The traditional concept that testosterone-dependent differentiation of male reproductive structures (internal and external genitalia) and Mullerian Inhibiting substance (MlS)-mediated regression of the female anlagen (Mullerian ducts) are parallel but independent processes has been challenged by recent data demonstrating cross-talk between these two pathways. In addition to causing Mullerian duct regression, MIS has been shown to play an integral role in controlling Leydig cell development by inhibiting proliferation of progenitor Leydig cells and modulating androgen biosynthesis. These testicular actions of MIS are critical for achieving an adequate adult endowment of Leydig cells and a normal capacity for androgen production- fundamental aspects of male reproductive health. We hypothesize that MIS has a critical role in the molecular switch that causes the cessation of Leydig cell proliferation and promotes acquisition of terminally differentiated function. Our long-term objectives are to understand how MIS elicits its actions in the Leydig cell in a developmental context. The Specific Aims of this grant are: 1. to identify the MIS type I receptors by experimental ablation of ALK function in Leydig cells;2. to identify and characterize the role of Smads in MIS signal transduction in the Leydig cell. This work will enhance our understanding of a novel pathway for control of cellular proliferation and provide insights into the pathogenesis of testicular tumorigenesis, which may lead to new molecular targets for control of unregulated Leydig cell proliferation and neoplastic transformation. Relevance to Public Health: Increased knowledge of the paracrine regulation of androgen synthesis and testicular development by MIS has therapeutic implications for management of children with disorders of testicular function and offers novel approaches for management of children with intersex conditions. Insights gained from this work may lead to alternative therapies for young men with testicular cancer and children with disorders of puberty and testicular function. Moreover, recent studies report a world-wide increase in male infertility and decrease in sperm counts. Our models may enable us to identify subtle defects in male reproductive health due to these endocrine disrupters. Because androgen production is so critical to male fertility, this work will improve our understanding of male reproductive health and fertility.