more frequent courtship sounds of higher frequency, providing gravid females with auditory information to supplement visual and other sensory cues that can be used to choose a mate.
interestingly, in another teleost fish, the midshipman, estradiol shifts auditory tuning in females towards higher frequencies as an adaptation for coupling of sender-receiver physiology  <cit> .
our finding of reproductive-related plasticity in steroid receptor expression in the ear of females raises the possibility that hormones may profoundly influence both female mate choice and the evolution of male signaling behaviors for acoustic communication.
future studies are needed, however, to determine the relative importance of auditory cues during courtship, and the effects of estradiol and other steroids on hearing ability in this and other species.
CONCLUSIONS
our data show the presence of multiple sex- and stress-related steroid receptors in the fish inner ear, and that their mrna levels vary with sex, internal physiological state  and social status.
this is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any single vertebrate, and provides support for the hypothesized conserved function of steroid receptor-mediated influence within the inner ear.
the next steps are to discover which cell types within the fish inner ear express different steroid receptor subtypes, whether observed changes in mrna expression are reflected in protein abundance, and what functional consequences these variations in receptor levels might have on auditory function and inner ear homeostasis.

 the peripheral and central auditory system of vertebrates is sensitive to sex- and stress-related steroid hormones, which can have profound effects on how an animal perceives acoustic information and ultimately behaves during social interactions. while steroids such as estradiol are known to influence hearing in many vertebrate taxa, the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. in mammals, females often have "better" hearing and later-onset of age-related hearing loss compared to males, as well as changes in hearing ability associated with the ovarian cycle and pregnancy. further, postmenopausal women who are on estrogen-based hormone replacement therapy have better hearing than those who are not, while progestin-based hrt can diminish hearing ability. these sex and female ovarian cycle variations in auditory ability are attributed to the protective effects of estrogen and may be partially related to estrogen receptor expression in the cochlea. this idea is supported by studies that demonstrate erβ knockout mice that have profound hearing impairment and hair cell loss, and changes in erα abundance in the ear that are correlated with fluctuating estrogen levels during pregnancy in rats. a role for estrogen in hearing also extends to non-mammalian vertebrates. in female midshipman fish, for example, estradiol shifts the tuning of primary afferent neurons that innervate the main hearing organ possibly to better encode the higher frequency components of the male's advertisement call during the breeding season. moreover, erα and aromatase, an enzyme necessary for estrogen biosynthesis, are found in the inner ear, which suggests direct action of estradiol at the periphery. in contrast to these well-known effects of estradiol on hearing function, relatively little is known about how androgens might influence hearing or whether androgen receptors are also expressed within the inner ear of vertebrates. physical or emotional stress and the subsequent release of glucocorticoid hormones can also regulate auditory sensitivity and inner ear homeostasis. moreover, glucocorticoids are used to treat several hearing disorders in humans such as hearing loss, tinnitus, meniere's disease and autoimmune disease because of their quick therapeutic response in the inner ear. glucocorticoid and mineralocorticoid receptors that respond to both cortisol and aldosterone are found in the inner ear of several mammalian species, but have not yet been described in other vertebrate taxa. the conserved function of cortisol in the stress response system suggests a possible similar hypothalamic-pituitary-adrenal -hearing link in non-mammalian vertebrates. for steroids to influence hearing at the periphery, steroid receptors must be present in the inner ear, but few studies have measured steroid receptor expression in the vertebrate ear. sex and/or stress-related steroid receptors have been localized to the inner ear in one fish, one bird, and several mammals, but little is known about how steroid receptor expression in the ear varies between the sexes within a species or across different reproductive states within a sex. many vertebrates, especially teleost fishes, express multiple subtypes of a particular steroid receptor, and the distribution and abundance of these different forms within a tissue are known to be important determinants of how estrogen, androgen, and corticosteroid hormones function. our current understanding of the prevalence of multiple er, ar, and cr isoforms within the ear of a single vertebrate species, however, is incomplete. indeed, only recently have investigators begun to study changes in receptor expression as a potential mechanism for auditory plasticity. to fully understand how steroid hormones might influence vertebrate inner ear function, we measured the expression patterns of all known ers, ars, and crs in a single species, along with circulating gonadal and stress hormone levels. cichlid fishes use multiple sensory cues to coordinate their complex social behaviors, and show great diversity in reproductive and parental care strategies, which makes them excellent models for analyzing how hormones influence sensory function. we examined steroid receptor expression in the inner ear of the african cichlid fish astatotilapia burtoni, a species that is endemic to shallow shore pools of lake tanganyika. a. burtoni live in a lek-like social system where males exist in one of two phenotypes: 1) dominant territorial males that are brightly colored, aggressively defend a spawning territory, and actively court and spawn with females; and 2) subordinate non-territorial males that school with and resemble females in coloration, perform subordinate behaviors, and do not court females. males can rapidly and reversibly switch between dominant and subordinate appearance and behavior depending solely on the composition of the social environment. importantly, this social transformation in males causes a suite of behavioral and physiological changes along the reproductive axis. females do not have a similarly organized social system, but typically school with subordinate males and enter the territories of dominant males only to eat and spawn. after spawning, females rear the developing young in their mouths for weeks before releasing them, and then physiologically recover before spawning again. while visual cues are critically important for social behaviors in this species, hydrodynamic and acoustic signals detected by the lateral line and inner ear are also likely essential for perception of social information. territorial male a. burtoni produce courtship sounds, which are a series of pulses emitted during quivering behaviors in the presence of a female, as well as hydrodynamic sounds associated with body movements during specific behavioral acts. these a. burtoni courtship sounds are very similar to those described in other cichlids and may provide crucial information on reproductive condition, dominance status, fish size, or location of feeding and spawning territories that complement visual cues. since auditory cues may be important for social behaviors in this species, the inner ear could be an important substrate for steroid-mediated auditory and social plasticity. our goal for this study was to test whether steroid receptor mrna expression in the main hearing organ of a teleost fish, the saccule, varied between sexes or across reproductive and social states within a sex, and whether these changes were correlated with circulating steroid levels. to our knowledge, this is the first comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any vertebrate. our data provide support for the hypothesized conserved action of steroid hormones in the vertebrate inner ear.  gsi and circulating steroid levels gsi differed among all three reproductive stages in females, where gravid females had mean values about four-fold greater than recovering females and ten-fold greater than brooding females. gsi was also two-fold greater in dominant males compared to subordinate males. circulating cortisol levels did not differ significantly among females in different reproductive states nor between male phenotypes. there was no correlation between serum cortisol levels and gsi for either males or females. serum e levels differed among all three female reproductive phases where gravid individuals had concentrations two-fold higher than recovering and over 10-fold higher than brooding individuals. in males, dominant individuals had serum e levels over 20-times higher than subordinate individuals. gsi was also positively correlated with circulating e levels in both females and males. circulating t and 11-kt levels also differed among all three female reproductive phases in the same pattern as e. dominant males had approximately 10-fold higher serum t and 11-kt levels compared to subordinate males. gsi was also positively correlated with serum t and 11-kt concentrations in both females and males. androgen receptor mrna expression in the saccule arα and arβ mrna levels differed among all three female reproductive phases where brooding individuals had the highest levels followed by recovering and then gravid animals. in contrast, there was no difference in either arα or arβ mrna levels of subordinate compared to dominant males. estrogen receptor and aromatase mrna expression in the saccule in females, erα mrna levels differed among all three reproductive phases with brooding individuals showing the highest levels, followed by recovering and then gravid animals. in males, subordinates had greater levels of erα compared to dominant animals. erβa mrna levels were lower in gravid females compared to both brooding and recovering individuals. subordinate males also had two-fold higher erβa levels compared to dominant males. erβb mrna levels did not differ among reproductive phases in females, nor between social states in males. aromatase expression in the saccule of females differed among all three reproductive phases in an identical pattern to that of erα. in males, there was no difference in mrna levels of aromatase between subordinate and dominant phenotypes. corticosteroid receptor mrna expression in the saccule gr mrna levels differed among the three female reproductive phases with the highest levels found in brooding females, followed by recovering and gravid individuals. gr1a and gr1b mrna levels were higher in recovering females compared to both brooding and gravid animals. mr levels were similar in brooding and recovering females, but both were greater than levels found in gravid females. in males, subordinate animals had higher levels of all four crs compared to dominant animals. steroid receptor mrna expression in the female brain the pattern of steroid receptor mrna levels in the female brain differed from that observed in the inner ear. brain arα mrna levels were greater in gravid females compared to both brooding and recovering females. gravid females also showed higher levels of erα than both brooding and recovering females. in contrast to the saccule, there was no difference among females in aromatase expression in the brain. gr and mr mrna levels were higher in brooding females compared to both gravid and recovering females. sex differences in saccular steroid receptor mrna levels on average, females had two to five-fold greater mrna levels of both ars compared to males. females also had higher levels of aromatase and erα, but not erβa or erβb, compared to males. in contrast, mrna levels of all four crs in the saccule were higher in males compared to females. female data were pooled from brooding, recovering and gravid individuals, while male data were pooled from subordinate and dominant individuals. u statistics and p-values are from mann-whitney rank sum tests and bold values indicate differences at p < correlations between receptor mrna levels and circulating steroid levels and gsi correlations between steroid receptor mrna levels in the saccule and circulating cs, e t, and 11-kt levels are summarized in table and table. there was a positive correlation between serum cs levels and gr1a expression in the saccule of males, but no other relationships between any gr type and cs levels were found in either sex. cs, cortisol; e estradiol; t, testosterone; 11-kt, 11-ketotestosterone. correlation coefficients and p-values are from pearson product moment or spearman rank tests. p-values in bold indicate significant relationships at p <. cs, cortisol; e estradiol; t, testosterone; 11-kt, 11-ketotestosterone. correlation coefficients and p-values are from pearson product moment or spearman rank tests. p-values in bold indicate significant relationships at p <. there was a negative correlation between serum e levels and both erα and erβa in the saccule of both males and females, but no relationship between e and erβb in either sex. further, circulating e levels were also negatively correlated with aromatase expression in the saccule of females but not males. there was a negative correlation between circulating androgen levels and arα, arβ, erα, erβa and aromatase in the saccule of females. in males, there was a negative correlation between circulating t levels and saccular expression of erβa and arα, but not arβ. circulating t levels were also positively correlated with serum e levels in both females and males. in contrast to the saccule, receptor mrna levels in the female whole brain samples were not well correlated with circulating steroid levels, with the exception of a positive correlation between arα and t. serum cs was not correlated with brain mrna levels of gr or mr. brain erα and aromatase mrna levels were also not correlated with either e or t. correlations between steroid receptor mrna levels in the saccule and gsi are summarized in table in females, gsi was negatively correlated with mrna levels of all sex-steroid receptor subtypes except erβb, as well as negatively correlated with the corticosteroid receptors gr and mr. in males, there was only a single negative correlation between gsi and gr1a. correlation coefficients and p-values are from pearson product moment or spearman rank tests. p-values in bold indicate significant relationships at p <. discussion our data on mrna expression patterns of steroid receptors in the saccule of a. burtoni demonstrate that 1) multiple steroid receptor subtypes are expressed in the inner ear of a single fish species; and 2) steroid receptor mrna levels vary with sex, social status in males, and reproductive condition in females. while previous studies in diverse taxa show the presence of some steroid receptors in both sensory and non-sensory tissues of the inner ear, their expression patterns are rarely quantified. our study confirms expression of multiple ers, ars, crs, and aromatase in the inner ear of a single vertebrate species, and reveals a previously undescribed plasticity in receptor transcript abundance related to both internal reproductive state and the external social environment. these data provide support for the hypothesis that, in addition to circulating steroid levels, auditory function and inner ear homeostasis could also be regulated by variations in receptor levels in the ear. future studies are needed, however, to determine whether or not these changes reflect functional significance. to our knowledge, this is the first study to demonstrate the presence of erβ, and multiple ars, grs and mr mrna in the inner ear of any non-mammalian vertebrate. our data indicate that steroid receptor expression, and therefore steroid sensitivity, in peripheral auditory structures may be a conserved feature among vertebrates. however, a whole-mount in situ hybridization study in zebrafish danio rerio did not detect er mrna within the inner ear, but did show abundant er expression within the morphologically and functionally similar lateral line neuromasts, which also raises the possibility of species-specific differences even within broad taxonomic groups. unique distribution profiles of multiple steroid receptor isoforms in the same species is common among vertebrates and suggests sub-functionalization. in teleost fishes, little is known about ligand-induced activation profiles of receptor subtypes in a single species, but there is evidence for both differential activation of ars, crs, and ers by the same ligand, as well as activation of a single receptor subtype by multiple ligands. this pattern of multiple interactions highlights the potential complexity of steroid-mediated transduction pathways, and given the profound organizational and activational effects of steroids in vertebrates, implicates the auditory system as an important target for all classes of steroid hormones. corticosteroid receptor mrna expression the corticosteroid signaling system in teleost fishes is complex, and in a. burtoni, all four types of crs are activated by both cortisol and aldosterone. expression levels of each cr subtype also differ among body tissues, which is indicative of functional specialization. here we show further differential expression of each cr subtype within the inner ear that is related to reproductive state in females and social status in males. however, there was little evidence that changes in cr expression in the ear are regulated by circulating cortisol levels, with the exception of the positive correlation between gr1a and serum cs levels in males. it is possible that cr expression is regulated by local glucocorticoid levels within the fluid of the inner ear that differs from circulating cortisol measures, or that crs are regulated independently of their ligands. alternatively, crs may be regulated by compounds in the corticosteroid biosynthetic pathway other than cs such as 11-deoxycorticosterone or some yet unidentified aldosterone-like hormone. glucocorticoids can both up-regulate and down-regulate synthesis of specific proteins within cochlear tissue of the rat, which highlights the importance of examining cr subtype distribution, abundance, and transduction pathways. in mammals, serum mineralocorticoid and glucocorticoid levels are correlated with na+, k+-atpase activity in the cochlea, but the hypothesis that na+, k+-atpase levels are regulated by gr or mr expression could not be verified. nevertheless, corticosteroids and cr expression are likely important for maintenance of ion balance and metabolic state of the inner ear across vertebrates, but the mechanisms remain to be elucidated. estrogen receptor and aromatase mrna expression saccular levels of erα, erβa and aromatase were lower in female a. burtoni undergoing ovarian recrudescence in preparation for spawning compared to mouth brooding females that put little investment into egg growth until the parental care period is over. however, it is important to note that changes in the cyp19b aromatase form may be different from the cyp19a form measured here, and requires future investigation. serum e levels in females were also negatively correlated with erα, erβa, and aromatase mrna levels in the saccule, indicating down-regulation of e2-sensitive pathways in the inner ear during ovarian recrudescence associated with high e production. high circulating estrogen levels during pregnancy are also associated with a down-regulation of ers in the inner ear of mother rats. unfortunately, the relationship between steroid receptor expression in the ear, circulating hormone levels, and hearing ability within a single species are largely unknown so it is difficult to comment on whether high estrogens and low er expression translate into changes in hearing. for example, in the midshipman fish, gravid females alter their auditory frequency tuning so they can better hear the male's advertisement call during the breeding season, a phenomenon mimicked by exogenous treatment with e or t. while these females have high circulating e levels just prior to the spawning period, and they do express erα and cyp19b aromatase in the saccule, it is not known whether this seasonal plasticity is regulated by changes in er expression in the ear. in mice however, aromatase knockouts show decreased erβ staining in the cochlea, and greater threshold shifts in response to acoustic trauma compared to wild-type individuals, but no difference in normal hearing thresholds. this indicates a very specific protective effect of erβ in response to trauma, which may also be species-specific. in a. burtoni, females can reproduce and brood young year-round and changes in inner ear steroid receptor expression are coupled to gonadal state and serum hormone levels, which suggests some potential yet unknown consequence on saccular maintenance or function. in contrast to erα and erβa, erβb mrna levels did not differ among females of different reproductive states or between males of different social status. erβb mrna levels in the ear were also not correlated with circulating e levels in either sex, which suggests this subtype probably does not serve as a substrate for feedback regulation from estrogens. in zebrafish, direct e exposure resulted in strong stimulation of erα expression, but reduced erβ and had little effect on erβ mrna in the liver, indicating that er subtypes are differentially regulated by the same ligand and likely serve distinct physiological functions. in mammals, the erβ subtype is important for maintaining hearing capabilities by preventing age-related hearing loss, and providing protection from acoustic trauma via a mechanism that involves brain-derived neurotrophic factor and possibly activation of intermediate filaments. the absence of status- and reproductive-related variations in erβb in a. burtoni suggests that this subtype may be important for regular maintenance of auditory function or homeostasis in non-mammalian vertebrates as well. androgen receptor mrna expression androgen receptors in the saccule of female a. burtoni differed among reproductive states, and females had two to three-fold greater ar mrna levels than males. ar mrna expression in the inner ear has only been demonstrated in one other vertebrate, the midshipman fish, so it is difficult to evaluate whether these reproductive- and sex-related variations seen in a. burtoni are present in other taxa. ar expression in the ear, however, was negatively correlated with circulating androgens in females suggesting down-regulation of ars by high androgen levels produced during oocyte growth and maturation. this ligand-receptor regulatory mechanism may also explain the elevated ar levels in the ear of females that have lower androgen levels compared to males. in contrast to the well-known effects of estrogens on auditory function, relatively little is known about how androgens may influence hearing or inner ear homeostasis. prenatal exposure to high levels of androgens are hypothesized to masculinize the cochlea and contribute to sex-differences in click-induced otoacoustic emissions in mammals, but since ars have not yet been found in the cochlea, it is not known whether these organizational effects are mediated directly by ars or possibly via ers after aromatization. in the midshipman fish, testosterone causes a shift in frequency tuning of saccular afferents that is similar to estradiol treatment, and coupled with the recent finding of ar in the ear of that species suggests direct androgen influence at the periphery. our discovery of multiple ars in the adult vertebrate inner ear and their plastic expression further supports the hypothesis that androgens can also have direct activational effects on the ear, but the mechanisms and impact on auditory function require further study. comparison of steroid receptor mrna expression between the saccule and brain the patterns of steroid receptor gene expression in the inner ear of a. burtoni differed from that observed in whole brain samples from the same female individuals. for example, gravid females with high plasma androgen levels had the lowest levels of ar in the saccule, but had the highest ar levels in the brain. this pattern was also true for males where sex-steroid receptor levels in the ear were higher in subordinate compared to dominant animals, but levels in sub-regions of the brain were higher in dominant compared to subordinate males. further, there were fewer correlations between circulating steroid levels and receptor mrna levels in the brain than there were in the inner ear. these data raise the possibility that steroid receptors in the brain and inner ear are regulated by different mechanisms. for example, receptor levels in the ear may be more influenced by peripheral steroid levels, while the brain may depend less on circulating hormones and more on local steroid production within specific brain regions to regulate receptors. it is also important to note that steroid receptors are found in many different brain nuclei and measures of receptor expression in whole brains, or large heterogeneous brain regions, may be very different from localized changes in specific processing centers. functional consequences in the cichlid fish a. burtoni, social status regulates reproductive opportunity and fertility. subordinate males have a suppressed brain-pituitary-gonad axis with small gnrh neurons, low levels of gonadotropin hormones, reduced testicular size, and low circulating levels of sex steroids compared to dominant reproductively active males. here we provide evidence that the peripheral auditory system is also a substrate for social regulation of gene expression. levels of erα, erβa, and all four crs were higher in the inner ear of subordinate compared to dominant males. however, the negative correlations between circulating sex-steroid levels and receptor expression in the ear indicates that it may not be social status per se that regulates this plasticity, but rather, transformation of the reproductive axis causes variations in serum hormone concentrations that in turn regulate receptor levels. while this mechanism may explain the pattern of higher er levels in the saccule of subordinate males, it does not rationalize the fact that levels of both ars are equivalent among male phenotypes despite subordinate animals having much lower serum androgen concentrations. thus it is possible that social cues do have direct effects on ar expression in the inner ear of males that is independent of ligand-mediated control from the general circulation. these data also suggest that the regulatory mechanisms of receptor expression in the ear may differ between the sexes such that receptor expression in females is influenced by internal physiological state, while in males, external social cues coupled with yet unknown pathways may play a larger role. subordinate a. burtoni males also have higher cr expression in the saccule, a difference which for subtypes other than gr1a is not explained by either gsi or circulating cs levels. social suppression alone may modulate inner ear cr expression directly, or act via mechanisms independent of the brain-pituitary-gonad or hypothalamic-pituitary-interrenal axis. in previous studies, serum cs levels often do not differ between subordinate and dominant a. burtoni males when examined at ≤ weeks in an established community, but subordinate males do have much higher cs levels after longer time periods. thus it is possible that circulating cs levels would better correlate with cr expression at later time points. examination of steroid receptor levels and serum steroid levels of males transitioning between subordinate and dominant states, or manipulation of plasma steroid levels independent of social status, will help resolve whether social status or reproductive state-related hormone concentrations regulate receptor expression in the ear. estradiol appears to improve hearing ability in all vertebrate models studied to date, particularly in females. in a. burtoni, gravid females have high circulating e levels, and were previously shown to prefer dominant over subordinate males, as well as smaller more active males over less active ones. in cichlids, the peak frequency of courtship sounds is related to fish body size, thus providing females with an honest signal for mate choice decisions. for example, more active small males would produce more frequent courtship sounds of higher frequency, providing gravid females with auditory information to supplement visual and other sensory cues that can be used to choose a mate. interestingly, in another teleost fish, the midshipman, estradiol shifts auditory tuning in females towards higher frequencies as an adaptation for coupling of sender-receiver physiology. our finding of reproductive-related plasticity in steroid receptor expression in the ear of females raises the possibility that hormones may profoundly influence both female mate choice and the evolution of male signaling behaviors for acoustic communication. future studies are needed, however, to determine the relative importance of auditory cues during courtship, and the effects of estradiol and other steroids on hearing ability in this and other species.  our data show the presence of multiple sex- and stress-related steroid receptors in the fish inner ear, and that their mrna levels vary with sex, internal physiological state and social status. this is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any single vertebrate, and provides support for the hypothesized conserved function of steroid receptor-mediated influence within the inner ear. the next steps are to discover which cell types within the fish inner ear express different steroid receptor subtypes, whether observed changes in mrna expression are reflected in protein abundance, and what functional consequences these variations in receptor levels might have on auditory function and inner ear homeostasis. 
gonadal and stress-related steroid hormones are known to influence auditory function across vertebrates but the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. we used qrt-pcr to measure mrna expression levels of multiple steroid receptor subtypes and aromatase in the main hearing organ of the inner ear in the highly social african cichlid fish astatotilapia burtoni, and tested whether these receptor levels were correlated with circulating steroid concentrations. sex-steroid receptor mrna levels were negatively correlated with circulating estradiol and androgens in both males and females, suggesting possible ligand down-regulation of receptors in the inner ear. our data suggest that changes in steroid receptor mrna expression in the inner ear could be a regulatory mechanism for physiological state-dependent auditory plasticity across vertebrates. we show that multiple steroid receptor subtypes are expressed within the main hearing organ of a single vertebrate species, and that expression levels differ between the sexes. this is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any single vertebrate. the presence of steroid receptors in the ear suggests a direct pathway for hormones to act on the peripheral auditory system, but little is known about which receptors are expressed in the ear or whether their expression levels change with internal physiological state or external social cues. in contrast, saccular changes in corticosteroid receptor mrna levels were not related to serum cortisol levels. we also show that steroid receptor subtype-specific changes in mrna expression are associated with reproductive phase in females and social status in males. circulating steroid levels and receptor subtype mrna levels were not as tightly correlated in males as compared to females, suggesting different regulatory mechanisms between sexes.
 gonadal and stress-related steroid hormones are known to influence auditory function across vertebrates but the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. the presence of steroid receptors in the ear suggests a direct pathway for hormones to act on the peripheral auditory system, but little is known about which receptors are expressed in the ear or whether their expression levels change with internal physiological state or external social cues. we used qrt-pcr to measure mrna expression levels of multiple steroid receptor subtypes and aromatase in the main hearing organ of the inner ear in the highly social african cichlid fish astatotilapia burtoni, and tested whether these receptor levels were correlated with circulating steroid concentrations.  we show that multiple steroid receptor subtypes are expressed within the main hearing organ of a single vertebrate species, and that expression levels differ between the sexes. we also show that steroid receptor subtype-specific changes in mrna expression are associated with reproductive phase in females and social status in males. sex-steroid receptor mrna levels were negatively correlated with circulating estradiol and androgens in both males and females, suggesting possible ligand down-regulation of receptors in the inner ear. in contrast, saccular changes in corticosteroid receptor mrna levels were not related to serum cortisol levels. circulating steroid levels and receptor subtype mrna levels were not as tightly correlated in males as compared to females, suggesting different regulatory mechanisms between sexes.  this is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mrna expression in the peripheral auditory system of any single vertebrate. our data suggest that changes in steroid receptor mrna expression in the inner ear could be a regulatory mechanism for physiological state-dependent auditory plasticity across vertebrates. 