Brain 5beta-reductase: a correlate of behavioral sensitivity to androgen.

Testosterone is converted in the dove (Streptopelia risoria) brain to 5 beta-reduced metabolites that do not affect behavior. In long-term castrated birds, which are relatively insensitive to the behavioral effects of testosterone, the activity of preoptic 5 beta-reductase is increased. The increase, which is specific to the preoptic area, is reversed by estrogen. Inactivation of testosterone by 5 beta-reduction may be involved in the control of brain sensitivity to androgen.

Biocompatible surfactants for water-in-fluorocarbon emulsions.

Drops of water-in-fluorocarbon emulsions have great potential for compartmentalizing both in vitro and in vivo biological systems; however, surfactants to stabilize such emulsions are scarce. Here we present a novel class of fluorosurfactants that we synthesize by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG). We demonstrate that these block copolymer surfactants stabilize water-in-fluorocarbon oil emulsions during all necessary steps of a drop-based experiment including drop formation, incubation, and reinjection into a second microfluidic device. Furthermore, we show that aqueous drops stabilized with these surfactants can be used for in vitro translation (IVT), as well as encapsulation and incubation of single cells. The compatability of this emulsion system with both biological systems and polydimethylsiloxane (PDMS) microfluidic devices makes these surfactants ideal for a broad range of high-throughput, drop-based applications.

Amphiphilic block copolymer nanotubes and vesicles stabilized by photopolymerization.

We report on a new method to stabilize nanotube and vesicle structures created from amphiphilic diblock copolymers by means of photopolymerization. Cross-linking with UV light exposure minimizes fluid disruption during stabilization. Additionally, the spatial control afforded by focusing or masking the initiating light source enables stabilization of distinct segments of individual nanostructures. This contribution demonstrates (1) that vesicles and nanotubes formed from poly(ethylene oxide)-block-polybutadiene are stabilized by exposure to UV light in the presence of a water-soluble photoinitiator and (2) that new nanotube geometries can be constructed by means of spot-curing, and (3) it reveals an application for photopolymerized nanotubes by showing electrophoresis of DNA through a UV-stabilized nanotube.

Hormonal control of behaviour: steroid action in the brain.

There have recently been significant advances in our understanding of the cellular action of steroids on brain mechanisms of behaviour. Brain cells contain steroid metabolizing enzymes whose activity is modified by environmental stimuli. Steroids have rapid effects on neurotransmitter receptors via cell membranes and modify the distribution of neuropeptide receptors in areas controlling behaviour. It has been known for some time that oestrogens have an effect on brain structure that can be related to behaviour in the sexually dimorphic avian song system. Recent work suggests that oestrogen may have a similar effect on the developing sexually dimorphic nuclei of the mammalian brain.

Brain aromatase expression after experimental stroke: topography and time course.

Brain aromatase has been shown to be increased in expression after neurotoxic damage and to exert neuroprotection via generation of local oestrogens. The present study investigates the topography and time course of brain aromatase expression after experimental stroke (middle cerebral artery occlusion (MCAO)). Ovariectomised stroke prone spontaneously hypertensive rats underwent distal MCAO by electrocoagulation. Immunohistochemistry revealed increased brain aromatase expression at 24h and 8 days in the cortical penumbra/peri-infarct zones with no increase evident at 2h or 30 days post-MCAO. Double label studies indicate that some of the increased aromatase expression is associated with astrocytic processes. Thus, this is the first evidence that aromatase protein is increased after MCAO and the location (peri-infarct), time course (within 24h) and cellular localisation (astrocytic) indicate the potential for aromatase to promote the survival of cells in the penumbra after experimental stroke by local synthesis of oestrogens.

Photoperiodic influences on sexual behavior in male Syrian hamsters.

The effect of photoperiodic conditions on sexual behavior was investigated in male Syrian hamsters that were either gonadally intact, or castrated and treated with low doses of testosterone throughout the experiment. Hamsters were exposed to long (LD 16:8) or short (LD 8:16) days for 7 weeks; for the next 8 weeks, either they were exposed to an intermediate daylength (LD 12:12), or daylength conditions remained unchanged. Sexual behavior was affected by photoperiod conditions in both gonadally intact animals and testosterone-treated castrates, but to different degrees. Intact males exposed to short days for 15 weeks exhibited gonadal regression, and their copulatory performance was impaired. The percentage of animals that intromitted or ejaculated was significantly reduced. Additional measures of sexual performance among the copulating males were also affected. In contrast, among the castrates with testosterone clamped at low but stable levels, the proportion of males that mounted, intromitted, or ejaculated was not affected by photoperiod. However, among the males that continued to copulate, sexual performance changes were present in the short-day castrates that resembled those displayed by the intact males. We infer that these behavioral effects in both hormonal conditions reflect primarily a difficulty in the attainment of intromission. Gonadal regression alone cannot easily account for the behavioral deficits of the intact males, because circulating testosterone levels at the end of the experiment were not significantly different between the gonadally intact hamsters and the castrated, testosterone-treated hamsters exposed continuously to short days. Males transferred from either long or short days to the intermediate-daylength condition responded behaviorally to this photoperiod as if it were a short day, that is, their ejaculatory frequency declined. We conclude that male hamsters exposed to photoinhibitory daylengths exhibit deficits in their sexual behavior, not only because endogenous levels of testosterone decrease, but also because the substrates on which this hormone acts become less responsive. We hypothesize that under physiological conditions, the episodic secretion of testosterone imposes constraints on the maintenance or restoration of copulation, and that the potent behavioral effects achieved by constant-release implants of testosterone may mask the presence of photoperiodically induced alterations in the hamster's sensitivity to this gonadal hormone.

Formation of behaviorally effective 17 beta-estradiol in the dove brain: steroid control of preoptic aromatase.

The preoptic area (POA) of the male dove is a known target area for separable behavioral actions of testosterone and 17 beta-estradiol (E2) and contains an active aromatase system. We have examined the regulatory influence of gonadal hormones on aromatase using an in vitro microassay that measures conversion of [1 alpha, 2 alpha-3H]testosterone to E2 in anatomically defined brain samples of individual animals. Preoptic aromatase activity, which is higher in males than females, is decreased (77%) in both short (30 day) and long term (180 day) castrates, indicating that gonadal hormones maintain POA aromatase activity. Basal levels of POA activity are not influenced by the period of hormonal deficit. Low levels of aromatase activity detected in area basalis are also unaffected by castration. Intramuscular testosterone propionate rapidly increases aromatase activity (within 12 h) specifically in POA of castrated males. The inductive effect of testosterone propionate in castrated doves is not increased by limited behavioral interactions in a test situation with sexually active females. A nonaromatizable androgen, 5 alpha-dihydrotestosterone, has no effect on POA aromatase activity, whereas the activity of this enzyme is restored to levels of sexually active males by systemic E2. Diethylstilbestrol has a similar, though less potent effect, indicating an estrogenic action on the enzyme. We conclude that circulating androgen modulates preoptic aromatase activity. The product of the reaction, E2, is also likely to be involved as part of a positive feedback system.

Androgens influence sexual differentiation of embryonic mouse hypothalamic aromatase neurons in vitro.

Estrogen formed perinatally in the brain from testicular androgen by aromatase is involved in the irreversible determination of male brain development. Perinatal sex differences in aromatase activity have been observed in the hypothalamus. Testosterone (T) is a major modulator for aromatase in the adult rat hypothalamus. However, it is not known whether circulating T influences aromatase neurons during fetal brain development. To study the influence of androgen exposure on embryonic neuronal aromatase, gender-specific primary cell cultures were prepared from embryonic day 15 mouse hypothalamus and cortex. Estrogen formation by cultured neurons was measured using an in vitro 3H2O product formation assay, and aromatase neurons were identified by immunocytochemistry using a highly specific antiserum. Aromatase activity (AA) per well and numbers of aromatase-immunoreactive (IR) neurons per microtubulus associated protein II-IR neurons x 10(5) were significantly higher in male hypothalamic cultures compared with female when grown in the absence of sex steroids. When AA was calculated per aromatase-IR neuron, no differences in enzyme activity were found between male and female. Therefore, the level of AA in individual male hypothalamic neurons is similar to the female, but a higher proportion of male neurons express aromatase. After T treatment, AA per well (P < or = 0.001) and AA/aromatase-IR cell (P < or = 0.005) in male and female hypothalamic cultures was significantly increased vs. controls. In addition, numbers of aromatase-IR neurons/microtubulus associated protein II-IR neurons x 10(5) were significantly higher after T exposure compared with controls (P < 0.001). Androgenic effects on hypothalamic AA and aromatase-IR cell numbers were dose-dependent and mediated via androgen receptor stimulation, since the observed effects were inhibited by the androgen-receptor antagonist flutamide. There was no effect of T on cortical AA or aromatase-IR cell numbers, indicating area-specific regulation of brain aromatase. We conclude that 1) sex differences in hypothalamic AA are due to a higher percentage of neurons expressing aromatase in males rather than to higher AA in individual male hypothalamic aromatase-IR cells, and 2) androgens influence the development of the fetal hypothalamic aromatase system. Because T influenced both the embryonic male and female hypothalamic neurons in culture, the developing mouse brain aromatase appears to be bipotential in response to androgen. The data suggest that environmental and genetic factors affecting androgen level and/or androgen receptor function in the developing brain could interfere with the sexual differentiation of estrogen forming neurons.

Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair.

Recent evidence indicates that 17beta-estradiol may have neuroprotective and neuroregenerative properties. Estradiol is formed locally in neural tissue from precursor androgens. The expression of aromatase, the enzyme that catalyses the conversion of androgens to estrogens, is restricted, under normal circumstances, to specific neuronal populations. These neurons are located in brain areas in which local estrogen formation may be involved in neuroendocrine control and in the modulation of reproductive or sex dimorphic behaviours. In this study the distribution of aromatase immunoreactivity has been assessed in the brain of mice and rats after a neurotoxic lesion induced by the systemic administration of kainic acid. This treatment resulted in the induction of aromatase expression by reactive glia in the hippocampus and in other brain areas that are affected by kainic acid. The reactive glia were identified as astrocytes by co-localization of aromatase with glial fibrillary acidic protein and by ultrastructural analysis. No immunoreactive astrocytes were detected in control animals. The same result, the de novo induction of aromatase expression in reactive astrocytes on the hippocampus, was observed after a penetrating brain injury. Furthermore, using a 3H2O assay, aromatase activity was found to increase significantly in the injured hippocampus. These findings indicate that although astrocytes do not normally express aromatase, the enzyme expression is induced in these glial cells by different forms of brain injury. The results suggest a role for local astroglial estrogen formation in brain repair.

Olfactory recognition in the male hamster: effect of non-aromatizable androgens, 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-triene-3-one (R 1881) and 5 alpha-dihydrotestosterone, in combination with oestrogen.

Intact male hamsters show olfactory behaviour (sniffing and licking) to novel females. After exposure to female vaginal secretions, they show less sniffing towards a novel female if she does not match previously encountered odours (Steel, 1984). Male hamsters, castrated and injected s.c. with various steroids, were tested for the amount of sniffing they directed towards novel and mismatching females. 5 alpha-Dihydrotestosterone propionate (DHTP), 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-triene-3-one (methyltrienolone, R1881) and oestradiol benzoate alone did not influence olfactory behaviour, while testosterone propionate (TP), DHTP plus oestradiol and R1881 plus oestradiol all increased sniffing to a novel female over the levels shown by saline-treated, castrated controls. Our results also indicated that only the aromatizable androgen (TP) or non-aromatizable androgens DHTP and R1881, in combination with oestradiol, reduced sniffing directed to a mismatching female after exposure to vaginal odour. We conclude that both androgenic and oestrogenic actions are required not only for olfactory behaviour, but also for olfactory recognition as indicated by a reduction in sniffing directed towards mismatching females. This suggests that aromatization of testosterone may be involved in the control of olfactory processes associated with reproductive behaviour.

Effects of intracranial androgen on the development of masculine ultrasonic vocalizations in the Mongolian gerbil (Meriones unguiculatus).

The effects of testosterone propionate (TP) on brain mechanisms involved in the sexual differentiation of ultrasonic vocalizations were examined in Mongolian gerbils (Meriones unguiculatus). Treatment of neonatal females with TP fully masculinized the rate of emission of the upsweep precopulatory ultrasound during adult sexual interactions with oestrous females. Intracranial implantation of small crystals of TP mixed with cholesterol (65 ng) into females 1-15 h after birth also masculinized the upsweep vocalization emitted in adulthood. Implants of TP positioned in the hypothalamic area had a significantly greater masculinizing effect than TP implants outside this region, or pure cholesterol implants. Two other sexually dimorphic vocalizations, the modulated (mainly precopulatory) and unmodulated (mainly copulatory) calls were masculinized by systemic TP, but intracranial TP had no significant masculinizing action on these calls. Genital structures of females which received neonatal injections of TP were strongly virilized in that their clitorides were lengthened and male-type cornified spines were present on the glans. Females which had received intracranial implants of TP were not virilized peripherally in adulthood. We conclude that testosterone or its metabolites have a direct hypothalamic effect on the development of masculine upsweep vocalizations. Because other vocalizations were insensitive to intracranial TP, the underlying neural tissues may have different thresholds of response to androgen.

Precopulatory behaviour in the male hamster: effect of the 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one.

Three independent components of hamster masculine behaviour (approaching, leaving and sniffing the female) have been shown to depend on both androgenic and oestrogenic action. The behavioural role of 5 alpha-reduced androgens was assessed by blocking 5 alpha-reduction of testosterone by means of 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one (4-MA) through slow-release silicone elastomer implants. Three dose levels of 4-MA were given to intact, sexually active males. The probability of approaching the female and the amount of sniffing directed to her were both decreased, while the probability of leaving the female was increased. Sniffing and the tendency to approach were affected at lower dose levels than was the tendency to leave. The higher dosage of 4-MA also abolished odour-based discrimination between females shown by normal males. These effects of 4-MA on behaviour were confirmed in castrated hamsters maintained on testosterone. The suppressive effects of 4-MA on behaviour were reversed by treatment with 5 alpha-dihydrotestosterone (DHT). We conclude that testosterone maintains sociosexual behaviour in part by conversion to its 5 alpha-reduced metabolites; components of this behaviour differ in their relative dependence on DHT. In this species, DHT is likely to be a behaviourally active metabolite of testosterone involved in the fine control of behaviour.

Behavioural action of androgen in the dove: effects of long-term castration on response specificity and brain aromatization.

Differences in the effectiveness of oestradiol-17 beta and testosterone on male courtship and vocal behaviour were examined in long-term castrated doves. Nest-orientated behaviour was restored by intramuscular injection of oestradiol-17 beta. Testosterone was effective in restoring aggressive courtship and vocal behaviour, but not for the nest-orientated behaviour. The effects of these hormones were separable, therefore, under conditions of prolonged androgen deficit, suggesting differences in their specificity of action. In-vitro assay of brain enzyme activity indicated that aromatization of testosterone to oestradiol-17 beta occurred in the preoptic area of long-term castrated doves. Preoptic aromatase activity of long- and short-term castrated doves did not differ. The ineffectiveness of testosterone in restoring nest-orientated behaviour in long-term castrated doves did not appear, therefore, to be due to a difference between the groups in the basal rate of testosterone aromatization in the preoptic area.

Effects of photoperiod on formation of oestradiol-17 beta in the dove brain.

The effects of photoperiod and castration on brain aromatase activity have been examined using an in-vitro radioassay. Formation of oestradiol-17 beta was lower in the preoptic area of male Barbary doves on a short daylength (6 h light:18 h darkness) than in males on a long daylength (14 h light:10 h darkness). This was a specific effect of photoperiod which did not influence aromatase activity in the anterior or posterior hypothalamic areas, and was not accompanied by changes in hormone-sensitive vocal behaviour. Production of 5 beta-dihydrotestosterone, 5 beta-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone by the preoptic area did not differ between birds on long or short days. Therefore, a short photoperiod does not appear to influence other pathways of androgen metabolism. In contrast to the effects of photoperiod, castration reduced oestradiol formation in both preoptic and hypothalamic areas. Intramuscular injection of testosterone propionate (TP) in intact males on short days did not restore the pattern of distribution of aromatase activity seen in males on long days. Preoptic aromatase activity was, however, restored by TP in castrated birds. We conclude that a short photoperiod influences both the activity of aromatase and the inductive effect of testosterone on enzyme activity in the preoptic area, which is known to be associated with the behavioural action of oestrogen in the dove. Photoperiod is likely to act both through changes in circulating androgen and by a direct action on preoptic cells.

Sex differences in plasma concentrations of steroids during the sensitive period for brain differentiation in the zebra finch.

Changes in plasma concentrations of sex steroids were examined in male and female zebra finch chicks during the sensitive period for differentiation of sexually dimorphic brain nuclei associated with the control of song. Using a chromatographic separation procedure and radioimmunoassay, androstenedione, testosterone and 5 alpha-dihydrotestosterone were detected in plasma at relatively high concentrations immediately after hatching. There were no sex differences in concentrations of these androgens. An oestrogen, oestradiol-17 beta, which is known to differentiate the song-control system, is raised specifically in the circulating plasma of male zebra finch chicks, and not in females. The surge in oestradiol, which occurs during the first week after hatching, coincides with the period when capacity for differentiation of the song system is maximal. Exposure of the male brain to oestradiol-17 beta could trigger neuronal differentiation.

Regulation of female brain aromatase activity during the reproductive cycle of the dove.

Oestrogen is formed in the female dove brain. The aim of this study was to determine whether (a) the catalytic properties of the brain aromatase are similar to the ovarian enzyme and (b) aromatase activity in the female brain changes during the reproductive cycle and is influenced by steroids and environmental stimuli. The results show that female preoptic aromatase has a higher substrate affinity than the enzyme in ovarian follicles (apparent Km: preoptic area, 7 nmol/l; ovarian follicles, 29 nmol/l), but a lower activity in the preoptic area (Vmax: preoptic area, 290 fmol/mg tissue per h; ovarian follicles, 843 fmol/mg tissue per h). In intact females with developing follicles, oestradiol-17 beta formation was higher in the posterior hypothalamus than the preoptic area. Females in a later stage of reproductive development (yolked follicles) had a different distribution of oestrogen formation with increased aromatase activity in the preoptic area. Preoptic and posterior hypothalamic aromatase activity of females paired with males for 10 days was positively correlated (r = 0.84, P = 0.0001; r = 0.75, P = 0.001 respectively) with ovarian development. Females with undeveloped ovaries which interacted with males had higher preoptic aromatase activity than visually isolated females with similar ovarian development, suggesting that behavioural stimuli have direct effects on brain aromatase activity which are independent of the ovary. Oestradiol benzoate treatment increased preoptic and posterior hypothalamic aromatase activity in intact and ovariectomized females, and testosterone propionate treatment increased anterior hypothalamic aromatase activity, but did not affect other areas, indicating that the distribution of induced aromatase activity is steroid-specific. Oestrogen treatment in ovariectomized or intact females did not replicate the maximal hypothalamic aromatase activity seen when the ovary contained yolked follicles. We conclude that brain aromatase activity is related directly to ovarian condition during the reproductive cycle of the female dove. As in the male, steroids have a role in the regulation of oestrogen formation in the female hypothalamus; behavioural stimuli are also likely to be involved in the control of the brain enzyme.

Gender-specific brain formation of oestrogen in behavioural development.

Steroid sex hormones have an organisational role in the development of brain mechanisms underlying gender-specific behaviour. Although peaks in gonadal androgen occur at developmental stages that coincide with sensitive periods for the differentiation of both structural sex differences in the brain and sexual behaviour, the factors that control the phasic effects of steroids are still not understood. Aromatase, converting androgen to oestrogen, is a key enzyme in development, and regulation of the activity of this enzyme is likely to be one of the factors determining availability of oestrogen effective for brain differentiation. Measurement of testosterone metabolism in vitro shows that in the mouse oestrogens are formed actively in the neonatal brain during male development. In cultured cells of the embryonic mouse hypothalamus there are sex differences in hypothalamic aromatase activity both during early embryonic and later perinatal development, with a higher capacity for oestrogen formation in the male than in the female. The sex differences are regionally specific, since no differences in aromatase activity are detectable in cultured cortical cells between male and female. Aromatase activity is neuronal rather than astroglial. Using a specific antibody to the mouse aromatase, immunoreactivity is also restricted to neuronal soma and neurites in hypothalamic cultures. Therefore, gender-specific differences in aromatase regulation are probably restricted to neurons. Testosterone increases oestrogen formation specifically in cultured hypothalamic neurones, but has no effect on cortical cells. Although there is a sex difference in early embryonic neuronal aromatase, aromatase activity appears to be sensitive to androgen only in later embryonic development. What determines the phasic sensitivity of the developing brain aromatase system to androgen has still to be determined.

Ontogeny of aromatase messenger ribonucleic acid and aromatase activity in the rat midbrain.

Estrogen formation catalyzed by neural aromatase is crucial for the sexual differentiation of the brain. Ontogenic expression of aromatase mRNA and aromatase activity were studied in male and female rat midbrains. Aromatase mRNA was transiently expressed in both sexes showing maximum levels on postnatal day (P)2 and being absent on P20 and in adults. Developmental expression of aromatase mRNA preceded that of aromatase activity. These data demonstrate that the capacity for estrogen formation is present during a distinct phase of midbrain development. Our findings suggest an active role for estrogens in the differentiation of midbrain neurons.

Inhibition of hypothalamic aromatase activity by 5 Beta-dihydrotestosterone.

Abstract A variable amount of circulating testosterone that reaches brain cells is converted to biologically inactive 5beta-reduced metabolites, namely, 5beta-dihydrotestosterone (5beta-DHT) and 5beta-androstane-3alpha,17beta-diol (5beta,3alpha-diol). In avian species, the production of inactive 5beta-DHT and 5beta,3alpha-diol is highest during embryonic and post-hatching life. In the present study, we have investigated the possibility that 5beta-reduction may not only correspond to a steroid inactivation pathway, but that 5beta-reduced metabolites of testosterone may exert direct inhibitory effects on enzymatic pathways producing biologically active steroids. When added to hypothalamic homogen-ates prepared from adult male doves, 5beta-DHT but not 5beta,3alpha-diol inhibits the activity of the aromatase enzyme, which converts testosterone to 17beta-oestradiol. During the first days after hatching, when the production of 5beta-reduced metabolites is high, the hypothalamic aromatase is also inhibited by 5beta-DHT. We conclude that a high 5beta-reductase activity during sensitive periods for sexual differentiation may protect the avian brain from the differentiating effects of circulating androgens by inhibiting the production of oestrogen.

Lateralization of a sexually dimorphic brain area associated with steroid-sensitive behavior in the male gerbil.

Emission rates of an androgen-sensitive male courtship vocalization were positively correlated with the volume of only the left hypothalamic sexually dimorphic area, pars compacta (SDApc) nucleus in sexually active male Mongolian gerbils. The asymmetric relationship between brain nucleus and vocal behavior was confirmed in testosterone (T)-treated castrated adult males but was eliminated by castration of males and did not exist in T-treated ovariectomized adult females. Interdependence between the left SDApc volume and emission rate was specific because no significant correlations were found between (a) nonvocal precopulatory or copulatory behaviors and left or right SDApc volumes and (b) a second sexually dimorphic brain area, the suprachiasmatic nucleus and vocal or other behavioral components. The asymmetric brain-behavior relationship depends on T effects in adult males and may be lateralized by perinatal T exposure.