Differentiation of spermatids into spermatozoa involves activation of transcription of unique genes and assembly of gamete-specific organelles including the acrosome and the flagellum. The objective of this proposal is to determine the role of the cAMP-dependent pathway during this differentiative process of the male gamete. We have made major progress in characterizing the enzymes involved in cAMP synthesis and degradation in round and elongating spermatids. Using the tools and models that we have developed, we now propose to study the mechanisms of regulation of these enzymes and the role of cAMP signaling in the spermatid-specific gene expression. The experiments described in this proposal are organized along two Specific Aims. With the first Specific Aim, we will address the role of adenylyl cyclases and phosphodiesterases in the control of cAMP levels in differentiating spermatids. The expression and subcellular localization of these enzymes as well as their retention in mature spermatozoa will be studied during spermatid development in vivo. The mechanisms of regulation of these enzymes will be investigated in a reconstitution system and in intact round and elongating spermatids. The second Specific Aim will be devoted to understanding how the cAMP signaling pathway is involved in the regulation of gene expression. In vitro studies will investigate how changes in cAMP concentration impact phosphorylation of cAMP-regulated transcription factors and expression of spermatid-specific genes. A genetic approach of transgenic overexpression or homologous recombination will be used to manipulate the cAMP- dependent pathway in vivo. Analysis of these in vivo models will elucidate how cAMP signaling controls gene expression and terminal differentiation of these germ cells. The proposed studies will further our understanding of regulation of spermatogenesis and sperm function. Moreover, they will help to identify novel targets for pharmacological manipulation of gamete production and fertility.