Temperature--Not Chromosomes--Determines the Gender of Certain Lower Vertebrates

在许多产卵的爬行动物中，卵的孵化温度决定了后代的性别，这一过程被称为温度依赖性性别决定（TSD）。温度如何刺激和抑制基因级联反应来决定性腺性别和通道性发育是本应用程序的重点。PI的工作假设是，孵化温度改变了胚胎的内分泌微环境，使类固醇激素成为性别决定的直接触发因素。本实验室开发了红耳滑龟作为动物模型系统，并证明雄性和雌性性腺发育是受类固醇、类固醇增强分子和类固醇干扰分子影响的独立途径。雌性决定是由雌激素引起的，而雄性决定是由非芳香性雄激素引起的。实验旨在确定温度是否通过作用于类固醇激素受体和一种关键的类固醇生成酶的基因编码来完成性别决定。PI将继续进行芳香化酶的克隆和测序。为了确定在性别决定的关键时期选定特定基因的表达模式和转录调控，PI将在温度敏感期开始，期间和之后的原位杂交和核糖核酸酶保护试验中使用同源反义探针检测雌激素受体和mENAs的芳香化酶。在TSD物种中，通过孵育温度、外源激素和其他因素操纵性别的能力提供了无与伦比的实验控制，从而能够比其他具有性染色体的羊膜脊椎动物物种更详细地分析性别决定过程中基因表达的正常模式。除了触发因素外，具有基因型性别决定的脊椎动物和患有创伤后应激障碍的脊椎动物之间的性别决定级联似乎是相似的。对创伤后应激障碍的研究使人们注意到温度和类固醇激素在羊膜脊椎动物的性发育中起着关键作用，因此有几个重要的原因。首先，长期以来，人们一直认为母体或胚胎来源的类固醇激素与哺乳动物和鸟类的性腺形成无关。对创伤后应激障碍爬行动物的研究表明，这个结论可能还为时过早。其次，由于TSD可能代表性染色体的进化前体，在恒温动物中，性别决定的潜在温度和类固醇效应可能存在，但部分或全部被掩盖。第三，在恒温动物中，温度作为类固醇激素作用的一个因素还没有得到充分的研究，尽管有大量研究表明激素反应和激素作用都明显依赖于温度。

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.