9509017 Spotila The sex of a developing embryo is determined by the expression of specific gene sequences at defined times during embryogenesis. In mammals, birds and some reptiles the initiating signal for the expression of genes that lead to maleness or femaleness is the presence of the Y chromosome (mammals) or the W chromosome (birds, some reptiles). In other reptilian species, however, the sex of the developing embryo is determined by the incubation temperature during a critical time of gestation. This proposal will use the common slider turtle (Trachemys scripta) a species that is not endangered, is readily obtainable, and is representative of many turtle species in that a higher incubation temperature (about 32 C) results in female hatchlings and a lower incubation temperature (about 26 C) results in male hatchlings, to investigate the phenomenon of temperature-dependent sex determination at the molecular level. Specifically, eggs will be incubated in the laboratory at 26 C or 32 C and embryos will be sacrificed at about 2-3 weeks of gestation, the time period that is critical for formation of gonadal tissue. RNA will be isolated from gonadal tissue and analyzed for differences in the sequences expressed at the two temperatures by a technique called Differential Display Polymerase Chain Reaction. RNA species that are expressed at 26 C, but not at 32 C may be involved in the male developmental pathway. Conversely, RNA species found at 32 C, but not at 26 C may be involved in female-specific developmental processes. It is the goal of this one-year project to compile a "catalogue" of gene sequences that are candidates for further analysis as critical factors in temperature-dependent sex determination. Understanding of this ecological phenomenon is important not only for the conservation of endangered reptile species, but also for the insight that might be gained as to how sex-determining mechanisms function in general. ***
