The goal of the project is to increase our understanding of the structure- function relationships of the oligosaccharide moieties on glycoprotein hormones and related molecules. The molecules under investigation are human chorionic gonadotropin (hCG), the subunits of hCG (hCG-alpha subunit and hCG-beta subunit), and free alpha-molecules associated with pregnancy or malignancy. We recently have shown that the free alpha-molecule purified from pregnancy urine stimulates secretion of prolactin from primary cultures of human decidual cells. These findings represent the first bioassay for pregnancy free-alpha molecules and indicate that free- alpha may be a glycoprotein hormone with a function that is independent of hCG. Establishment of this bioassay will allow us to investigate the role of carbohydrate moieties in free-alpha function. Carbohydrate modifications, resulting in a variety of branched oligosaccharide structures, occur on all glycoproteins prior to secretion. These modifications can affect virtually every aspect of the molecules's behavior, including receptor binding and signal transduction, yet the underlying regulatory mechanisms remain elusive. We have investigated changes in the oligosaccharide moieties as a function of the following parameters: synthetic environment and gestational development. With respect to synthetic environment, interaction of subunits can either promote or restrict modifications on the oligosaccharide structures produced in the final secreted protein. When hCG-beta subunit associates with hCG-alpha subunit during its carbohydrate processing, hCG-beta prevents addition of some branches to hCG-alpha subunit, whereas free-alpha molecules receive the additional branches. The presence of these branched structures on free-alpha prevent it from combining with hCG-beta to form intact hormone. In our investigation of gestational development, we found that glycosylation changes as a function of gestational age. Molecules produced in late pregnancy are more highly branched and are extensively more fucosylated than those of early pregnancy. We plan to examine how the differences observed in glycosylation affect the bioactivity of these molecules.