Steroid regulation of brain aromatase expression in glia: female preoptic and vocal motor nuclei

Expression of the enzyme aromatase, which converts androgens to estrogens, is known to be regulated by gonadal steroids in brain areas linked to reproduction and related behaviors in several groups of vertebrates. Previously, we demonstrated in a vocal fish, the plainfin midshipman, that both males and females undergo seasonal changes in brain aromatase mRNA expression in the preoptic area (POA) and the dimorphic sonic/vocal motor nucleus (SMN) that parallel seasonal variation in circulating steroid levels and reproductive behavior. We tested the hypothesis that steroids are directly responsible for seasonal modulation of aromatase in females because they show the most dramatic fluctuations of testosterone (T) and 17beta-estradiol (E2) throughout the year. Adult female midshipmen were ovariectomized and administered T, E2, or blank (control) implants. We then quantified aromatase mRNA expression within the POA and SMN by in situ hybridization. Both T- and E2-treated females had elevated mRNA expression levels in both brain areas compared to controls. T affected aromatase expression in a level-dependent manner, whereas E2 showed a decreased effect at higher circulating levels. This study demonstrates that seasonal differences in brain aromatase expression in female midshipman fish may be explained, in part, by changes in levels of circulating steroids.     

Phenotypic plasticity and divergence in gene expression

The extent to which phenotypic plasticity, or the ability of a single genotype to produce different phenotypes in different environments, impedes or promotes genetic divergence has been a matter of debate within evolutionary biology for many decades (see, for example, Ghalambor et al. 2007 ; Pfennig et al. 2010 ). Similarly, the role of evolution in shaping phenotypic plasticity remains poorly understood (Pigliucci 2005 ). In this issue of Molecular Ecology, Dayan et al. ( 2015 ) provide empirical data relevant to these questions by assessing the extent of plasticity and divergence in the expression levels of 2272 genes in muscle tissue from killifish (genus Fundulus) exposed to different temperatures. F. heteroclitus (Fig.  1 A) and F. grandis are minnows that inhabit estuarine marshes (Fig.  1 B) along the coasts of the Atlantic Ocean and Gulf of Mexico in North America. These habitats undergo large variations in temperature both daily and seasonally, and these fish are known to demonstrate substantial phenotypic plasticity in response to temperature change (e.g. Fangue et al. 2006 ). Furthermore, the range of F. heteroclitus spans a large latitudinal gradient of temperatures, such that northern populations experience temperatures that are on average ~10°C colder than do southern populations (Schulte 2007 ). By comparing gene expression patterns between populations of these fish from different thermal habitats held in the laboratory at three different temperatures, Dayan et al. ( 2015 ) address two important questions regarding the interacting effects of plasticity and evolution: (i) How does phenotypic plasticity affect adaptive divergence? and (ii) How does adaptive divergence affect plasticity?     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Seasonal aromatase activity in the brain of the male red-sided garter snake

We investigated regional and seasonal variations in neural aromatase activity (AA), the enzyme that converts androgens into estrogens, to examine a possible indirect role of testosterone (T) in mediating spring reproductive behavior of red-sided garter snakes, a species exhibiting a dissociated reproductive pattern. Neural AA in male snakes varied significantly among brain regions. Additionally, there were significant interactions between brain region and season. In the spring, actively courting males had greater AA in the olfactory region (O) compared to the septum/anterior-hypothalamus preoptic area (S/AHPOA), nucleus sphericus (NS) and midbrain (Mb). Fall animals collected as they returned to the den prior to winter dormancy had significantly greater AA in the S/AHPOA compared to all other regions. These findings were consistent using either regional (gross) dissection or punch microdissection, which allowed us to separate the S and AHPOA. There were no significant differences in AA production between the S and AHPOA. This study provides the first documentation of seasonal and regional variations in AA in a snake brain and suggests that aromatization of androgens may play a role in regulating reproduction in red-sided garter snakes. During spring mating, elevated AA in the O may activate pathways essential for detection of courtship pheromones, while increased AA in the S and AHPOA of fall animals suggests that circulating androgens play an indirect role in programming critical neural pathways involved in reproduction. Thus, as in many other vertebrates, estrogenic metabolites of testosterone may be a critical hormonal component regulating reproductive behavior in this dissociated breeder.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

Distribution of aromatase activity in cultured neurons and glia cells

Aromatase plays a crucial role in the mechanism of action of testosterone in the central nervous system. Nevertheless, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain. In the experiments described here the presence of aromatase (assayed by the tritiated water method) has been evaluated in the two main cellular components of the brain: neurons and glia. Neurons, mixed glial cells, type 1 astrocytes, were obtained in cultures; oligodendrocytes were prepared by gradient ultracentrigugation. The results indicate that, among the different cells tested, only neurons possess a significant degree of aromatase activity, while the enzymatic activity is extremely low in mixed glial cell and in astrocyte preparations. Oligodendrocytes seem to be completely inactive in this respect.     

Regulation of TLR2 expression by prostaglandins in brain glia

TLR have emerged as important primary sensors for diverse stimuli and are increasingly implicated in various diseases. However, the molecular mechanisms underlying the regulation of the TLR system remain poorly understood. In this study, we report that some PGs may control TLR-mediated inflammatory events through modulation of TLR2 expression in brain immune cells. We first found that 15-deoxy-Delta12,14-PG J(2) (15d-PGJ(2)) markedly altered the expression of TLR2 but not TLR4, TLR1, and TLR9 at the message and protein levels in activated glia. Down-regulation of TLR2 expression and downstream events of TLR2 activation, including phagocytosis by 15d-PGJ(2), were also observed in cells treated with representative TLR2 ligands such as lipoteichoic acid and Pam(3)CSK(4). We further revealed that certain 15d-PGJ(2)-related PGs such as 15d-PGD(2) and PGD(2) also suppressed the ligand-stimulated increase of TLR2 expression, whereas PGE(2) and arachidonic acids did not. Interestingly, TLR2 expression was down-regulated even when such PGs were added at several hours after stimulator treatment. These findings appear to be independent of peroxisome proliferator-activated receptor gamma and D prostanoid receptors (DPs) because potent synthetic peroxisome proliferator-activated receptor gamma agonists, selective DP1 agonist, or DP2 agonist did not mimic the effects of such PGs on TLR2 expression. Taken together, our results suggest that 15d-PGJ(2), 15d-PGD(2), and PGD(2) may play notable roles as modulators of the TLR2-mediated inflammatory events, and provide new insight into the resolution of inflammation in the brain.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and thesexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.     

Seasonal expression of androgen receptors, estrogen receptors, and aromatase in the canary brain in relation to circulating androgens and estrogens

Songbirds have a complex neural network for learning and production of song, namely the neural song system. Several nuclei of the song system contain androgen receptors (AR), and the neostriatal nucleus HVc also contains alpha type estrogen receptors (ER). Many songbird species show seasonal changes in both song and the neural song system that are correlated with seasonal variations in the circulating levels of gonadal steroids. However, there is increasing evidence that the sensitivity of the song system to gonadal steroids also changes seasonally. This could involve changes in the expression and activity of steroid receptors and steroid-metabolizing enzymes, such as the estrogen-synthesizing enzyme aromatase (AROM). The seasonal regulation of brain AR, ER, and AROM has not been studied before in the same individual songbirds. In this work, we compared plasma levels of androgens and estrogens, the expression level of AR-, ER-, and AROM-mRNA in the telencephalon, and brain AROM activity in male canaries between autumn (November) and spring (April) periods of high singing activity. Plasma levels of androgens and estrogens were higher in April than in November. The expression level of ER in HVc was higher in November than in April. In contrast, the expression level of AROM in the caudomedial neostriatum was higher in April than in November. However, we found no seasonal differences in the level of expression of AR and the volume of HVc as delimited by AR expression. Thus, AR expression in HVc was not correlated with circulating androgen levels. This study shows that both steroid-dependent and -independent seasonal factors regulate the action of gonadal hormones on the song system. In addition, we report a new site of AROM expression in the songbird brain, the nucleus interfacialis.     

Signaling between glia and neurons: focus on synaptic plasticity

Glial cells are now emerging from the shadows cast by their more excitable CNS counterparts. Within the developing nervous system, astrocytes and Schwann cells actively help to promote synapse formation and function, and have even been implicated in synapse elimination. In the adult brain, astrocytes respond to synaptic activity by releasing transmitters that modulate synaptic activity. Thus, glia are active participants in brain function. Many questions remain about the identity of glial-neuronal signals and their significance.     

More than a sidekick: glia and homeostatic synaptic plasticity

Homeostatic synaptic plasticity is thought to have a crucial role in stabilizing the activity of neurons and networks, but the mechanisms are poorly understood. In a recent study, Stellwagen and Malenka have shown that synaptic scaling can be induced by activity-dependent changes in release of the cytokine tumor necrosis factor-alpha (TNF-alpha) and, surprisingly, that the source of TNF-alpha is glia rather than neurons. In addition to provide insight into the mechanisms of homeostatic plasticity, these data argue for the first time for an equal partnership between glial cells and neurons in the generation of an important form of synaptic plasticity.     

Adaptive divergence in body size overrides the effects of plasticity across natural habitats in the brown trout

The evolution of life-history traits is characterized by trade-offs between different selection pressures, as well as plasticity across environmental conditions. Yet, studies on local adaptation are often performed under artificial conditions, leaving two issues unexplored: (i) how consistent are laboratory inferred local adaptations under natural conditions and (ii) how much phenotypic variation is attributed to phenotypic plasticity and to adaptive evolution, respectively, across environmental conditions? We reared fish from six locally adapted (domesticated and wild) populations of anadromous brown trout (Salmo trutta) in one semi-natural and three natural streams and recorded a key life-history trait (body size at the end of first growth season). We found that population-specific reaction norms were close to parallel across different streams and Q_ST was similar – and larger than F_ST – within all streams, indicating a consistency of local adaptation in body size across natural environments. The amount of variation explained by population origin exceeded the variation across stream environments, indicating that genetic effects derived from adaptive processes have a stronger effect on phenotypic variation than plasticity induced by environmental conditions. These results suggest that plasticity does not “swamp” the phenotypic variation, and that selection may thus be efficient in generating genetic change.     

Seasonal butterfly design: morphological plasticity among three developmental pathways relative to sex,flight and thermoregulation

The temperate‐zone butterfly Pararge aegeria can use three developmental pathways corresponding to different seasonal cohorts: (1) development with a pupal winter diapause resulting in early spring adults; (2) development with a larval winter diapause resulting in late‐spring adults and (3) direct development resulting in summer or second generation adults. In order to test adaptive predictions, we compared variation in flight‐ and thermoregulation‐related morphology among adult males and females from the three pathways using both field data (i.e. wild‐caught butterflies) and experimental breeding data (i.e. reared under different photoperiod regimes). Morphological patterns among the pathways were largely similar in the field and rearing data. Seasonal patterns differed between the sexes for most traits, including (relative) size measures and wing colour. Our results suggest sex‐related, adaptive seasonal plasticity for morphological traits related to flight behaviour in a multivoltine insect.     

Differential expression of P450 aromatase during gonadal sex differentiation and sex reversal of the newt Pleurodeles waltl

A better understanding of vertebrate sexual differentiation could be provided by a study of models in which genetic sex determination (GSD) of gonads can be reversed by temperature. In the newt Pleurodeles waltl, a P450 aromatase cDNA was isolated from adult gonads, and the nucleotide or deduced amino acid sequences showed a high level of identity with various vertebrate species. In adults, aromatase expression was found in gonads and brain. In developing gonads, the expression was found to fit with the thermo-sensitive period (TSP) and was detected in both ZZ and ZW larvae, as well as in ZW submitted during the whole TSP to a masculinizing temperature. In the latter individuals, in situ hybridization and semi quantitative RT-PCR showed that, at the end of TSP, aromatase expression was at the same level than in normal ZZ larvae and was significantly lower than in normal ZW ones. Furthermore, temperature-induced down regulation did not occur when heating was performed at the end of TSP. Our results confirm the importance of aromatase regulation in female versus male differentiation and demonstrate that a down regulation of aromatase expression is involved in the process of sex reversal.