GENDER BIAS IN MAMMALIAN DNA REPLICATION DURING DEVELOPMENT

项目摘要 DNA复制是物种繁殖过程中最重要的事件。这一过程中的缺陷可能 导致疾病，包括出生(发育)缺陷和癌症。当DNA复制是 在遗传或环境上受到损害时，会出现一种称为“复制应激”(RESS)的状态 可能会导致上述有害后果。广泛的生物化学基础研究 DNA复制已经在单细胞生物体和培养细胞中进行，而不考虑 在哺乳动物等高等生物中可能存在的潜在性别差异。这样做的目的是 该项目旨在了解DNA复制的普遍过程如何受制于戏剧性的有性 哺乳动物胚胎发育中的二形性。初步研究发现，雌性小鼠胚胎 当MCM2-7的水平(微染色体维护)显著更容易致死时 2，3，4，5，6，7)DNA复制许可和解旋酶蛋白基因减少，解旋酶 动荡不安。随后的研究表明，偏向女性的致命性立即开始发生。 在性别决定后，与X-失活缺陷无关。转基因介导的转化 将女性胚胎移植给男性或注射睾丸素逆转了性别偏见的致命性， 说明这一现象与第二性征有关。进一步的实验 提示睾丸激素通过其抗炎活性使女性胚胎得以挽救， 布洛芬，一种非类固醇抗炎药(NSAID)， 也挽救了缺乏MCM的雌性胚胎。这个项目试图阐明细胞和 这个哺乳动物性别二型性的新例子的机制基础。目标一号将测试 在缺乏MCM的动物中，雌性偏见的胚胎致死是由于DNA的性别差异 复制本身，或对内在RESS的差异敏感性。目标2将检验这样的假设：女性 胚胎超敏反应与压力引起的炎症有关，而雄性胚胎受到保护。 由于睾丸激素的抗炎活性，它们在性别决定后产生高水平的睾丸激素。 目标3将探索初步数据的基础，这些数据表明性别偏见的一个关键因素 现象是母体环境；只有具有内在压力的水坝才会优先失去雌性 胚胎。这将通过将高危受精卵基因型转移到养母体内来完成 是野生型，缺乏MCM，或缺乏抗炎细胞因子IL-10。总的来说， 这些研究将影响我们对RESS、炎症和 与母体和/或胎儿炎症反应有关的不良妊娠结局 由基因或环境引起的。

Project Abstract DNA replication is the most essential event in the propagation of a species. Defects in this process can cause diseases including birth (developmental) defects and cancer. When DNA replication is compromised genetically or environmentally, a state known as “replication stress” (RESS) occurs that can lead to the aforementioned deleterious outcomes. Extensive basic research on the biochemistry of DNA replication has been conducted in single celled organisms and cultured cells, without regard for potential gender differences that may exist in higher organisms such as mammals. The goal of this project is to understand how the universal process of DNA replication is subject to dramatic sexual dimorphism in mammalian embryogenesis. Preliminary studies found that female mouse embryos were dramatically more prone to lethality when levels of the MCM2-7 (Minichromosome maintenance 2,3,4,5,6,7) DNA replication licensing and helicase proteins were genetically reduced and the helicase destabilized. Subsequent studies revealed that the female-biased lethality began occurring immediately after sex determination, and was not related to defects in X-inactivation.Transgene-mediated conversion of female embryos to males or testosterone administration reversed the gender-biased lethality, indicating that the phenomenon is related to secondary sexual characteristics. Further experiments suggested that testosterone enabled female embryo rescue by virtue of its anti-inflammatory activity, a possibility supported by the observation that ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), also rescued MCM-deficient female embryos. This project seeks to elucidate the cellular and mechanistic basis of this novel example of mammalian sexual dimorphism. Aim 1 will test whether female- biased embryo lethality in MCM-depleted animals is due to gender differences in DNA replication per se, or differential sensitivity to intrinsic RESS. Aim 2 will test the hypothesis that female embryo hypersensitivity is related to RESS-induced inflammation, while males embryos are protected by the anti-inflammatory activity of testosterone they produce in high levels following sex determination. Aim 3 will explore the basis of preliminary data showing that a key contributing factor in the sex bias phenomenon is the maternal environment; only dams with intrinsic RESS preferentially lost female embryos. This will be accomplished transferring at-risk genotypes of zygotes into foster mothers that are genotypically wild-type, MCM-deficient, or deficient for the anti-inflammatory cytokine IL-10. Overall, these studies will can impact our understanding of the relationships between RESS, inflammation, and adverse pregnancy outcomes related to maternal and/or fetal inflammatory responses that are genetically or environmentally induced.