Cryptorchidism, or undescended testis, is one of the most common congenital defect in newborn boys. If left untreated, this condition causes infertility and drastically increases the chance of testicular cancer in adulthood. The key aim of this project is to establish the precise network of genetic and biochemical events underlying testicular descent during development. The proposal is based on recent identification of the testicular hormone Insulin-like 3, and its receptor, GREAT, as the critical regulators of gubernacular differentiation. Using mouse crsp mutant we have identified the GREAT gene, showed that it encodes a G protein-coupled receptor, and demonstrated that GREAT is the only cognate receptor for INSL3 in vivo. Mutation analysis of the two genes in cryptorchid patients revealed functionally deleterious mutations, which may be responsible for the abnormal phenotype. A series of transgenic mouse mutants produced in our laboratory provides a unique basis to address specific questions regarding the role and mechanisms of INSL3 signaling in vivo. Furthermore, the fact, that the INSL3 receptor is a GPCR, makes it an attractive target for future therapeutic development. We are going to explore the hypothesis, that INSL3 signaling pathway is a key regulator of testicular descent. To test this hypothesis we propose the following Specific Aims 1: To identify genetic factors leading to the activation of INSL3/GREAT expression during development; 2. To identify downstream target genes controlled by INSL3; 3. To identify cellular pathways involved in INSL3/GREAT signaling; 4. To determine mechanisms of GREAT receptor activation and its desensitization. As a result, we will identify a sequence of genetic events leading to testicular descent and characterize the mechanisms of INSL3/GREAT cellular signaling. The proposed studies will provide a better understanding of the testicular descent and its regulation during male development.