It is widely believed that the Sertoli cells generate a special milieu required for the completion of spermatogenesis. A large body of evidence suggests that this special environment is probably produced in response to numerous stimuli, e.g., hormones and growth factors, as well as by a reciprocal effect of germ cells on Sertoli cells. These data imply that Sertoli cells must be able to process a myriad of extracellular signals distinct in composition and in the message transduced. Thus, the Sertoli cell must contain the necessary mechanisms to sort out and efficiently translate signal transduction events to effect normal spermatogenesis. Extracellular signal effects are mediated via cell surface localized receptors. Following occupancy, the receptor-ligand complex is internalized and processed intracellularly. Although the itinerary of the interiorized receptor-ligand complex is known for numerous hormones, the mechanism by which the intracellular sorting is effected remains obscure. However, it is likely that the mechanism does involve the integral membrane proteins that provide the vesicle framework of endocytic organelles that include endosomes, lysosomes and Golgi. Thus, it is important to know what the proteins are that make up the membrane containers of the endocytic organelles. What is the pathway(s) that these proteins take prior to their final destination? Do they contain a signal that makes them organelle specific? What is the nature of this signal; is it a primary amino acid sequence? Where is the signal read? Finally, how are these proteins regulated at the level of the gene? To address these questions it is proposed that the identity of the endocytic organelle membrane components be elucidated. Monoclonal antibodies (mAb) will be generated and used as probes to study in detail the composition of endocytic organelle containers, their biosynthesis and degradation, and the potential regulatory mechanisms that control such phenomena. The mAb will then be used to purify the integral membrane proteins (by affinity chromatography) which will be used to produce polyclonal antibodies (pAb). The pAb will be used to study further the proteins at two levels for complexity: 1) At the ultrastructural level, using frozen ultrathin section methodology, the disposition of the integral membrane proteins will be determined. This methodology permits co-localization of antigens. 2) At the molecular level, the genes of these proteins will be cloned and sequenced to probe the potential mechanism of gene regulation in Sertoli cells in relation to the cycle of the seminiferous epithelium.
