High Levels Of Aromatase Activity Research Articles

Sex Differences in the Rapid Control of Aromatase Activity in the Quail Preoptic Area

Adult male quail show high levels of aromatase activity in the preoptic area-hypothalamus (POA-HYP), which parallels the high number of aromatase-immunoreactive cells and elevated mRNA concentrations detected in this brain region by in situ hybridisation. Interestingly, females display considerably lower aromatase activity than males but have almost equal numbers of aromatase-immunoreactive cells and express similar levels of aromatase mRNA. Aromatase activity in the male POA-HYP can be rapidly regulated by calcium-dependent phosphorylations, in the absence of changes in enzyme concentration. In the present study, we investigated whether aromatase activity is differentially regulated by phosphorylations in males and females. A linear increase in accumulation of aromatisation products was observed in both sexes as a function of time but the rate of conversion was slower in females. Saturation analysis confirmed the lower maximum velocities (V(max) ) in females but indicated a similar affinity (K(m) ) in both sexes. Aromatase activity in females reacted differentially to manipulations of intracellular calcium. In particular, chelating calcium with ethylene glycol tetraacetic acid (EGTA) resulted in a larger increase of enzymatic activity in males than in females, especially in the presence of ATP. A differential reaction to kinase inhibitors was also observed between males and females (i.e. a larger increase in aromatase activity in females than in males after exposure to specific inhibitors). These findings suggest that the nature of aromatase is conserved between the sexes, although the control of its activity by calcium appears to be different. Additional characterizations of intracellular calcium in both sexes would therefore be appropriate to better understand aromatase regulation.

Fish with thermolabile sex determination (TSD) as models to study brain sex differentiation

As fish are ectothermic animals, water temperature can affect their basic biological processes such as larval development, growth and reproduction. Similar to reptiles, the incubation temperature during early phases of development is capable to modify sex ratios in a large number of fish species. This phenomenon, known as thermolabile sex determination (TSD) was first reported in Menidia menidia, a species belonging to the family Atherinopsidae. Since then, an increasing number of fish have also been found to exhibit TSD. Traditionally, likewise in reptiles, several TSD patterns have been described in fish, however it has been recently postulated that only one, females at low temperatures and males at high temperatures, may represent the “real” or “true” TSD. Many studies regarding the influence of temperature on the final sex ratios have been focused on the expression and activity of gonadal aromatase, the enzyme involved in the conversion of androgens into estrogens and encoded by the cyp19a1a gene. In this regard, teleost fish, may be due to a whole genome duplication event, produce another aromatase enzyme, commonly named brain aromatase, encoded by the cyp19a1b gene. Contrary to what has been described in other vertebrates, fish exhibit very high levels of aromatase activity in the brain and therefore they synthesize high amounts of neuroestrogens. However, its biological significance is still not understood. In addition, the mechanism whereby temperature can induce the development of a testis or an ovary still remains elusive. In this context the present review is aimed to discuss several theories about the possible role of brain aromatase using fish as models. The relevance of brain aromatase and therefore of neuroestrogens as the possible cue for gonadal differentiation is raised. In addition, the possible role of brain aromatase as the way to keep the high levels of neurogenesis in fish is also considered. Several key examples of how teleosts and aromatase regulation can offer more insight into basic mechanisms of TSD are also reviewed.

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Estrogen production and metabolism in endometriosis.

Aromatase activity is absent in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis, which gives rise to strikingly high levels of aromatase activity in this tissue. Both aromatase expression and activity are stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. Another abnormality in endometriosis, that is, deficient 17beta-HSD type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.

Anatomical Distribution and Cellular Basis for High Levels of Aromatase Activity in the Brain of Teleost Fish: Aromatase Enzyme and mRNA Expression Identify Glia as Source

Although teleost fish have higher levels of brain aromatase activity than any other vertebrate group, its function remains speculative, and no study has identified its cellular basis. A previous study determined aromatase activity in a vocal fish, the plainfin midshipman (Porichthys notatus), and found highest levels in the telencephalon and lower levels in the sonic hindbrain, which was dimorphic between and within (males) sexes. We have now localized aromatase-containing cells in the midshipman brain both by immunocytochemistry using teleost-specific aromatase antibodies and by in situ hybridization using midshipman-specific aromatase probes. Aromatase-immuno-reactivity and mRNA hybridization signal are consistent with relative levels of aromatase activity in different brain regions: concentrated in the dimorphic sonic motor nucleus, in a band just beneath the periaqueductal gray in the midbrain, in ventricular regions in the hypothalamus, and highest levels in the telencephalon especially in preoptic and ventricular areas. Surprisingly, double-label immunofluorescence does not show aromatase-immunoreactive colocalization in neurons, but instead in radial glia throughout the brain. This is the first study to identify aromatase expression mostly, if not entirely, in glial cells under normal rather than brain injury-dependent conditions. The abundance of aromatase in teleosts may represent an adaptation linked to continual neurogenesis that is known to occur throughout an individual's lifetime among fishes. The localization of aromatase within the intersexually and intrasexually dimorphic vocal-motor circuit further implies a function in the expression of alternative male reproductive phenotypes and, more generally, the development of natural, individual variation of specific brain nuclei.

Phosphorylation processes mediate rapid changes of brain aromatase activity

The enzyme aromatase (also called estrogen synthase) that catalyzes the transformation of testosterone (T) into estradiol plays a key limiting role in the action of T on many aspects of reproduction. The distribution and regulation of aromatase in the quail brain has been studied by radioenzyme assays on microdissected brain areas, immunocytochemistry, RT-PCR and in situ hybridization. High levels of aromatase activity (AA) characterize the sexually dimorphic, steroid-sensitive medial preoptic nucleus (POM), a critical site of T action and aromatization for the activation of male sexual behavior. The boundaries of the POM are clearly outlined by a dense population of aromatase-containing cells as visualized by both immunocytochemistry and in situ hybridization histochemistry. Aromatase synthesis in the POM is controlled by T and its metabolite estradiol, but estradiol receptors alpha (ERα) are not normally co-localized with aromatase in this brain area. Estradiol receptor beta (ERβ) has been recently cloned in quail and localized in POM but we do not yet know whether ERβ occurs in aromatase cells. It is therefore not known whether estrogens regulate aromatase synthesis directly or by affecting different inputs to aromatase cells as is the case with the gonadotropin releasing hormone neurons. The presence of aromatase in presynaptic boutons suggests that locally formed estrogens may exert part of their effects by non-genomic mechanisms at the membrane level. Rapid effects of estrogens in the brain that presumably take place at the neuronal membrane level have been described in other species. If fast transduction mechanisms for estrogen are available at the membrane level, this will not necessarily result in rapid changes in brain function if the availability of the ligand does not also change rapidly. We demonstrate here that AA in hypothalamic homogenates is rapidly down-regulated by exposure to conditions that enhance protein phosphorylation (addition of Ca 2+, Mg 2+, ATP). This inhibition is blocked by kinase inhibitors which supports the notion that phosphorylation processes are involved. A rapid (within minutes) and reversible regulation of AA is also observed in hypothalamic explants incubated in vitro and exposed to high Ca 2+ levels (K +-induced depolarization, treatment by thapsigargin, by kainate, AMPA or NMDA). The local production and availability of estrogens in the brain can therefore be rapidly changed by Ca 2+ based on variation in neurotransmitter activity. Locally-produced estrogens are as a consequence available for non-genomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide/neurotransmitter than previously thought.

Distribution of Aromatase Activity in the Brain and Peripheral Tissues of Passerine and Nonpasserine Avian Species

Many behavioral effects of testosterone on hypothalamic and limbic brain areas are mediated by the action, at the cellular level, of estrogens derived from local testosterone aromatization. Aromatase activity and cells containing the aromatase protein and mRNA have accordingly been identified in the brain areas involved in the control of behavior. The presence of an unusually high level of aromatase activity has been detected in the telencephalon of one songbird species, the zebra finch (Taeniopygia guttata), and it is suspected that this high telencephalic aromatase may be a specific feature of songbirds but this idea is supported only by few experimental data. The distribution of aromatase activity in the brain of zebra finches and of one nonsongbird species, the Japanese quail (Coturnix japonica), was compared with the distribution of aromatase activity in the brain of four species of free-living European songbirds, the chaffinch (Fringilla coelebs, Fringillidae), willow warbler (Phylloscopus trochilus, Sylviidae), great tit (Parus major, Paridae), and pied flycatcher (Ficedula hypoleuca, Muscicapidae). High levels of enzyme activity were observed in the diencephalon of all species. The high levels of aromatase activity that had been observed in the zebra finch telencephalon and were thought to be typical of songbirds were also present in the four wild oscine species but not in quail. None of these songbird species had, however, a telencephalic aromatase activity as high as that in the zebra finch, which may represent an extreme as far as the activity of this enzyme in the telencephalon is concerned. Measurable levels of aromatase activity were also detected in all songbird species in the liver and in the three other brain areas that were assayed, the optic lobes, cerebellum, and brain stem, with the exception of the cerebellum in willow warblers and quail, but no detectable activity was observed in the testes, muscle, and adrenals of all species. Additional studies will be needed to identify the functional significance of estrogen synthesis in areas that are not classically known to be implicated in the control of reproduction. Within a given species, the birds that had the highest plasma testosterone levels also displayed the highest levels of diencephalic aromatase activity and the interspecies differences in the two variables were positively related. This raises the possibility that the absolute level of diencephalic aromatase represents a species-specific characteristic under the control of plasma testosterone levels. There was, in contrast, no correlation between the aromatase activity in the telencephalon and the plasma testosterone levels but the enzyme activity was correlated with the plasma levels of luteinizing hormone. These data bring additional support to the idea that the diencephalic and telencephalic aromatases are controlled by independent mechanisms.

Aromatase inhibition in JAr choriocarcinoma cells by 7alpha-arylaliphatic androgens.

The JAr choriocarcinoma cell cultures have demonstrated high levels of aromatase activity and have been useful for assaying a wide variety of aromatase inhibitors for aromatase inhibition in intact cells. Recently, several 7alpha-arylaliphatic androgens have shown effective inhibition of human placental microsomal aromatase in vitro, with apparent Ki values ranging from 10 to 20 nM. A series of 7alpha-arylaliphatic androst-4-ene-3,17-dione compounds demonstrated potent competitive inhibition, and 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were enzyme-activated irreversible inhibitors. Both series of these potent inhibitors were investigated for the ability to inhibit aromatase activity in JAr cells by measuring the conversion of [1beta-3H]-androstenedione to 3H2O and unlabelled estrone. JAr cell cultures were incubated for 2 h at 37 degrees C with the aromatase inhibitors at concentrations of 10 pM to 10 microM, the percentage of enzyme inhibition was determined, and IC50 values for inhibitors were calculated. Both series of synthetic compounds demonstrated good to excellent aromatase inhibition, and the most effective inhibitors in both series were those compounds with a phenylpropyl substituent at the 7alpha-position of the steroid nucleus. The 7alpha-arylaliphatic androst-4-ene-3,17-diones exhibited inhibition of JAr aromatase activity with IC50 values from 300 to 434 nM. More potent aromatase inhibition was observed with the 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones, which exhibited IC50 values from 64 to 232 nM. Enhanced efficacy of steroidal enzyme-activated irreversible inhibitors compared to competitive inhibitors was observed in these studies and is consistent with previous reports. These results suggest that JAr choriocarcinoma cells with high levels of aromatase activity may be useful in differentiating steroidal aromatase inhibitors exhibiting different mechanisms of enzyme inhibition. In summary, the 7alpha-phenylpropyl androsta-1,4-diene-3,17-dione analogs, which are enzyme-activated irreversible inhibitors, demonstrated the most effective inhibition of aromatase activity present in the JAr cell cultures among the various 7alpha-arylaliphatic androgens.

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity.

The transformation of testosterone into estradiol in the brain plays a key role in several behavioral and physiological processes, but it has been so far impossible to localize precisely the cells of the mammalian brain containing the relevant enzyme, viz., aromatase. We have recently established an immunohistochemical technique that allows the visualization of aromatase-immunoreactive cells in the quail brain. In this species, a marked increase in the optical density of aromatase-immunoreactive cells is observed in subjects that have been treated with the aromatase inhibitor, R76713 or racemic Vorozole. This increased immunoreactivity, associated with a total blockade of aromatase activity, has been used as a tool in the present study in which the distribution of aromatase-immunoreactive material has been reassessed in the brain of mice pretreated with R76713. As expected, the aromatase inhibitor increases the density of the immunoreactive signal in mice. Strongly immunoreactive cells are found in the lateral septal region, the bed nucleus of the stria terminalis, the central amygdala, and the dorso-lateral hypothalamus. A less dense signal is also present in the medial preoptic area, the nucleus accumbens, several hypothalamic nuclei (e.g., paraventricular and ventromedial nuclei), all divisions of the amygdala, and several regions of the cortex, especially the cortex piriformis. These data demonstrate that, contrary to previous claims, aromatase-immunoreactive cells are present in all brain regions that have been shown previously to contain high aromatase activity.

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity

Aromatase-immunoreactive cells are present in mouse brain areas that are known to express high levels of aromatase activity

Aromatase Activity in Developing Guinea Pig Brain: Ontogeny and Effects of Exogenous Androgens1

The formation of estrogens from androgens by aromatase in the developing brain is an important step in the sexual differentiation of many species. We characterized aromatase activity (AA) in a high-speed pellet of brain tissue from fetal guinea pigs. The apparent substrate affinity (approximately 17 nM) was comparable to reported values in other species. Aromatase activity was highest in the amygdala (AMG) and preoptic area (POA), with lesser amounts in the septum (SEPT) and medial basal hypothalamus (MBH). Activity was low but measurable in parietal cortex (CTX). In the AMG, POA, SEPT, and MBH, AA was highest in early gestation (Days 35-40) and showed a steady decline through development. No sex difference in AA was apparent. We also determined the effects of administration of exogenous androgens to pregnant females on brain AA in the fetus. Testosterone propionate (5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) caused a significant increase (p < 0.05) in AA found in the MBH and CTX. Administration of dihydrotestosterone propionate (2.5 mg/day on Days 30-39 followed by 1 mg/day on Days 40-50) significantly stimulated AA in SEPT, MBH, and CTX. These data demonstrate that the fetal guinea pig brain contains high levels of AA during the critical period of sexual differentiation. Treatment with high levels of exogenous androgens consistently induces AA in the MBH and CTX. These latter effects may be among the mechanisms through which exogenous androgens act on the developing brain.

Distribution and regulation of estrogen-2-hydroxylase in the quail brain

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens.

Distribution and steroid dependence of aromatase enzyme immunoreactivity in limbic nuclei of the female musk shrew brain

In the female musk shrew ( Suncus murinus) neural aromatization of testosterone to estradiol is critical for the expression of sexual behavior. To localize the brain regions capable of aromatization, we used immunocytochemistry to map the distribution of aromatase enzyme. Aromatase immunoreactivity (AROM-ir) has a discrete distribution primarily limited to the lateral septum (LS), central nuclei of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST). In these nuclei the intensity of immunoreactivity varies with hormonal status. Ovariectomy (OVX) significantly reduces the optical density of AROM-ir neurons in all nuclei as compared with brains of normal females. Combined OVX and adrenalectomy (ADX) further reduces optical density readings in AROM-ir cells in the LS and BST, as compared with readings from the brains of OVX animals. Normal and ovariectomized females implanted with testosterone had qualitatively equivalent AROM-ir. High levels of aromatase activity have been measured in the preoptic area and hypothalamus in a number of mammals, including the musk shrew. However, in this experiment AROM-ir was absent in these areas. We present several hypotheses to account for this discrepancy between previously reported biochemical data and these histological data. In summary, these data suggest that limbic nuclei may play a role in the expression of sexual behavior in female musk shrews.

Aromatase Activity in Adult Guinea Pig Brain is Androgen Dependent1

Androgen metabolism in target tissues constitutes an important step for understanding hormone action. The in situ aromatization of androgen represents one of these metabolic events. We characterized aromatase activity (AA) in a microsomal preparation of brain tissue from adult guinea pigs since earlier reports questioned its presence in neural tissues of this species. Analyses revealed an apparent substrate affinity (approximately 17 nM) that was equivalent in adult males and females. However, adult male brains contained greater quantities of AA than female brains. Specifically, AA in the preoptic area (POA: p less than 0.05) and the medial basal hypothalamus (MBH; p less than 0.01) was greater in males than in females. AA was concentrated in the limbic system and hypothalamus (amygdala greater than POA greater than septum greater than MBH), whereas low levels were consistently measured in cortical tissue. In vitro estrogen formation was significantly lower in POA (p less than 0.05) and MBH (p less than 0.01) after castration. After dihydrotestosterone treatment, AA returned to levels equal to or greater than those observed in intact males. These data indicate that AA does exist in the guinea pig brain and is modulated by androgens through the androgen receptor. The presence of high levels of aromatase activity may suggest a role for locally formed estrogens in brain function in this species.

The expression of a functional cDNA encoding the chicken cytochrome P-450arom (aromatase) that catalyzes the formation of estrogen from androgen.

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.

Central regulation of reproduction in teleosts

As in other vertebrates, reproduction in teleosts depends upon interactions taking place along the brain-pituitary-gonads axis. At the central level, these interactions involve at least three types of factors:A gonadotrophin-releasing factor which has recently been isolated from chum salmon brain extracts. This decapeptide, whose structure is (Trp(7)-Leu(8))-LHRH, appears to have a widespread distribution among teleosts, and is less active that LHRH or LHRH analogues in releasing gonadotrophin from the teleost pituitary. Immunohistochemical and quantitative studies have demonstrated that Gn-RH neurons are mainly located in the ventral telencephalon and the preoptic area, while projections are found in the entire brain and the pituitary gland.A gonadotrophin release-inhibiting factor has been demonstrated in the anterior preoptic region of the goldfish and a large set of data suggests that dopamine has GRIF activity in goldfish, and in other teleost species, by direct action on the gonadotrophs. Accordingly, a dopaminergic preoptico-hypophyseal pathway could be demonstrated in the goldfish brain.Sex steroids exert, depending on the dosages, either a negative feedback in sexually mature fish or a positive feedback in immature fish. Such a positive feedback is caused by estrogens and aromatizable androgens. Accordingly, the brain of teleosts contains high levels of aromatase activity in particular in the telencephalon and anterior hypothalamus. The distribution of estrogens concentrating cells within the brain is consistent with possible interactions with Gn-RH or catecholaminergic neurons at the level of certain brain territories.These data are discussed in relation with the functional significance of different brain areas where interactions between these different factors possibly take place, in particular the terminal nerve, the ventral telencephalon, the preoptic area and nucleus lateralis tuberis.

Androgen aromatization and 5 alpha-reduction in ferret brain during perinatal development: effects of sex and testosterone manipulation.

Ferrets of both sexes were killed 8 or 5 days before expected parturition as well as 7, 15, 30, or 51 days after birth, and the activities of aromatase (using 19-[3H]hydroxyandrostenedione as substrate) and of 5 alpha-reductase (using [3H] testosterone as substrate) were assayed in whole homogenates of preoptic area plus anterior hypothalamus (POA), mediobasal hypothalamus (MBH), temporal lobe (TL), and cerebral cortex. Aromatase and testosterone 5 alpha-reductase activities were also measured in these regions in adult gonadectomized male and female ferrets. Compared with adults of both sexes in which aromatase activity was low in all brain regions studied, fetal ferrets had high levels of aromatase activity in POA plus MBH and in TL. At these prenatal ages, aromatase activity in POA plus MBH was significantly higher in males than in females. Aromatase activity in POA, MBH, and TL remained high in both sexes on postnatal days 7, 15, and 30, before declining by postnatal day 51. Cortical aromatase activity was uniformly low across all perinatal ages. The existence of a sex difference in aromatase activity in fetal POA plus MBH cannot be explained by a concurrent sex difference in circulating testosterone. Administration of testosterone to pregnant female ferrets over days 30-41 of gestation caused 150- to 350-fold increases in maternal plasma concentrations of testosterone and 2- to 5-fold increases in fetal plasma testosterone. However, aromatase activity was not affected in the POA and MBH of fetuses or mothers, although activity was significantly increased in the TL of mothers given testosterone. Furthermore, castration of neonatal or adult breeding males decreased plasma androgen levels by factors of 8 and 480, respectively, but resulted in only modest reductions in POA, MBH, and TL aromatase activity (a significant reduction occurred only in the adult male TL). Relatively high levels of testosterone 5 alpha-reductase activity were found in all brain regions across all perinatal ages, as well as in gonadectomized adult ferrets; there was no sex differences at any postnatal age studied. Prenatally, males had higher levels of 5 alpha-reductase activity than females only on day -8 in the POA plus MBH. The results show that estrogen and 5 alpha-reduced androgens can be synthesized in the brains of ferrets of both sexes during the perinatal period of sexual differentiation. A functional role for this neural metabolism of androgen remains to be demonstrated in this carnivorous species.

Biochemical evidence for aromatization of androgen to estrogen in the pituitary

We previously reported that substantial quantities of estrogen were present in the pituitary of the marine teleost ( Myoxocephalus) following perfusion of the isolated head with radiolabeled androgen. In order to determine whether this estrogen was synthesized in situ or was derived from that formed in brain areas with high levels of aromatase activity, we used an explant culture system. Pituitary and brain (preoptic area/anterior hypothalamus) were incubated separately for 20 hr with [ 3H]androstenedione. Alternatively, the two tissues were cultured together, either as an intact unit or after stalk section. [ 3H]Estrone and estradiol-17β were measured in the media and tissue fragments after ether extraction, thinlayer chromatography, and phenolic partition. The authenticity of the products was validated by reverse isotope dilution and recrystallization. Sculpin brain and pituitary in isolated cultures were independently capable of converting radiolabeled androgen to estrogen (estradiol > estrone). Aromatase activity, as indicated by estrogen production per unit wet weight or per unit protein, was greater in pituitary than in brain cultures. Both tissue types, when compared to the medium, retained estrogen against a concentration gradient. Culturing the brain and pituitary together, with or without stalk section, did not alter total estrogen yields or tissue concentration of formed estrogen. It is unlikely that aromatase activity in isolated pituitary cultures could be ascribed to fiber terminals originating in the hypothalamus; however, the cell type responsible for estrogen production remains to be determined. We conclude that, in this teleost, a significant portion of the estrogen reaching target cells in the pituitary may be synthesized in situ from circulating androgen. Hence, control of the aromatization reaction would be one means of regulating gonadotropin secretion. A reexamination of aromatase activity in the pituitary of mammals and other vertebrates is warranted.