Our recent work provided unexpected but convincing evidence that 125I-human chorionic gonadotropin (125I-hCG), binds (specific) not only to plasma membranes (PM), but also to nuclear membranes (NM), lysosomes (LY), rough endoplasmic reticulum (RER) and three (light, medium and heavy) subfractions of golgi (GFs) of bovine corpora lutea. Some of the intracellular binding sites undoubtedly represent those in the biosynthetic (RER & GFs) and degradative (LY) pathways while the others (NM) do not fit into this scheme. Even for RER, GFs, and LY binding sites, some functional significance, in addition to biosynthesis and catabolism, cannot be ruled out. The presence of intracellular binding sites for gonadotropin raises three very important questions a) What are the properties of intracellular binding sites and how do they compare with those of PM? b) Knowing gonadotropin enters luteal cells (from the work of several investigators) where it goes once it enters the luteal cell, nature of the entered gonadotropin, mechanism and sequence of entry. c) Does intracellular binding represent only bio-synthesis and catabolism and/or is there some functional significance to this? The aim of this grant proposal is to seek answers to these questions. The use of luteal tissue from cows, throughout pregnancy, and from superovulated rats will provide flexibility and convenience in accomplishing the stated goals. Purified PM, NM, LY, RER and GFs will be prepared and their purity assessed by marker enzymes and electron microscopy (EM). These fractions will be used in studying binding properties, hCG effects on functions established as normal to these organelles, receptor profiles during luteal growth and demise and during down regulation induced by injecting PGF2Alpha. The subcellular fractionation and electron microscopy - autoradiography on bovine luteal slices preincubated with 125I-hCG, will be used to obtain answer to question b). In summary, the studies outlined in the proposal are very important and concept changing not only for gonadotropin action, but may also be applicable to other agents believed to bind and act through PM receptors exclusively.