Eugene Spaziani - US grants

Two hormones control the cycles of growth, molting and regeneration in arthropod animals (insects and crustaceans). In crustaceans, one of the hormones is ecdycsone, chemically a steroid, that directly controls the cycles. Ecdysone is secreted by glands (Y-organs) in the body cavity. Secretion by the Y-organs is controlled in turn by the second hormone, molt-inhibiting hormone (MIH) which is chemically a protein and is secreted by specialized nerve cells in the brain. This system is analogous to that in the human and other vertebrates (adrenal cortex, ovary and testis) that secrete steroid hormones and that are governed by protein hormones from the brain/pituitary gland. However, the crustacean system is simpler in cell structure and metabolism. For this reason, the system is being used as a model for studying the details of how cells take up cholesterol (the universal precursor of steroid hormones) and process it for hormone synthesis, and how neural hormones control these events. The focus of this project is to uncover the mechanism by which cholesterol enters Y-organ cells in light of findings that cholesterol is presented to the cells bound up in high- density lipo-protein (HDL). Specific receptors for HDL on the cell surface, how the complex is broken down in the cell and how and where the freed cholesterol is processed into hormone will be examined. Attendant studies will explore how MIH modifies cell function to slow or stop hormone biosynthesis. In this regard, crustaceans appear to be unique among animals; a theoretically significant aspect of this model is that the brain hormone (MIH) regulates negatively, whereas in all other known systems the corresponding hormone is stimulatory. Techniques will include use of radioactive tracers, the electron microscope, the ultracentrifuge and high-pressure liquid chromatography. This research embraces the fields of neurobiology, endocrinology and developmental biology in addressing basic questions on coordination of bodily functions. Results should have eventual application of clinical medicine in the areas of steroid endocrinology, reproductive physiology, pathologies of glands and atherosclerosis (arising from defects in cholesterol metabolism). Also, knowledge gained will add to understanding the arthropod molting/growth cycle, a developmental process that dominates the existance of this abundant, economically important group of animals.

Growth, molting and regeneration in crustaceans is controlled by steroid hormones (ecdysteroids) from a pair of glands (Y-organs) in the body cavity. The Y-organs in turn are regulated by a protein hormone (MIH) from the brain. This system is comparable to human glands (testis, ovary, adrenal) that secrete steroid hormones (testosterone, estrogen, cortisone) and are controlled by protein hormones from pituitary and brain. We have found that crustaceans, like humans, make steroid hormones from cholesterol. Accordingly, we use crustacean glands as a simplified model to study how cholesterol enters cells and is converted to hormones, and how the process is regulated by the external proteins. We already know that all cholesterol in crustacean blood is bound to high-density lipoprotein (HDL), and that there are receptors on the surface of Y-organ that recognize and bind the HDL. Using radioactively labeled cholesterol and HDL, the electron microscope and the ultracentrifuge, we now will find how cholesterol enters the cells, how it is processed if still bound to HDL and where in the cells the processing takes place. Part of the study is to determine how MIH controls cholesterol entry; e.g., does MIH change the number of receptors for HDL? We will also attempt to work out the details of the biosynthesis of ecdysteroids, by using the mass spectrograph to identify the individual steps in the conversion of cholesterol to the hormones. This project embraces the fields of neurobiology, endocrinology and development in addressing basic questions on neuroendocrine coordination of functions.