In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). How temperature both stimulates and inhibits genetic cascades to determine gonadal sex and channel sexual development is the focus of this application. The PI's working hypothesis is that incubation temperature modifies the endocrine microenvironment of the embryo such that steroid hormones serve as the proximate trigger for sex determination. This laboratory has developed the red-eared slider turtle as an animal model system and demonstrated that male and female gonadal development are separate pathways influenced by steroids, steroid-augmenting molecules, and steroid-interfering molecules. Female determination is caused by estrogens whereas male determination is caused by nonaromatizable androgens. Experiments are designed to determine if temperature accomplishes sex determination by acting on genes coding for steroid hormone receptors and aromafast, a key steroidogenic enzyme. The PI will continue the cloning and sequencing of the aromatase. To identify the patterns of expression and transcriptional regulation of selected specific genes during the critical periods of sex determination, the PI will utilize homologous antisense probes for estrogen receptor and aromatase of mENAs in in situ hybridization and ribonuclease protection assays at the beginning, during, and following the temperature-sensitive period. The ability to manipulate sex in TSD 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 amniote vertebrate species having sex chromosomes. Except for the trigger, the sex determining cascade appears to be similar between vertebrates having genotypic sex determination and those with TSD. The work on TSD draws attention t o the fact that temperature and steroid hormones can play a pivotal role in sexual development in an amniote vertebrate and is thus 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 homeotherms. Third, temperature has not been adequately investigated as a factor in steroid hormone action in homeotherms despite numerous studies documenting how both hormone responsiveness and hormone action are markedly dependent on temperature.
