Pei-San Tsai - US grants

The goal of this Research Planning Grant (RPG) is to examine the temporal pattern of endogenous gonadotropin-releasing hormone (GnRH) release in the bullfrog, Rana catesbeiana. Ranid flogs are highly unique in that they are the only tetrapods known to date to display a true physiological preference for continuous, rather than pulsatile, GnRH input for the maintenance of gonadotropin secretion. Based on this observation, it is hypothesized that unlike mammals and birds, reproduction in ranid frogs is driven by continuous release of endogenous GnRH. This hypothesis will be tested directly in this Research Planning Grant by investigating the temporal pattern of GnRH release from bullfrog hypothalamic slices using an in vitro perfusion system. The RPG results will provide the physiological basis for future investigations into the mechanisms by which continuous GnRH stimulation supports the overall reproductive functions. In addition, these data will facilitate the understanding of the dynamics of a potentially very novel hypothalamic-pituitary system.

9733237 Tsai Gonadotropin-releasing hormone (GnRH) is central to the initiation and maintenance of reproduction in vertebrates. It stimulates the release of two hormones from the pituitary that directly regulate the growth of ovaries and testes and the production of sex steroid hormones, such as estrogen and testosterone. Defects in GnRH production and/or release will invariably lead to sterility. Despite this hormone's importance in the reproduction of vertebrates, very little is known about when GnRH originally arose in evolution and when it began to assume the role of a central reproductive regulator. Dr. Tsai has shown that GnRH-like molecules exist in a protostome, Aplysia californica. The evolutionary implication of these results is extremely exciting since it suggest that GnRH may have already been present in a primitive invertebrate ancestor that gave rise to diverse group of vertebrates and invertebrates. Such extraordinary conservation over the course of evolution opens up the door to many possibilities regarding the functional roles that GnRH had assumed over the course of evolution. Dr. Tsai will integrative molecular, physiological and evolutionary approaches to examine structural and functional conservation of GnRH. As part of this CAREER award, she will use the same multidisciplinary approach to design a new laboratory course on comparative reproductive biology. Moreover, Dr. Tsai will actively recruit students interested in addressing evolutionary questions with molecular techniques from regional campuses to initiate research projects. The information gained from her research program will have profound implications concerning how the system of reproductive regulation presently seen in humans and other vertebrates has evolved over many millions of years.

A grant has been awarded to Dr. David Stock, Dr. William Friedman, Dr. Pei-San Tsai, and Dr. Robert Boswell at the University of Colorado to establish a common-use confocal microscopy facility. The research of these investigators in the broad areas of developmental and/or evolutionary biology requires three-dimensional characterization of anatomical structures and domains of gene expression in relatively thick specimens. Traditional histological methods for such characterization are laborious, prone to artifacts, and cannot be applied to living specimens. Confocal microscopy is ideal for such applications, as it allows collection of data from single focal planes ("optical sections") which can then be assembled by a computer into a three-dimensional reconstruction. This technique does not require physical sectioning of samples and can therefore be used with living material. The major item of equipment to be purchased is a laser scanning confocal microscope. Two additional pieces of equipment, an epifluorescence compound microscope and a fluorescence stereomicroscope, will be purchased to facilitate the preparation of specimens for analysis with the confocal microscope. These microscopes, along with an existing scanning electron microscope, will be housed in a facility open to all researchers at the University of Colorado.

The common-use imaging facility to be established at the University of Colorado is expected to contribute to intra- and interdepartmental interactions. Developmental and Evolutionary Biology, the research fields of the users of this facility, are both split between two separate departments at the University of Colorado, with the majority of developmental biologists located in the Department of Molecular, Cellular & Developmental Biology and the majority of evolutionary biologists in the Department of Environmental, Population & Organismic Biology. Interactions between these departments have been minimal in the past, despite common interests highlighted by the recent rise of the subdiscipline of Evolutionary Developmental Biology. A facility bringing together researchers on the basis of shared technology is expected to encourage the exchange of ideas among developmental biologists of all types as well as between developmental and evolutionary biologists. In addition to representing a full spectrum of questions in and approaches to Developmental and Evolutionary Biology, potential users of this facility employ a diversity of organisms ranging from animals to plants. As these groups of organisms represent independent inventions of multicellular development, interactions among plant and animal biologists catalyzed by the planned facility may bring into sharper focus universal versus labile features of development and evolution. Students in particular should benefit from such interactions, as they are likely to be the ones spending the most time in the facility. Their educational experience will further be enhanced by the incorporation of confocal microscopy into an existing histology course, as well as by periodic training sessions aimed at attracting new users. In summary, in an era of increasing specialization in research and training throughout Biology, a common use confocal microscopy facility is seen as a means of exposing researchers in Developmental and Evolutionary Biology to a diversity of questions, approaches and organisms.

Gonadotropin-releasing hormone (GnRH) is a neurohomrone responsible for the activation and maintenance of reproduction. Neurons that synthesize GnRH must undergo complex maturational processes during development to become a functional system capable of supporting reproduction. Once mature, the GnRH system also needs to remain functional for an appropriate duration to ensure the propagation of offspring. Thus, one's reproductive health is critically dependent on factors that orchestrate the formation and maintenance of the GnRH system. The goal of the proposed study is to understand how a group of signaling molecules, fibroblast growth factors (FGFs), and their receptors (FGFRs) regulate the developmental maturation and postnatal functionality of the GnRH system. A number of transgenic mouse models, each lacking a distinct component of the FGF signaling system, will be used to investigate if these deficiencies result in the aberrant formation or maintenance of the GnRH system, ultimately leading to sterility. In vitro culture methods, morphological analysis, gene expression studies, electrophysiology, and whole animal manipulation will be utilized for this purpose. This research is highly relevant to public health because mutations on one of the FGFRs lead to human disorders characterized by reproductive failure. Understanding how FGFs and FGFRs regulate the GnRH system could provide important insights into the nature of GnRH system disruption in these individuals. In addition, it will reveal if mutations on other genes encoding FGFs/FGFRs are also candidates for these human disorders.

[unreadable] DESCRIPTION (provided by applicant): Gonadotropin-releasing hormone (GnRH) neurons are central to the initiation and maintenance of reproductive function in diverse vertebrates. During development, GnRH neurons enter sequential stages to mature into a functional network capable of supporting reproduction in adulthood. These stages include cell fate specification in the olfactory placodes, migration into the forebrain, and targeting of axons to the median eminence for hormone release. Signals that trigger GnRH neuronal entrance into these stages, as well as factors that regulate maturation within each stage remain largely unknown. We hypothesize that neurotrophic factors provide time-specific signals to drive the progression of GnRH neurons development. In this proposal, we will use a candidate neurotrophic factor approach and focus on the actions of a family of growth factors shown to have profound neurotrophic activities in cultured primary and immortalized GnRH neurons: the fibroblast growth factors (FGFs). Further, the actions of insulin-like growth factor I (IGF-I), a neurotrophic factor previously shown to act independently and/or collaboratively with FGF-2, will be investigated. This proposal will utilize a combination of existing transgenic models and primary GnRH neuron cultures to address the following Aims: to determine 1) if receptors for FGFs are expressed in a time-specific manner during development, 2) if FGFs alter GnRH progenitor cell expansion and the emergence of GnRH neurons, 3) if FGFs alter the migration of GnRH neurons into the forebrain, 4) if FGFs promote GnRH axon targeting, and 5) if IGF-I acts independently or synergistically with FGFs to promote the survival of GnRH neurons. Together, results from these Aims will provide important clues regarding how a neuroendocrine system critical for vertebrate reproduction develops and matures with the guidance of neurotrophic factors. Further, these results will aid in the understanding of cellular and molecular basis of developmental reproductive abnormalities that result from GnRH deficiency. [unreadable] [unreadable]

Functional and Structural Evolution of Gonadotropin-Releasing Hormone

Successful reproduction in all vertebrates depends upon the action of gonadotropin-releasing hormone (GnRH). Because GnRH is present in diverse animals ranging from mammals to tunicates, it is hypothesized that GnRH may have already been present 630 million years ago in an ancestor that gave rise to vertebrates and invertebrates. If proven, GnRH could be one of the most ancient hormones to persist through evolution. As such, its evolutionary pattern could reveal much about how an important hormone changed its function and structure over time. Dr. Tsai's recent data that a GnRH-like molecule is present in the sea slug, Aplysia californica, proved that GnRH arose very early during evolution. This project utilizes a combination of molecular, cellular and whole organism approaches to characterize the receptor for Aplysia GnRH (ap-GnRH) and to understand what ap-GnRH does in the sea slug. This project will also allow Dr. Tsai to train graduate and undergraduate students interested in hormone evolution and strengthen collaborative ties with other trainees in the Ecology and Evolutionary Biology Department. In sum, this project will contribute substantially to our understanding of how evolutionary selection pressures drive changes in hormone structures and functions, and how these changes allow the organism to adapt better to the environment.

Evolution is driven, in part, by changes in the function of molecules that control the physiology of the organism. Studying these changes is crucial for understanding the unique adaptations developed by organisms to survive the test of time. One molecule whose change over time can influence evolution is a hormone called gonadotropin-releasing hormone (GnRH). GnRH activates reproduction to ensure the propagation of all vertebrate species. In invertebrates, GnRH is assumed to also activate reproduction although consistent support for such an assumption is lacking. It is hypothesized that invertebrate GnRH is not universally important to reproductive activation in invertebrates. Instead, invertebrate GnRH may assume a wide range of functions in neural, motor, and metamorphic control. The project investigates non-reproductive function of GnRH and a related peptide in an invertebrate animal, the sea slug Aplysia californica. These studies may reveal non-reproductive roles of GnRH critical to the survival of invertebrate species.

To study the non-reproductive function of GnRH and the related peptide, adipokinetic hormone, a combination of whole organism studies and molecular and biochemical techniques will be used. The investigators will address the following aims: 1) Examine the role of GnRH in metamorphic development, 2) Perform in vitro functional analysis of GnRH receptor isoforms, investigate their distribution, and 3) Investigate the function of adipokinetic hormone. The work challenges a vertebrate-centric notion that invertebrate hormones essentially function as their vertebrate counterparts. This conceptual shift is important for advancing our knowledge on how changes in hormone function contribute to the survival, adaptation, and evolution of metazoans. For broader impacts, this project will provide Dr. Tsai with opportunities for: (1) undergraduate training, (2) minority recruitment, (3) inter-departmental and trans-Pacific collaborations, (4) information dissemination via the development of a novel website that uses humor and cartoons to engage K-12 students in comparative reproduction, and (5) outreach activities to showcase sea slugs to middle school students. Data collected from these studies will be shared through peer-reviewed publications and the Invertebrate Brain Platform http://invbrain.neuroinf.jp/modules/htmldocs/IVBPF/Top/index.html.

? DESCRIPTION (provided by applicant): Gonadotropin-releasing hormone (GnRH) neurons drive reproductive activation in all vertebrates. Fibroblast growth factor (Fgf) signaling is required for the early development of GnRH neurons, but its role in the postnatal maintenance of GnRH neurons is less clear. Recent data from the PI's lab reveal that Fgf signaling deficiency leads to a significant loss of postnatal GnRH neurons in mice, but this loss can be reversed by early environmental intervention in the form of opposite-sex (OS) housing. These novel results suggest the postnatal GnRH system, like the cognitive brain, is highly plastic and responsive to experience-altering environmental stimuli. The overarching goals of this proposal are to examine the cellular processes responsible for the loss of postnatal GnRH neurons in Fgf signaling-deficient animals, and to identify molecular mechanisms and upstream cues triggered by OS housing to reverse GnRH neuronal loss in Fgf signaling- deficient mice. These goals will be accomplished by three specific aims. Aim 1 will use a lineage tracing technique to test if Fgf signaling deficiency leads sequentially to the de-dedifferentiation and then death of postnatal GnRH neurons. Aim 2 will first use RNA-seq to identify genes differentially regulated by OS housing. Following, the candidate factors will be validated by qPCR and as permitted by mouse models, tested in whole animals to confirm their roles in reversing the postnatal GnRH neuronal loss. Aim 3 will determine if pheromones and elevated androgens are two upstream cues triggered by OS housing to reactivate the failing GnRH system. Collectively, the proposed studies will be important for demonstrating that the postnatal reproductive brain is not a static structure. Instead, its integrity and function are dynamically modulated by environmental cues and perhaps one's lifestyle choices and experience. This information can be used to improve the reproductive health of GnRH-deficient humans by manipulating the highly plastic postnatal GnRH system via suitable environmental means.