In preliminary studies, a crude preparation of collagenase dispersed interstitial cells upon purification by the method of unit gravity sedimentation using Percoll or Metrizamide gradient yielded two types of interstitial cells. Although their origin is undetermined, the cell types are morphologically distinct and respond to human chorionic gonadotropin (hCG) and human luteinizing hormone (hLH) with distinct biochemical characteristics. To confirm and extend these observations, following specific aims are set: (1) Attempts will be made to further purify these cell preparations by a combination of cell separating techniques such as with unit gravity and gradient/centrifugation techniques to study in depth their morphological and biochemical characteristics in an attempt to understand the nature, origin, viability/integrity and biochemical functionality of each cell type in interstitium responsive to LH/hCG. (2) Studies will be directed to assure that the cells so purified in vitro are present in in vivo and exhibit similar morphological and biochemical characteristics to those determined earlier (specific aim #1). For this purpose autoradiographic studies will be performed to detect the binding patterns of these cells in vivo during the period of target cell activation. (3) A perifusion system will be developed to study the influence of hormone, cAMP or ethanol upon the production of either cAMP, testosterone, or both. Along with these measurements, the dissociated receptors in the eluate will also be measured to explore their association with biological responsive parameter. (4) And finally, attempts will be made to determine if a correlation is found between either receptor occupancy and biological response, or receptor dissociation and biological response in purified cell subfractions. The overall information obtained will facilitate our understanding of the polypeptide hormone action and provide sufficient background information to establish cell culture systems in an attempt to isolate receptor components involved in recognition of LH/hCG by these cell subfractions.
