Many egg-laying reptiles lack sex chromosomes, depending instead upon the incubation temperature of the egg to determine the sex of their offspring, a process known as temperature-dependent sex determination (TSD). How temperature both stimulates and inhibits genetic cascades to determine the type of gonad and direct sexual differentiation is the focus of this application. This investigator and his colleagues have developed the red-eared slider turtle (Trachemys scripta elegans) as an animal model system to study TSD. These researchers have demonstrated that male and female gonadal development are the result of separate genetic cascades influenced by steroid hormones, and that specific enzymes activate or inhibit steroid hormone effects on sex determination. In the red-eared slider system, female (or ovary) determination involves estrogens whereas male (or testis) determination involves nonaromatizable androgens. Mammals and turtles share a common evolutionary history and recent findings indicate that despite a difference in the trigger (environmental temperature vs. sex chromosomes), the red-eared slider shares many characteristics of sexual development with the mammalian system, including the same genes involved along the sex determination pathway. Experiments are designed to identify the genes involved in testis formation and then to determine how temperature and sex hormones accomplish this feat. For example, relative levels of gene expression will be measured using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Localization of gene expression will be analyzed with in situ hybridization. The ability to manipulate sex in a primitive vertebrate species by incubation temperature, exogenous hormones, and other agents provides unparalleled experimental control, thereby enabling more detailed analysis of the normal pattern of gene expression during sex determination than is possible with other amniotes (reptiles, birds and mammals are known as the amniote or higher vertebrates) having sex chromosomes. This work is important for several reasons. First, it has long been assumed that steroid hormones of maternal or embryonic origin are not involved in gonad formation in mammals and birds. The work with TSD reptiles indicates that this conclusion may be premature. Second, since TSD may represent the evolutionary precursor to sex chromosomes, potential temperature and steroid effects in sex determination may be present, but partly or wholly masked, in warm-blooded vertebrates. Third, temperature has not been adequately investigated as a factor in steroid hormone action at the genetic level in warm-blooded animals despite numerous studies documenting how both hormone responsiveness and hormone action are markedly dependent on temperature.