Gloria V. Callard - US grants

This is a Research Opportunities for Women Career Advancement Award. The PI will use this money to take a hands-on sabbatical leave to develop molecular genetic and other new techniques.

Circulating androgen derived from gonadal secretions is converted to estradiol (neuroestrogen) within the brain and pituitary. This transformation, which is catalyzed by an enzyme complex termed aromatase (P450arom), is essential for the full expression of androgen actions on brain sex differentiation, sex behavior and reproductive neuroendocrinology. Because the brain and pituitary are structurally and functionally complex and the quantity of neuroestrogen is very small, it has been difficult to determine how P450arom is regulated, its precise cellular and subcellular location, and the cellular targets and mechanisms by which neuroestrogen exerts its actions. Dr. Callard has identified an animal model, the goldfish, in which P450arom activity is 100-to-100- times higher than in conventional laboratory species. Moreover, brain androgen receptors are correspondingly elevated. Based on this correlation and results of experiments during the last project period, she postulates a functional interrrelationship between P450arom and androgen receptors in which locally synthesized estrogen acts as a positive feedback regulator of quantifiable endpoints for elucidating the parahormonal role of neuroestrogen in brain and pituitary. To this end, she proposes to isolate and characterize cDNA probes for the goldfish P450arom and androgen receptor genes, to develop antibodies for measuring their protein products, and to apply these new tools to unanswered questions using the goldfish model.

Epidemiological studies indicate that male gametes and the complex, hormone-regulated processes by which germ cell proliferation, development and survival are controlled, may be threatened. By virtue of a long biological half-life, cadmium (Cd) exemplifies a trace environmental pollutant with potential as a cumulative toxicant. Although spermatogenesis is exquisitely sensitive to Cd toxicity, which may be related to increased permeability of the blood-testis barrier and high constitutive expression of metallothionein (Mt), attempts to pinpoint sensitive stages, cell-types and mechanisms of action have been hampered by the complex testicular organization of common laboratory mammals. A technically advantageous alternative is the shark testis, which facilitates analysis of spermatogenesis stage-by-stage in vivo and allows intact germinal units (spermatocysts), comprising stage-synchronized germ cell/Sertoli cell clones, to be isolated for experimentation and analysis in vitro. Initial studies indicate that Cd increases permeability of a functional barrier in meiotic and postmeiotic stages but is preferentially concentrated in tissues at premeiotic stages, where it increases synthesis of a Mt-like protein, increases the percentage of germinal clones undergoing programmed cell death (apoptosis), and increases secretion of SGP-2, a frequent marker of apoptosis in somatic cells. We propose to examine the hypothesis that Cd has direct access to germ cells in early developmental st ages, is taken up by an active Cd-accumulating mechanisms that is further amplified by enhanced mt expression, and activates a cell death program by perturbing normal control mechanisms. Cd-mediated defects early in development would have profound consequences for all subsequent stages and the final number of mature spermatozoa. Using the shark testis model and in vivo and in vitro approaches, we will (1) define kinetics of Cd uptake and retention stage-by-stage and identify cellular and nuclear vs. cytosolic sites of accumulation; (2) relate Cd exposure to intracellular levels and to Cd effects on barrier permeability, Mt and SGP- 2 expression, and to the extent and timing of apoptosis; (3) elucidate the relationship between apoptosis and DNA replication/repair and reproductive/hormonal status in response to Cd; (4) initiate studies to determine pathways by which Cd activates apoptosis in the stem cell/spermatogonial germ cell population; and (5) evaluate the utility of cultured premeiotic spermatocysts for identifying spermatogenic toxicants that affect programmed (apoptotic) vs. unregulated (necrotic) death of male germ cells.

Steroidal and non-steroidal estrogen-like chemicals (xenoestrogens, XE), and agents that otherwise impact estrogen-mediated regulatory pathways, are widespread in the general environment and common contaminants at Superfund sites. XE are prototypical "endocrine disruptors" and likely candidates as neurodevelopmental toxicants. Cytochrome P450 aromatase (P450arom, estrogen synthetase) and estrogen receptors (ER), the two major players in estrogen signaling, are expressed in many regions of the CNS prior to birth and throughout life. Estrogens are known to have organizational effects in the developing CNS and have been implicated in the regulation of brain structure and function postnatally and in the adult organism, including humans. Zebrafish (Danio rerio) are an advantageous experimental model for investigating the role of XE as neurodevelopmental toxicants and, together with Fundulus heteroclitus, an indigenous telosteam species, have potential as sentinels for neuroactive XE in the aquatic environment. Compared to other vertebrates, teleost fish have exaggerated levels of neural P450arom and retain a remarkable potential for neurogenesis and neuroregeneration. Moreover, separate and distinct CYP19 gene loci encode different P450arom isoforms in neural and non-neural tissues, which allows recognition of brain-specific processes. The brain P450arom (-B) isoform is transcribed from the earliest stages of embryogenesis and is estrogen- inducible. The estrogen response system in zebrafish is mediated by ERalpha, ERbeta, and a novel third isoform (ERgamma). Proposed studies will investigate the following proposition: (a) XE present in the environment disrupt P450arom/ER-dependent processes of neurodevelopment, neuroplasticity and neuroregeneration; and (b) effects of XE, even at low doses and transient exposures, are amplified by a regulatory cascade involving estrogen-induced enhancement of P450aromB and changes in neural ER expression. Specific aims are to: (1) develop and validate a whole animal, in vitro screening system using the brain form of P450arom as a mechanism-based molecular marker of neuroactive XE; (2) define XE-induced alterations in temporal, spatial, quantitative and isoform-specific patterns of P450arom and ER expression in the developing and adult CNS; (3) determine the consequences of inappropriate estrogen signaling for CNS development neuroplasticity and neural functions; and (4) characterize the molecular mechanisms of XE actions on estrogen mediated signaling pathways in the developing CNS.

Steroida! and non-steroidal estrogen-like chemicals (environmental estrogens, EE) that mimic or block hormonal estrogen actions on estrogen receptors (ER), and dioxin-like chemicals that interact with arylhydrocarbon receptors (AhR), are widespread in the environment and common contaminants at Superfund sites. ER mediated signaling is known to have a critical role in neurodevelopmental programming and in the maintenance of neuroplasticity and repair in the adult and aging brain. Although ER and AhR/ARNT signaling pathways converge at multiple points in peripheral tissues, the normal role of AhR signaling in the CNS, and brain specific mechanisms and effects of ER-AhR interactions are largely unknown. Here we investigate the hypothesis that EE and dioxins are neurotoxicants, by virtue of their ability to disrupt neural estrogen signaling and estrogen responsive genes in the CNS. A corollary of this hypothesis is that irreversible chemical effects on estrogen dependent neurodevelopmentaf programming alter responses to hormonal estrogen and subsequent EE and dioxin exposures, and dysregulate estrogendependent components of neuroplasticity and self-repair in the adult and aging brain. Specific aims are to: (1) Determine separate and convergent actions of ER and AhR signaling pathways on gene expression during developmental programming, and in the adult and aging brain; (2) Determine corresponding changes in estrogen responsive neural genes and cellular processes of neurodevelopment, neuroplasticity and repair; (3) Characterize estrogen-dioxin actions and interactions on developmentally programmed alternative splicing decisions in the CNS; (4) Identify cis-elements and transacting cellular factors that mediate estrogen and dioxin actions and interactions on the neural cyp19B gene promoter.