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Genotoxic effects of L1 retrotransposition trigger oocyte elimination during MPI

L1 逆转录转座的基因毒性作用触发 MPI 期间卵母细胞的消除

基本信息

批准号：
9262757
负责人：
Marla Elizabeth Tharp
金额：
$ 4.4万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9262757
关键词：
ATM function Affect Attenuated Automobile Driving Birth Cell Death Cells Complementary DNA DNA DNA Damage DNA Double Strand Break DNA damage checkpoint Dangerousness Data Developmental Process Elements Embryonic Development Ensure Family Planning Female Fertility Future Genetic Recombination Genomic Instability Health Planning Human Hybrids Influentials Longevity Meiosis Meiotic Prophase I Meiotic Recombination Messenger RNA Methods Molecular Monitor Mus Nucleosides Oocytes Ovarian Ovary Pachytene Stage Phosphotransferases Population Process Production RNA RNA-Directed DNA Polymerase Reaction Reproductive Health Retrotransposition Retrotransposon Reverse Transcriptase Inhibitors Reverse Transcription Ribonucleases Role Signal Transduction Site System Testing Woman Work Zidovudine Zygonema ataxia telangiectasia mutated protein checkpoint kinase 2 endonuclease fetal genetic manipulation genotoxicity improved in vivo mouse model mutant mouse model prevent public health relevance reproductive response sensor small molecule inhibitor transposon/insertion element

项目摘要

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.