Joan C. King - US grants

Secretion of the hypothalamic neuropeptide luteinizing hormone releasing hormone is critical to reproduction in all mammals. The neural afferents that modulate the activity of LHRH neurons are not known, although gonadal steroids and the catecholamines, especially norepinephrine, are potent stimulators of LHRH secretion. Activity of LHRH neurons may be modulated by neural systems either by direct innervation of LHRH neurosecretory cells or indirectly through interneurons. Nonetheless, these influences must ultimately reach LHRH neurons to influence synthesis and/or neurosecretion. It is the purpose of these studies to identify and morphologically characterize contacts that are afferent to those LHRH neurons that are critical to ovulation in mammals. First, it is hypothesized that there are groups of LHRH neuronal cell bodies that abruptly change their content of immunoreactive LHRH in relationship to ovulation, while other groups do not; and that these "dynamic" and "static" groups of cells are present within the forebrain of mammalian species that are spontaneous (monkeys and rats), induced (ferrets) or seasonal ovulators (bats). Second, it is hypothesized that synaptic input to "dynamic" but not "static" groups of LHRH neurons changes in relation to ovulation and/or development. Finally, it is hypothesized that among mammals exhibiting different mechanisms of ovulation, afferent contacts to those LHRH neurons that are critical to ovulation, will be morphologically similar; while afferents to other LHRH neurons, whose function is specifically related to the pattern of ovulation, will differ. Afferent synapses will be examined for their plasticity, and, regardless of the degree of plasticity, for morphological parameters in pre- and post- ovulatory females compared to that in prepuberal females and adult males. These studies will utilize light and electron microscopic immunocytochemistry to study relationships between LHRH neurons themselves, between LHRH neurons and catecholamine neurons and between LHRH neurons and other afferents of unspecified origin.

One impairment in the function of neuroendocrine cells in aging animals appears to be a deficit in neurosecretion. Further, it is thought that this deficit results, in part, from declining activity of neurons that is characteristic of aging animals. However, relatively little is known about the relationship between neural activity and biosynthesis of neuropeptides. Biosynthesis of several neuropeptides involves initial synthesis of larger molecular weight precursors that are subsequently modified to yield smaller molecular weight peptides which are physiologically active. We propose to examine products of biosynthesis and maturation of a neuropeptide, luteinizing hormone releasing hormone (LHRH). Age-related alterations in the function of LHRH neurons are thought to significantly contribute to the reproductive decline apparent in aging rodents. We propose to, first, identify molecular weight species of LHRH and, secondly, to quantify their occurrence in extracts from the preoptic/hypothalamic area of young and aging rats. To accomplish this we will use a combination of techniques including: sizing columns, reverse phase high performance liquid chromatography and radioimmunoassay. Since it is not known how gonadal steroids influence the biosynthesis and/or post-translation of LHRH, rats will be sacrificed in several hormonal conditions and every attempt will be made to equalize hormonal environments of young and old animals. These studies constitute a novel and unique approach to conceptualize the intraneuronal balance of biosynthesis and maturation of hypothalamic neuropeptides in aging animals.

This is a proposal to establish a Reproductive Core Center on the Health Sciences Campus of Tufts University. Over the last ten years, Tufts University has amassed a group of 18 researchers in reproduction whose primary affiliations span 6 departments. The research programs of these investigators are broad-based and are multidisciplinary in their approach, including reproductive behavior, physiology, cell biology, including cell-matrix interactions, molecular biology, and the generation of transgenic animals by both DNA microinjection and embryonic stem cell methods. Many within the group have long-standing collaborations. The specific aim of the Center is to enhance the quality, productivity and cost-effectiveness of 26 funded research projects investigating the control of reproduction and fertility. Centralized facilities, consultation, training, guidance and services will be provided by three scientific cores. The Protein Probe and Immunometric Assay Core will produce characterized labelled protein probes, perform steroid assays, and provide training and advice on the purification of proteins and on immunoassay procedures. The Histochemistry and Hybridization Core will provide centralized facilities, equipment and advice on preparing tissues for immunohistochemistry, in situ hybridization, and for the generation and labelling of nucleic acid probes. The Imaging Core, will provide equipment, facilities, training and guidance for the quantitative analysis of: 1) gels, blots and films, and 2) isolated cells and tissues, processed for in situ hybridization or immunocytochemistry, using state-of-the-art image analysis protocols. A second aim of the Center is to foster scientific interactions between Center Investigators, and other Principal Investigators on the Health Sciences Campus and in the academic community of the Boston area. Reproductive researchers in the Boston community will be included in our Seminar Series and in our External Advisory Committee. Communication on campus will be encouraged via a Newsletter and postings of Center Events on the Centralized Computer System. The development of a Reproductive Core Center on the Health Sciences Campus is timely given the breadth of the research and the diverse and complementary technologies used by this group of investigators at Tufts University.