Modulation of cognition-specific cortical activity by gonadal steroids: A positron-emission tomography study in women

There is considerable evidence from animal studies that gonadal steroid hormones modulate neuronal activity and affect behavior. To study this in humans directly, we used H215O positron-emission tomography to measure regional cerebral blood flow (rCBF) in young women during three pharmacologically controlled hormonal conditions spanning 4–5 months: ovarian suppression induced by the gonadotropin-releasing hormone agonist leuprolide acetate (Lupron), Lupron plus estradiol replacement, and Lupron plus progesterone replacement. Estradiol and progesterone were administered in a double-blind cross-over design. On each occasion positron-emission tomography scans were performed during (i) the Wisconsin Card Sorting Test, a neuropsychological test that physiologically activates prefrontal cortex (PFC) and an associated cortical network including inferior parietal lobule and posterior inferolateral temporal gyrus, and (ii) a no-delay matching-to-sample sensorimotor control task. During treatment with Lupron alone (i.e., with virtual absence of gonadal steroid hormones), there was marked attenuation of the typical Wisconsin Card Sorting Test activation pattern even though task performance did not change. Most strikingly, there was no rCBF increase in PFC. When either progesterone or estrogen was added to the Lupron regimen, there was normalization of the rCBF activation pattern with augmentation of the parietal and temporal foci and return of the dorsolateral PFC activation. These data directly demonstrate that the hormonal milieu modulates cognition-related neural activity in humans.

Attenuated sensitivity to neuroactive steroids in γ-aminobutyrate type A receptor delta subunit knockout mice

γ-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the δ subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the δ subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Firefly “femmes fatales” acquire defensive steroids (lucibufagins) from their firefly prey

Female fireflies of the genus Photuris, the so-called firefly “femmes fatales,” prey on male fireflies of the genus Photinus. The females are able to entrap the males by faking the flash signal characteristics of the Photinus female. We found that by feeding on Photinus males, Photuris females gain more than nutrients. They also acquire defensive steroidal pyrones called lucibufagins, which are contained in Photinus but which Photuris fireflies are unable to produce on their own. Photuris females that eat Photinus males or lucibufagin are rejected by Phidippus jumping spiders. Lucibufagin itself proved to be a deterrent to such spiders. Field-collected Photuris females contain lucibufagin in varying amounts. The more lucibufagin they contain the more unacceptable they are to Phidippus.

Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.

Ergosteroids: induction of thermogenic enzymes in liver of rats treated with steroids derived from dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7 alpha-hydroxy-DHEA, 7 beta-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7 alpha-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens.

Dehydroepiandrosterone: A potential signalling molecule for neocortical organization during development

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEAS) are the most abundant steroids produced by the human adrenal, but no receptors have been identified for these steroids, and no function for them has been established, other than as precursors for sex steroid synthesis. DHEA and DHEAS are found in brains from many species, and we have shown that enzymes crucial for their synthesis, especially P450c17 (17α-hydroxylase/c17,20 lyase), are expressed in a developmentally regulated, region-specific fashion in the developing rodent brain. One region of embryonic expression of P450c17, the neocortical subplate, has been postulated to play a role in guiding cortical projections to their appropriate targets. We therefore determined if products of P450c17 activity, DHEA and DHEAS, regulated the motility and/or growth of neocortical neurons. In primary cultures of mouse embryonic neocortical neurons, DHEA increased the length of neurites containing the axonal marker Tau-1, and the incidence of varicosities and basket-like process formations in a dose-dependent fashion. These effects could be seen at concentrations normally found in the brain. By contrast, DHEAS had no effect on Tau-1 axonal neurites but increased the length of neurites containing the dendritic marker microtubule-associated protein-2. DHEA rapidly increased free intracellular calcium via activation of N-methyl-d-aspartate (NMDA) receptors. These studies provide evidence of mechanisms by which DHEA and DHEAS exert biological actions, show that they have specific functions other than as sex steroid precursors, mediate their effects via non-classic steroid hormone receptors, and suggest that their developmentally regulated synthesis in vivo may play crucial and different roles in organizing the neocortex.

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.

Developmental exposure to vasopressin increases aggression in adult prairie voles

Although the biological roots of aggression have been the source of intense debate, the precise physiological mechanisms responsible for aggression remain poorly understood. In most species, aggression is more common in males than females; thus, gonadal hormones have been a focal point for research in this field. Although gonadal hormones have been shown to influence the expression of aggression, in many cases aggression can continue after castration, indicating that testicular steroids are not completely essential for the expression of aggression. Recently, the mammalian neuropeptide arginine vasopressin (AVP) has been implicated in aggression. AVP plays a particularly important role in social behavior in monogamous mammals, such as prairie voles (Microtus ochrogaster). In turn, the effects of social experiences may be mediated by neuropeptides, including AVP. For example, sexually naïve prairie voles are rarely aggressive. However, 24 h after the onset of mating, males of this species become significantly aggressive toward strangers. Likewise, in adult male prairie voles, central (intracerebroventricular) injections of AVP can significantly increase intermale aggression, suggesting a role for AVP in the expression of postcopulatory aggression in adult male prairie voles. In this paper, we demonstrate that early postnatal exposure to AVP can have long-lasting effects on the tendency to show aggression, producing levels of aggression in sexually naïve, adult male prairie voles that are comparable to those levels observed after mating. Females showed less aggression and were less responsive to exogenous AVP, but the capacity of an AVP V1a receptor antagonist to block female aggression also implicates AVP in the development of female aggression.

Endocrine modulation of the neurotoxicity of gp120: Implications for AIDS-related dementia complex

HIV infection often involves the development of AIDS-related dementia complex, a variety of neurologic, neuropsychologic, and neuropathologic impairments. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120, which damages neurons via a complex glutamate receptor- and calcium-dependent cascade. We demonstrate an endocrine modulation of the deleterious effects of gp120 in primary hippocampal and cortical cultures. Specifically, we observe that gp120-induced calcium mobilization and neurotoxicity are exacerbated by glucocorticoids, the adrenal steroids secreted during stress. Importantly, this deleterious synergy can occur between gp120 and synthetic glucocorticoids (such as prednisone or dexamethasone) that are used clinically in high concentrations to treat severe cases of the Pneumocystis carinii pneumonia typical of HIV infection. Conversely, we also observe that estradiol protects neurons from the deleterious actions of gp120, reducing toxicity and calcium mobilization.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.

On roads not taken in the evolution of protein catalysts: Antibody steroid isomerases that use an enamine mechanism

Reactive immunization has emerged as a new tool for the study of biological catalysis. A powerful application resulted in catalytic antibodies that use an enamine mechanism akin to that used by the class I aldolases. With regard to the evolution of enzyme mechanisms, we investigated the utility of an enamine pathway for the allylic rearrangement exemplified by Δ5-3-ketosteroid isomerase (KSI; EC 5.3.3.1). Our aldolase antibodies were found to catalyze the isomerization of both steroid model compounds and steroids. The kinetic and chemical studies showed that the antibodies afforded rate accelerations up to a factor of 104 by means of an enamine mechanism in which imine formation was the rate-determining step. In light of our observations and the enzyme studies by other workers, we suggest that an enamine pathway could have been an early, viable KSI mechanism. Although this pathway is amenable to optimization for increased catalytic power, it appears that certain factors precluded its evolution in known KSI enzymes.

Role of brain allopregnanolone in the plasticity of γ-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of γ-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl− uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for γ2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also α1,α2, α3, α4, β1, β2, β3, and γ2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5α-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl− uptake and the decrease of γ2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.