Mechanisms of Sex Determination in Zebrafish

描述（由申请人提供）：人类生殖健康取决于性腺的发育，而性腺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启动了一个尚不完全了解的网络，该网络指导性腺成为睾丸而不是卵巢。在非哺乳脊椎动物中，网络中的其他基因决定性别，在某些物种中，环境因素，如温度或社会线索，可能会影响性发育。该项目的总体目标是了解基因因素如何与环境相互作用，从而使两性之间的平衡偏向一方。研究的重点是斑马鱼，这是一种遗传上易于控制的脊椎动物，其性别决定很容易向一个方向或另一个方向倾斜。斑马鱼性腺最初发育为双电位器官，所有幼鱼中都有少量原始生殖细胞最初发育为卵母细胞。在一些个体中，卵母细胞死亡，性腺变成睾丸；在其他情况下，卵母细胞存活下来，性腺变成卵巢。一个关键的问题是，什么样的遗传或环境信号会导致卵母细胞在某些个体中死亡，而在另一些个体中存活？研究这个问题的一个重要工具是，基因中的一种隐性突变会导致女性向男性的性别逆转。初步实验表明，纯合突变的幼年性腺中存在过多的细胞死亡，并提出以下假设：1)环境和遗传因素影响幼年卵母细胞的存活，从而2)改变卵母细胞来源的信号强度，从而促进周围体细胞的形成；3)芳香化酶的产生，这种酶能将睾丸激素转化为雌激素，从而保护卵母细胞，使性腺倾向于雌性；5)由于假设的卵母细胞来源信号数量不足，性腺变成了睾丸。目的1是确定F2定位杂交中与性别表型相关的遗传因素。目标2是通过比较斑马鱼和medaka（一种具有遗传性别决定的物种）的全雄性种群与含有雌性的兄弟种群（Aim 2a）的转录组，对性别特异性表达基因进行全基因组搜索，并研究候选性别决定基因、类固醇受体基因和细胞外信号基因的表达模式（Aim 2b）。目的3是通过了解阻断维黄酸信号进入减数分裂是否会导致性别逆转（目的3a）以及原始生殖细胞数量是否控制斑马鱼和medaka的性别发育（目的3b）来测试该假设的其他组成部分。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（medaka），这项工作具有重要意义，因为它有可能识别新的基因和新的基因功能。结果将有助于更好地了解脊椎动物性别平衡机制的分子遗传学性质，从而有助于我们了解当前人类生殖疾病增加的可能机制，包括睾丸发育不良综合征和多囊卵巢综合征，这些疾病在发达国家越来越多地观察到。

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.