Gloria V. Callard

Aromatase and 5α-reductase in the teleost brain, spinal cord, and pituitary gland

The distribution of aromatase and 5α-reductase was investigated in the brain, spinal cord, and pituitary of adult goldfish (Carassius auratus) and toadfish (Opsanus tau) of both sexes. Tissue homogenates were incubated with [3H]androstenedione in the presence of an NADPH-generating system and, following validation of assay conditions, radiolabeled products (estradiol, estrone, 5α-androstanedione), were measured as an index of enzyme activity. Neuroendocrine tissue of both species produced exceptionally large amounts of estrogen, thus confirming previously observed differences between teleosts and other vertebrates. By contrast, 5α-reductase levels resembled the vertebrate norm. In general, aromatase was concentrated in the pituitary and various forebrain regions, especially the hypothalamus/preoptic area; however, estrogen yields from the medulla and anterior spinal cord of toadfish were high compared to adjacent midbrain, hindbrain, and cord regions. This same neural region in toadfish, but not in goldfish, is known to control a sex dimorphic behavior, the courtship boatwhistle. In contrast to aromatase, 5α-reductase was more uniformly distributed throughout the brain, although somewhat higher activity was obtained in the pituitary. High levels of aromatase in the neuroendocrine tissues of teleosts recommend them as animal models for further studying the enzyme, its regulation, and its role in governing androgen-dependent responses in central targets.

Isolation of a goldfish brain cytochrome P450 aromatase cDNA:: mRNA expression during the seasonal cycle and after steroid treatment

To investigate the molecular basis and physiological regulation of exceptionally high levels of aromatase (P450arom) activity in the brain of teleost fish, a 2927 bp P450arom cDNA encoding a 510 amino acid protein was isolated from a goldfish brain cDNA library. The brain-derived cDNA had 53% and 61-62% sequence identity when compared with human placental and fish ovarian P450arom forms, respectively, and higher homologies in conserved functional domains. Goldfish brain poly(A) RNA was translatable in vitro to a 56 kDa P450arom immunoprecipitation product. Northern blot analysis using the brain cDNA revealed a major 3.0 kb transcript of high abundance in brain (FB, forebrain > M/HB, mid/hindbrain), but no signal in ovary, testis or liver. P450arom mRNA varied seasonally in brain, with a peak at the onset of gonadal regrowth (February) that preceded the annual rise in enzyme levels and was 4-fold (FB) or 50-fold (M/HB) higher than during reproductive inactivity (July-December). Known markers of neurogenesis and estrogen action in brain (28S rRNA, beta-actin and beta-tubulin transcripts) each had unique seasonal patterns which differed from P450arom mRNA. In vivo steroid treatment showed that estrogen and aromatizable androgen increase FB and M/HB levels of P450arom mRNA 8- and 4-fold, respectively. P450arom mRNA in pituitary and retina had a different regulation. Southern analysis provided no evidence for multiple genes encoding the brain derived cDNA or for brain-specific gene amplification. Results imply that high accumulated levels of P450arom mRNA are the major determinant of high measured enzyme activity in goldfish brain, and that physiological regulation of mRNA expression in the natural environment is mediated by aromatization of androgen to estrogen.

Localization of aromatase in synaptosomal and microsomal subfractions of quail (Coturnix coturnix japonica) brain.

The subcellular distribution patterns of aromatase, 5 alpha- and 5 beta-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional constituents were identified by marker enzymes. Aromatase was concentrated 8-fold in the 100,000 g pellet (P3) along with a 3-fold enrichment in the microsomal marker NADPH-cytochrome c reductase (NCR) a result consistent with glandular tissues. In addition, aromatase was enriched 2-fold in the 11,000 g pellet (P2) and, owing to its large size, this fraction accounted for more total activity than P3. Although P2 contained the mitochondrial marker succinate dehydrogenase (SDH), treatment with Triton X-100 to solubilize membranes and release occluded enzymes increased measured NCR and the cytosol marker lactate dehydrogenase (LDH) 2- and 4-fold, respectively--evidence that this fraction was composed of mitochondria plus synaptosomes (pinched-off nerve terminals). To further explore the location of aromatase in the 11,000 g fraction, P2 was exposed to hypotonic buffer, a treatment known to cause lysis of synaptosomes, and then separated into three fractions P2a (11,000 g pellet), P2b (100,000 g pellet) and P2s (100,000 g supernatant). Aromatase colocalized with the microsomal marker NCR (13- and 4-fold increase, respectively) in the 100,000 g (P2b) pellet which was, however, devoid of mitochondrial enzyme activity. We infer from this that a significant portion of aromatase in brain is associated with smooth membranes present inside synaptosomes. 5 beta-Reductase in quail brain subfractions was enriched 6-fold in the 100,000 g supernatant together with a 4-fold enrichment in the cytosolic marker LDH.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatization mediates aggressive behavior in quail

Although testosterone (T) stimulates aggressive and reproductive behaviors in males of many vertebrate species, it is now known that the full expression of T action in the brain requires aromatization to estradiol (E2) and subsequent interaction of locally formed E2 with nuclear estrogen receptors. In experiments reported here, we used a behavioral test which quantifies the response of an individual male Japanese quail (Coturnix coturnix japonica) to the visual stimulus of a conspecific. We have called this behavior aggression because it shares many features in common with traditional measures of aggression, e.g., predicting dominance and subordinance. Nevertheless, the behavior probably also combines a complex steroid-sensitive masculine behavior. The advantage of this test is that it allows the discrimination of individual differences in masculine behavior but avoids fighting and sexual encounters per se, thereby reducing effects of learning, a problem with previous tests of avian aggression. In addition, this test has been applied usefully to identify neuroendocrine correlates to male behavior. Using this test, the arousal of reproductively inactive males (hereafter referred to as aggression) is activated by administration of T or estradiol benzoate (EB), but not by 5 alpha-dihydrotestosterone (DHT). T-induced aggression was blocked by the aromatase inhibitor 4-hydroxyandrostenedione (OHA), an effect partially reversed by treatment with EB. In addition, OHA or the estrogen receptor blocker CI-628 reduced aggressiveness of reproductively active males whereas the androgen receptor blocker flutamide had no effect. Results with the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 alpha-carboxyamide (4-MA) were equivocal. Additionally, treatment of reproductively inactive quail with T or E2 but not DHT increased aromatase activity in the hypothalamus-preoptic area (HPOA). We conclude, therefore, that T to E2 conversion is essential for the activation of aggressiveness in this species. Although locally formed estrogen exerts its effects on aggression in part by increasing activity of aromatase per se, analysis of the time course of behavioral induction or suppression by the various treatments suggests that the response has multiple components, including both short latency, receptor-independent and long latency, receptor-dependent events.

Seasonal changes in testicular structure and function and the effects of gonadotropins in the freshwater turtle, Chrysemys picta.

Abstract Plasma testosterone was measured at stages during the reproductive cycle of control male turtles ( Chrysemys picta ) and following hypophysectomy and/or mammalian FSH or LH injection. Testosterone levels were highest in April and December when large Leydig cells were observed in the interstitium but the epithelium was inactive. During active spermatogenesis (July, October) plasma steroid levels were basal, and not significantly reduced by hypophysectomy. While LH injections had no effect, FSH was a potent stimulus to plasma testosterone in both intact and hypophysectomized turtles. Histologic observations during the testicular cycle and after hypophysectomy indicated that the tubular epithelium was dependent on endogenous pituitary hormone secretion but the interstitium did not atrophy during the experimental period. Injections of FSH in May increased testicular weight and caused disappearance of vacuoles and granules from apical portions of the Sertoli cell with a consequent reduction in cell height. Neither FSH nor LH had obvious effects on the morphology of the interstitium in control or hypophysectomized turtles either in July or November.

Estrogen synthesis in vitro and in vivo in the brain of a marine teleost (Myoxocephalus)

Conversion of radiolabeled substrate to estrone and estradiol was studied in a marine teleost, Myoxocephalus octodecimspinosus (longhorn sculpin), a species previously shown to have exceptionally high brain aromatase activity. In vitro aromatization of 3H-labeled androstenedione was greatest in homogenates of the preoptic area/anterior hypothalamus, medial telencephalon, and lateral telencephalon, in that order, and when measured in individual fish, marked sex and seasonal differences were observed. Relatively little estrogen was produced by the inferior lobes of the hypothalamus, thalamus, optic lobes, basal midbrain, cerebellum, and medulla. Sculpin testis was aromatase negative, and at no time did ovarian activity per unit weight exceed that in the brain. Addition of 11β-hydroxy- and 11-ketotestosterone to forebrain homogenates did not alter estrogen yields. Following perfusion of a sculpin head preparation with 3H-labeled 19-hydroxyandrostenedione, estrone was isolated from the efferent perfusate and estrone and estradiol recovered from the brain and pituitary. The authenticity of formed estrogen in this experiment was verified by derivative formation and recrystallization. Estrogen concentration was highest in the preoptic area/hypothalamus, somewhat lower in the medial and lateral telencephalon, and lowest in the remaining brain regions. Pituitary estrogen per unit weight was at least 12 times higher than in any brain region and, in contrast to the brain in which estrone predominated, the greatest proportion of total estrogen was estradiol. No authentic estrogen was recovered from non-neural tissues (liver, kidney, heart, muscle, skin, and gills) after perfusion. All of the above findings show that the highest levels of aromatase activity correspond neuroanatomically to the reported distribution of estrogen-binding cells and the location of reproductive control centers in the teleost brain. In these regions, a correlation between CNS estrogen production and reproductive status (gender and season) is further evidence for a functional interrelationship. The presence of relatively large amounts of estrogen in the sculpin pituitary following perfusion with radiolabeled androgen suggests that, at this level of phylogeny, the brain may regulate pituitary function by supplying estrogen directly, or the pituitary itself is capable of aromatization. Whether or not brain-derived estrogen makes a significant contribution to the peripheral estrogen pool is still open to question.

Evolutionary and functional significance of two CYP19 genes differentially expressed in brain and ovary of goldfish

Remarkably high levels of cytochrome P450 aromatase (P450arom) enzyme are expressed in the brains of teleost fish when compared to the ovaries of the same fish, or to the brain or ovaries of other vertebrates. Northern analysis using a full-length P450arom cDNA from a goldfish brain library indicates high accumulated levels of CYP19 mRNA in the brain but fails to detect P450arom mRNA in the ovary. The possibility of different brain and ovarian mRNA variants was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) amplification of ovarian RNA using degenerate primers led to the isolation of a 243 bp P450arom cDNA fragment with approximately 20% of nucleotide and amino acid replacements when compared to the brain cDNA. Southern analysis with sequence-specific probes indicated two distinct CYP19 loci, and this was confirmed by PCR-restriction enzyme analysis of genomic DNA. Corresponding brain- and ovary-type genomic sequences were identified in a second, diploid fish species (zebrafish), evidence that two genes are not caused per se by tetraploidy in goldfish. RT-PCR analysis of different tissues with sequence-specific primers showed high levels of the brain mRNA variant and much lower levels of the ovarian variant in neural tissues with high enzyme activity. In contrast, the ovary expressed low levels of the ovarian mRNA variant exclusively. The data imply that the expression of two CYP19 genes in goldfish is controlled by distinct regulatory mechanisms. Further studies are required to determine whether the two genes lead to functionally distinct isozymes.

In vivo steroid regulation of aromatase and 5α-reductase in goldfish brain and pituitary

Abstract The full expression of testosterone (T) actions in neuroendocrine tissues requires aromatization and 5α-reduction to estradiol (E2) and 5α-dihydrotestosterone (DHT), respectively. Recently, we documented striking changes in aromatase and 5α-reductase during the annual reproductive cycle of goldfish ( Carassius auratus ). To investigate possible regulatory effects of sex steroids, goldfish were implanted with hormone-filled silastic capsules for 2–5 weeks. Conversion of [ 3 H]androstenedione to estrone or 5α-androstanedione by homogenates of anterior hypothalamus/preoptic area, remaining telencephalon, and whole pituitary (PIT) was used to estimate aromatase and 5α-reductase, respectively. Gonadosomatic index and plasma E2, T, and DHT were monitored as an index of reproductive status and capsule effectiveness. In reproductively inactive fish in which plasma steroids and aromatase were basal (October), E2 or T increased aromatase activity in brain of both sexes but stimulated activity in PIT of females only; DHT was not effective. In a subsequent experiment initiated close to the spawning peak and prior to the seasonal decline in plasma steroids and brain aromatase (April), T increased or maintained brain aromatase in a time-dependent manner. 5α-Reductase activity was unaffected by steroid treatment in both reproductively active and inactive fish. These results indicate that variations in circulating steroids are responsible, at least in part, for changes in brain aromatase during the annual reproductive cycle of goldfish and provide the first evidence for steroid control of pituitary aromatase. The steroid specificity of the induction suggests that an estrogen receptor mechanism is involved. Product (E2)-induced increases in aromatization during the spawning period may be part of a positive feedback loop leading to neural E2 accumulation and culminating in ovulation/spermiation and breeding behavior.

Aromatization is cyclic AMP-dependent in cultured brain cells.

When added to primary cultures of adult turtle (Chrysemys) brain, dibutyryl cyclic AMP increases estrogen yields from [3H]testosterone in a dose- and time-dependent manner, although rat FSH, rat LH, and dibutyryl cyclic GMP are relatively ineffective. A phosphodiesterase inhibitor (MIX) by itself increases estrogen yields from the substrate and potentiates the response to dibutyryl cyclic AMP.

Molecular and cellular physiology of aromatase in the brain and retina

Due to exceptionally high brain aromatase activity, teleost fish are advantageous for studying neural aromatase regulation, localization, and physiology. To determine the molecular mechanism of enhanced expression, we have isolated, cloned and sequenced a 3 kb full-length aromatase cDNA from a goldfish (Carassius auratus) brain library using a human placental aromatase cDNA as probe. The deduced sequence of goldfish aromatase is 510 amino acids (predicted Mw, 58 kDa) with 69% overall sequence similarity, when compared to human placental aromatase, and higher homologies in presumptive functional domains. A major 3 kb mRNA species was abundant in brain and low or non-detectable in non-neural tissues, reflecting the order of enzyme activities. To determine the cellular basis of high enzyme activity in goldfish brain, a human placental aromatase antibody was used to immunolocalize labeled cells. This antibody immunoprecipitated a single 56 kDa in vitro translation product of goldfish brain poly(A+)RNA and revealed discrete clusters of intensely stained neurons, processes, and terminals concentrated in, but not limited to, reproductive brain centers. Close proximity of aromatase- and androgen receptor-positive neurons in certain regions provides anatomic evidence of a functional relationship between direct and indirect pathways of neural androgen action. Aromatase-positive neurons and fibers formed interconnected networks in novel loci (e.g. retina-->optic tract-->optic tectum), and catalytic activity was confirmed biochemically in these tissues, indicating that neuroestrogen may have a role in visual input and integration. Availability of goldfish-specific nucleotide and antibody probes will facilitate further studies using this model.