Genotoxic effects of L1 retrotransposition trigger oocyte elimination during MPI

 描述(由申请人提供)：胎儿卵母细胞磨损(FOA)的神秘过程负责在小鼠出生前选择性地消除超过三分之二的减数分裂前期I(MPI)卵母细胞。虽然已经考虑了许多假说来解释这种巨大的卵母细胞丢失，但其潜在的机制仍然不清楚。LINE-1反转录转座子活性的作用最近在FOA(Malki等人，2014)中被描述，因为它表明卵母细胞中L1表达的增加与它们的优先消除相关。这一数据表明，L1逆转录转座的过程导致了触发FOA的遗传毒性效应。 为了确定这个触发因素，我们将考虑L1 ORF2p的内在逆转录酶(RT)活性。研究表明，通过使用核苷类RT抑制剂AZT来阻断RT活性，FOA大大减弱，这表明L1逆转录的中间产物-RNA：DNA杂交物和单链DNA-对细胞是危险的。我将开发一种方法来检测这些中间体，并使用无法降解RNA：DNA杂交物的突变小鼠模型来识别哪个是FOA的驱动因素。在MPI中，DNA损伤反应(DDR)被认为是感知程序性DNA断裂，并在厚膜中期DNA损伤检查点消除不可修复的损伤的卵母细胞。由于我们知道FOA主要发生在MPI的早期阶段(瘦体和合体)，我们建议DDR机制也可以检测到来自L1 TPRT的信号作为危险信号，并在典型的厚壁中期检查点之前淘汰具有过多L1 TPRT中间体或TPRT相关损害的卵母细胞。我打算通过独立干扰DNA断裂核心传感器、ATM激酶和下游效应检查点激酶2的活性来牵涉到DDR在FOA中的作用，并在MPI的早期阶段监测DDR对下游信号和卵母细胞数量的影响。我们的发现将揭示DDR在MPI早期阶段消除卵母细胞中的新功能，而不仅仅是在粗线期中期检查点。此外，通过确定FOA的基因毒性触发因素，我们在理解FOA方面取得了重大进展，这对人类生殖健康和生育具有重大影响。

 DESCRIPTION (provided by applicant): The enigmatic process of fetal oocyte attrition (FOA) is responsible for the selective elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth in mice. While numerous hypotheses have been considered for explaining this massive oocyte loss, the underlying mechanism remains unknown. A role for LINE-1 retrotransposon activity has been recently described in FOA (Malki et al., 2014) upon showing that increased L1 expression in oocytes correlates with their preferential elimination. This data suggests that the process of L1 retrotransposition results in a genotoxic effect that triggers FOA. To identify this trigger, we will consider the intrinsic reverse transcriptase (RT) activity of L1 ORF2p. It has been shown that by blocking RT activity using the nucleoside RT inhibitor AZT, FOA is greatly attenuated, suggesting that intermediates of L1 reverse transcription - RNA:DNA hybrids and ssDNA - are dangerous to the cell. I will develop a method to detect these intermediates and identify which is the driver of FOA using a mutant mouse model that fails to hydrolyze RNA:DNA hybrids. In MPI, the DNA damage response (DDR) is known to sense programmed DNA breaks and eliminates oocytes with irreparable damage at a mid-pachynema DNA damage checkpoint. Since we know that FOA occurs primarily in earlier stages of MPI (leptonema and zygonema), we propose that DDR machinery can also detect signals from L1 TPRT as dangerous and cull oocytes with excess L1 TPRT intermediates or TPRT- associated damage prior to the canonical mid-pachynema checkpoint. I intend to implicate the DDR in FOA by independently disrupting activity of the core sensor of DNA breaks, ATM kinase and downstream effector checkpoint kinase 2 and monitor effects on downstream signaling of the DDR and oocyte number in early stages of MPI. Our findings will uncover a new function of the DDR in oocyte elimination during early stages of MPI rather than solely at the mid-pachytene checkpoint. Further, by identifying the genotoxic trigger of FOA, we are making significant advances in our understanding FOA that have great implications for human reproductive health and fertility.