Genotoxic effects of L1 retrotransposition trigger oocyte elimination during MPI

 描述（由申请方提供）：胎儿卵母细胞磨损（FOA）的神秘过程导致小鼠出生前选择性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设来解释这种大量的卵母细胞损失，但其潜在的机制仍然未知。LINE-1反转录转座子活性的作用最近已在FOA中描述（Malki等人，2014），表明卵母细胞中L1表达增加与其优先消除相关。这些数据表明，L1反转录转座的过程导致了引发FOA的遗传毒性效应。 为了确定这种触发因素，我们将考虑L1 ORF 2 p的内在逆转录酶（RT）活性。已经表明，通过使用核苷RT抑制剂AZT阻断RT活性，FOA大大减弱，这表明L1逆转录的中间体- RNA：DNA杂合体和ssDNA -对细胞是危险的。我将开发一种方法来检测这些中间体，并使用不能水解RNA：DNA杂交体的突变小鼠模型来确定FOA的驱动因素。在MPI中，已知DNA损伤反应（DDR）感测程序性DNA断裂并在粗线中期DNA损伤检查点消除具有不可修复损伤的卵母细胞。由于我们知道FOA主要发生在MPI的早期阶段（细丝体和接合丝体），因此我们提出DDR机制也可以检测来自L1 TPRT的信号作为危险信号，并在典型的粗丝体中期检查点之前剔除具有过量L1 TPRT中间体或TPRT相关损伤的卵母细胞。我打算通过独立地破坏DNA断裂的核心传感器ATM激酶和下游效应检查点激酶2的活性，并在MPI的早期阶段监测对DDR和卵母细胞数量的下游信号传导的影响，从而在FOA中牵连DDR。我们的研究结果将揭示一个新的功能的DDR在卵母细胞消除在MPI的早期阶段，而不仅仅是在粗线期中期检查点。此外，通过确定FOA的遗传毒性触发因素，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力具有重大影响。

 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.