Mechanisms of Sex Determination in Zebrafish

说明(申请人提供)：人类生殖健康有赖于性腺发育，而性腺发育在子宫内可能受到未知遗传和环境因素的不利影响。在哺乳动物中，Y染色体基因SRY启动了一个不完全被理解的网络，该网络引导性腺成为睾丸而不是卵巢。在非哺乳动物脊椎动物中，网络中的其他基因决定性别，在某些物种中，温度或社会线索等环境因素可能会偏向性别发育。这个项目的广泛目标是了解遗传因素如何与环境相互作用，使天平倾向于某一性别。研究的重点是斑马鱼，这是一种基因易驯化的脊椎动物，性别决定可以很容易地倾向于一个或另一个方向。斑马鱼的性腺最初发育为双潜能器官，所有幼鱼的原始生殖细胞最初都发育为卵母细胞。在一些个体中，卵母细胞死亡，性腺变成睾丸；在另一些个体中，卵母细胞存活，性腺变成卵巢。一个关键的问题是，是什么遗传或环境信号导致假定的卵母细胞在一些个体中死亡，在另一些个体中存活？探索这一问题的一个重要工具是FANCL中的一种隐性突变，它导致了女性到男性的性别逆转。初步实验表明，在纯合突变的幼年期性腺中存在过度的细胞死亡，并提出了以下假设：1)环境和遗传因素影响幼年期卵母细胞的存活，从而2)改变卵母细胞来源的信号的强度，该信号在周围的体细胞中促进3)芳香化酶的产生，4)芳香化酶的产生，4)将睾酮转化为雌激素的酶，后者保存卵母细胞，并使性腺偏向女性命运；5)假设的卵母细胞来源的信号量不足，性腺就变成了睾丸。目的1是在F2作图杂交中鉴定与性别表型相关的遗传因素。目的2是通过比较具有遗传性别决定的斑马鱼和青竹的全雄性种群和含有雌性的同胞种群(目标2a)，在全基因组范围内寻找性别特异表达的基因，并研究候选性别决定基因、类固醇受体基因和细胞外信号转导基因的表达模式(目标2b)。目标3是通过了解阻止维甲酸信号进入减数分裂是否会导致性别逆转(目标3a)以及原始生殖细胞数量是否控制斑马鱼和青蛙的性发育(目标3b)来检验该假说的其他组成部分。这项拟议的工作对于通过比较具有性别分化微妙平衡的脊椎动物(斑马鱼)和具有已知遗传雄性决定因素的脊椎动物(青海)来识别新基因和新基因功能的潜力具有重要意义。研究结果将有助于更好地理解脊椎动物性别平衡机制的分子遗传学本质，从而有助于我们理解目前人类生殖疾病增加的可能机制，包括在发达国家越来越多地观察到的睾丸发育不良综合征和多囊卵巢综合征。 公共卫生相关性：了解打破男女性别决定平衡的生物学机制，对于了解最近源于子宫的人类生殖疾病的增加至关重要，这些疾病包括男性的不孕不育和睾丸癌以及女性的多囊卵巢综合征。某些基因构成可能特别容易受到破坏。对具有精细平衡的性别决定机制的脊椎动物物种的研究可能会让我们深入了解环境和遗传因素如何打破人类的性别决定平衡。虽然斑马鱼性腺发育的许多特征与人类相似，但斑马鱼的性别决定更不稳定；此外，在斑马鱼中，可以使用遗传工具来剖析性别决定的机制，这应该确定与人类生殖健康相关的新基因和新基因功能。

DESCRIPTION (provided by applicant): Human reproductive health depends on gonad development, which can be adversely affected in utero by unknown genetic and environmental factors. In mammals, the Y-chromosome gene SRY initiates an incompletely understood network that directs the gonad to become a testis rather than an ovary. In nonmammalian vertebrates, other genes in the network determine sex, and in some species, environmental factors such as temperature or social cues can bias sex development. This project's broad objective is to learn how genetic factors, interacting with the environment, can tip the balance towards one sex or another. Work focuses on zebrafish, a genetically tractable vertebrate in which sex determination can be easily tipped in one direction or the other. The zebrafish gonad initially develops as a bipotential organ, with a few primordial germ cells in all juveniles initially developing as oocytes. In some individuals, oocytes die and the gonad becomes a testis; in others, oocytes survive and the gonad becomes an ovary. A key question is, what genetic or environmental signals cause presumptive oocytes to die in some individuals and to survive in others? An important tool to probe this question is a recessive mutation in fancl that causes female-to-male sex reversal. Preliminary experiments show excess cell death in homozygous mutant juvenile gonads and suggest the hypothesis that 1) environmental and genetic factors affect oocyte survival in juveniles, and thereby 2) alter the strength of an oocyte-derived signal that promotes, in surrounding somatic cells, 3) the production of aromatase, the enzyme that 4) converts testosterone to estrogen, which preserves oocytes and biases the gonad toward a female fate; 5) with insufficient quantities of the hypothesized oocyte-derived signal, the gonad becomes a testis. Aim 1 is to identify genetic factors linked to sex phenotype in F2 mapping crosses. Aim 2 is to conduct a genome-wide search for genes expressed sex specifically by comparing transcriptomes of all-male populations of zebrafish and medaka, a species with genetic sex determination, to female-containing sibling populations (Aim 2a) and to investigate expression patterns of candidate sex determination genes, steroid receptor genes, and extracell signaler genes (Aim 2b). Aim 3 is to test additional components of the hypothesis by learning whether blocking the retinoic acid signal for entry into meiosis can cause sex reversal (Aim 3a) and whether primordial germ cell number controls sex development in zebrafish and medaka (Aim 3b). The proposed work has significance for it's potential to identify new genes and new gene functions by comparing a vertebrate with a delicate balance for sex differentiation (zebrafish) to one with a known genetic male determinant (medaka). Results will contribute to a better understanding of the molecular genetic nature of the vertebrate sex-balance mechanism, and thus contribute to our understanding of possible mechanisms for the current increase in human reproductive disease, including testicular dysgenesis syndrome and polycystic ovary syndrome increasingly observed in developed countries. PUBLIC HEALTH RELEVANCE: Understanding the biological mechanisms that tip the balance of sex determination between male and female is essential to understand recent increases in human reproductive disorders originating in the womb, including sterility and testis cancer in men and polycystic ovary syndrome in women. Certain genetic make- ups may be especially prone to disruption. Investigations of vertebrate species with finely balanced sex determination mechanisms may provide insight into how environmental and genetic factors tip the sex determination balance in humans. Although many features of zebrafish gonad development are similar to those in humans, zebrafish sex determination is more labile; furthermore, in zebrafish, genetic tools are available to dissect the mechanisms of sex determination, which should identify new genes and new gene functions relevant for human reproductive health.