To explore how chromosomes prepare for the meiotic divisions, we are examining the function of the C. elegans Myt1 ortholog. Myt1 belongs to the Wee1 family of kinases and is thought to down regulate Cdk1 during the cell cycle. RNAi studies with the Myt1 ortholog, wee-1.3, result in infertility. Mothers injected with dsRNA quickly become infertile;the oocyte chromosomes are no longer paused in diakinesis of meiosis I. These chromosomes have many hallmarks of being mitotic;they stain with a number of mitotic marker antibodies. Oocyte maturation also appears to be precocious. We propose that WEE-1.3 normally functions to keep maternal CDK-1 inactive during oogenesis, and that upon fertilization, CDK-1 becomes activated to allow for the meiotic and mitotic divisions of the embryo. In the absence of WEE-1.3, CDK-1 becomes precociously active and drives oocyte maturation and chromosome maturation in immature oocytes that are not fully differentiated. These oocytes fail to be fertilized presumably because they have not synthesized all the proper oocyte/embryo products they need for further development or because they have precociously triggered their block to polyspermy. We have recently constructed and expressed transgenes coding for WEE-1.3::GFP fusion proteins. These transgenes are expressed throughout the germline on the nuclear envelope, and reflect endogenous WEE-1.3 expression as they rescue a wee-1.3 null deletion mutant. These transgenic animals will be useful in identifying genes that regulate WEE-1.3 localization by screening genetic mutants or using RNAi for perturbations in the WEE-1.3::GFP expression pattern. To determine genes that interact with wee-1.3, we performed an RNAi suppressor screen utilizing 1874 embryonic lethal clones from the OpenBiosystems RNAi library. From this screen we identified 149 genes that when co-depleted with wee-1.3 result in a restoration of fertility. We have confirmed the identity of these suppressor genes and begun to characterize the mechanisms through which they suppress the sterility of wee-1.3 RNAi utilizing both RNAi approaches and available mutants. The strength of this approach is its potentiality for identifying novel components of both the cell cycle and oocyte meiotic maturation. We are currently focusing on the characterization of two specific genes that have been implicated in human disease, but that have not been previously known to function during oocyte maturation or germline development. The first gene, ETR-1, encodes an RNA-binding protein that has been previously characterized as being muscle-specific in the worm. The human homolog is implicated in myotonic muscular dystrophy and homologs in other organisms have been linked to fertilization defects. Notably, our data supports a novel role for ETR-1 in germline development and/or function. The second gene, PRX-5, encodes an ortholog of the human receptor for type 1 peroxisomal targeting signal protein (PXR1) which results in Zellweger syndrome or neonatal adrenoleukodystropy when mutated. Our data implicates both of these genes as playing previously undiscovered roles in oocyte maturation and how they function during meiosis is something that we are actively pursuing.
