Temperature-Dependent Male Sex Determination in the Red-Eared Slider Turtle

许多产卵的爬行动物缺乏性染色体，而是依靠卵的孵化温度来决定后代的性别，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，以确定性腺的类型和直接的性别分化是本应用的重点。本研究员及其同事将红耳滑龟（Trachemys scripta elegans）作为研究创伤后应激障碍的动物模型系统。这些研究人员已经证明，雄性和雌性性腺发育是受类固醇激素影响的单独遗传级联反应的结果，并且特定的酶激活或抑制类固醇激素对性别决定的作用。在红耳滑块系统中，雌性（或卵巢）的测定涉及雌激素，而雄性（或睾丸）的测定涉及非芳香化雄激素。哺乳动物和海龟有着共同的进化史，最近的研究表明，尽管触发因素（环境温度与性染色体）不同，但红耳滑龟与哺乳动物系统在性发育方面有许多共同的特征，包括在性别决定途径中涉及的相同基因。实验旨在确定与睾丸形成有关的基因，然后确定温度和性激素是如何完成这一壮举的。例如，将使用定量逆转录聚合酶链反应（RT-PCR）和Northern blot分析来测量基因表达的相对水平。基因表达的定位将通过原位杂交进行分析。在原始脊椎动物物种中，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动物（爬行动物、鸟类和哺乳动物被称为羊膜动物或高等脊椎动物）更详细地分析性别决定过程中基因表达的正常模式。这项工作之所以重要，有几个原因。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，在温血脊椎动物中，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，在温血动物的遗传水平上，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，尽管大量的研究记录了激素反应和激素作用是如何明显依赖于温度的。

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.