Faithful transmission of the genome through gametes is critical for fertility, health of offspring, and success of the species. Errors in meiosis can lad to aneuploidy, chromosome aberrations, or gene mutations. The vast majority of spontaneous abortions due to aneuploidy are traceable to Meiosis I defects that occur during oogenesis. Although "checkpoint" mechanisms exist to stimulate repair of meiotic errors and eliminate irreparably defective meiocytes, their molecular identities and mechanisms are poorly characterized or unknown in humans or mammals. We have used genetic analyses of mice to identify the key checkpoint responsible for responding to DNA double strand breaks (DSBs) in oocyte meiosis. DSBs are enzymatically induced at the beginning of meiosis in order to stimulate meiotic recombination - a process essential for proper chromosome segregation upon cell division. Failure to repair the DSBs triggers a checkpoint that results in oocyte elimination. We identified checkpoint kinase 2 (CHK2;CHEK2) as an essential component of this response. Startlingly, female mice that are oocyte-deficient from a mutation that disrupts DSB repair can have their fertility restored by concurrent mutation of Chk2, yielding apparently normal offspring. We also found that both programmed and induced DSBs trigger CHK2-dependent phosphorylation of p63 (TRP63) in diplotene oocytes. These and other data establish CHK2 as essential for DNA damage surveillance in female meiosis, and we propose that the DNA damage checkpoint pathway is: ATR>CHK2>p63. However, Chk2 is not essential for elimination of DSB-bearing spermatocytes, indicating sexual dimorphism. This proposal builds on these findings with the following basic and translational objectives: 1) To validate and mechanistically characterize putative upstream (ATR) and downstream (p63) components of the oocyte DNA damage checkpoint using conditionally mutant mice and molecular analyses;2) Determine the identity of the male DNA damage checkpoint;3) Determine the fate of DSBs in recombination-defective, rescued oocytes by whole genome sequencing of offspring and genetic analyses of potential alternative DNA repair pathways. This is important for assessing the safety of checkpoint inhibition for reasons of assisted reproduction or fertility preservation in cancer patients;and 4) Determine if CHK2 drug inhibitors can protect oocytes from death due to cancer treatments, and in a manner that doesn't cause birth defect-causing mutations. In sum, this project will elucidate the genetic quality control mechanisms operate in mammalian gametes. Arguably, this is one of the most important processes for the health and success of our species and others.

Public Health Relevance

Meiosis is the critical process leading to formation of sperm and eggs. Errors that occur in meiosis, primarily those involving the proper behavior of chromosomes during this process, underlie birth defects, subfertility/sterility and failed pregnancies. This project studies the molecular identity of genetic quality control mechanisms called checkpoints that normally guard against production of defective eggs and sperm, and will delineate how these mechanisms function. We will exploit this knowledge to identify inhibitors of these checkpoints that can be used to protect the eggs of female cancer patients from chemotherapies that would otherwise destroy the eggs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM045415-23
Application #
8579452
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Janes, Daniel E
Project Start
1992-02-01
Project End
2017-07-31
Budget Start
2013-09-01
Budget End
2014-07-31
Support Year
23
Fiscal Year
2013
Total Cost
$314,062
Indirect Cost
$107,812
Name
Cornell University
Department
Other Basic Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Bolcun-Filas, Ewelina; Rinaldi, Vera D; White, Michelle E et al. (2014) Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway. Science 343:533-6
Luo, Yunhai; Hartford, Suzanne A; Zeng, Ruizhu et al. (2014) Hypersensitivity of primordial germ cells to compromised replication-associated DNA repair involves ATM-p53-p21 signaling. PLoS Genet 10:e1004471
Qiao, Huanyu; Prasada Rao, H B D; Yang, Ye et al. (2014) Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination. Nat Genet 46:194-9
Schimenti, Kerry J; Feuer, Sky K; Griffin, Laurie B et al. (2013) AKAP9 is essential for spermatogenesis and sertoli cell maturation in mice. Genetics 194:447-57
Li, Xin Chenglin; Bolcun-Filas, Ewelina; Schimenti, John C (2011) Genetic evidence that synaptonemal complex axial elements govern recombination pathway choice in mice. Genetics 189:71-82
Bolcun-Filas, Ewelina; Bannister, Laura A; Barash, Alex et al. (2011) A-MYB (MYBL1) transcription factor is a master regulator of male meiosis. Development 138:3319-30
Chuang, Chen-Hua; Wallace, Marsha D; Abratte, Christian et al. (2010) Incremental genetic perturbations to MCM2-7 expression and subcellular distribution reveal exquisite sensitivity of mice to DNA replication stress. PLoS Genet 6:e1001110
Ward, Jeremy O; Reinholdt, Laura G; Motley, William W et al. (2007) Mutation in mouse hei10, an e3 ubiquitin ligase, disrupts meiotic crossing over. PLoS Genet 3:e139
Shima, Naoko; Alcaraz, Ana; Liachko, Ivan et al. (2007) A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nat Genet 39:93-8
Browning, V L; Chaudhry, S S; Planchart, A et al. (2001) Mutations of the mouse Twist and sy (fibrillin 2) genes induced by chemical mutagenesis of ES cells. Genomics 73:291-8

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