Sexually reproducing organisms generate gametes through meiosis, the process by which the genetic material of the cell is halved to form a haploid sperm or egg. Although essential for all animal life, meiosis is strikingly error-prone, with an estimated 5-25% of all human conceptions resulting in an aneuploid embryo, often leading to severe developmental defects and pregnancy complications that effect millions of individuals every year. Therefore, it is critical to investigate the core molecular mechanisms that define progression through meiosis to further understand what checkpoints may exist to sense errors. This proposal investigates the control of
meiotic progression using two powerful meiotic models, the lab mouse Mus musculus and the nematode Caenorhabditis elegans. Meiosis I proceeds through pairing, synapsis, and crossing over (CO) of homologous chromosomes during prophase, and only once these events have occurred correctly can homologs align appropriately on the meiotic spindle and then segregate equally at the first meiotic division. However, it is unclear how successful CO formation is sensed by cell cycle machinery to progress meiosis into M-phase. Proper CO formation requires orthologous cyclin-like domain containing proteins CNTD1, in mouse spermatocytes, and COSA-1, in C. elegans oogenesis. Interestingly, CNTD1 interacts with CDC34, a ubiquitin E2 conjugating enzyme that acts with the Skp1-Cullin-F-Box (SCF) family of E3 ubiquitin ligases. A known target of CDC34 in mitotic systems is the cell cycle M-phase inhibitor, WEE1. In CNTD1 knockout mouse spermatocytes, CDC34 is absent from nuclei and WEE1 persists aberrantly through until the end of meiotic prophase, suggesting a role for CNTD1-promoted crossover maturation in WEE1 degradation and resulting progression into M-phase. This leads to the hypothesis that progression from meiotic prophase I to M-phase I is licensed through direct WEE1 ubiquitination by SCFCDC34 in a crossover-dependent manner that is conserved across eukaryotes. This proposal presents two specific aims: 1) Is SCFCDC34-mediated ubiquitination of WEE1 critical for progression from meiotic prophase I to M-phase I? WEE1 ubiquitination and WEE1/CDC34 chromatin localization will be directly tested in both wild type and crossover deficient mutant mice. An assay will be developed to directly visualize the germ line abundance of the WEE1 ortholog in C. elegans, WEE-1.3, to test the role of WEE-1.3 in C. elegans meiotic progression. 2) What is the biochemical mechanism of SCFCDC34 mediated WEE1 ubiquitination? The molecular components of the SCFCDC34 complex will be elucidated in mouse spermatocytes through mutant analysis of putative complex members, and in C. elegans through a RNAi screen. This work will be performed in the lab of Dr. Paula Cohen in the Cornell University College of Veterinary Medicine, providing extensive training in mammalian reproductive biology, specifically in the early meiotic events during gametogenesis. This research strategy coupled with the applicant’s training and career development plans will provide a unique approach towards investigation of meiosis through using complementary model organisms