A fundamental gap exists in understanding the role of RNP granule dynamics during the decline of oocyte quality that occurs in old-aged or meiotically-arrested oocytes. Without a better understanding of the regulation of oocyte quality in old-aged oocytes, the ability to assist a larger number of women with age-related infertility problems will be hampered. The long-term goal is to identify the critical mechanisms that maintain oocyte quality when fertilization is delayed, as for old-aged or meiotically- arrested oocytes. The primary objectives of this application are to identify direct regulators of RNP granule assembly and dissociation and to determine the molecular function of RNP granules. The central hypothesis of this proposal is that RNP granule-mediated regulation of mRNA metabolism promotes oocyte quality. This hypothesis has been formulated based on exciting preliminary data produced in the PI's lab. The overall approach is to investigate germ line RNP granule dynamics and function using the nematode C. elegans. The approach is innovative because it takes advantage of a multicellular system with naturally-occurring RNP granule assembly and dissociation in oocytes. Preliminary results from an RNAi screen identify 143 candidate regulators of RNP granule assembly that provide an excellent foundation for these studies.
The first aim i s to identify direct regulators of RNP granule assembly from among the 143 candidates. Maturation rate assays and immunofluorescence experiments will be used to identify regulators of RNP granule assembly that do not also affect oocyte meiotic maturation rates, and those that control the localization of multiple RNP granule components.
The second aim i s to determine the relative roles of the microtubule and actin cytoskeleton networks in regulating RNP granule assembly and dissociation. Live-imaging experiments will examine the cytoskeleton as RNP granules assemble and dissociate. A targeted analysis of microtubule motor protein mutants and WASP complex mutants will identify additional regulators of RNP granule assembly.
The third aim i s to determine if RNP granules promote oocyte quality by modulating mRNA metabolism. mRNA levels, transcriptional activation, mRNA polyadenylation, and de- repression of translation will be assayed when RNP granule assembly is disrupted. Taken together, these studies will provide insight into the mechanisms of RNP granule assembly, dissociation, and function. This project is significant because it tests the novel, central hypothesis that RNP granule components play a protective role in maintaining the quality of meiotically arrested oocytes by modulating mRNA metabolism. Due to the strong conservation of RNP granule components, the results are likely to have far-reaching impact in invertebrates and vertebrates. It is expected the results will have relevance to understanding the basis for infertility that occurs as women age.
The proposed project is relevant to human health because the results will lay a foundation for understanding the basis for infertility and likelihood of havng children with developmental disorders that increases as women age. The broad goal of this project is to identify the mechanisms used by eggs (oocytes) to maintain their integrity when they must wait a prolonged period to be fertilized. The proposed experiments test the hypothesis that large RNP granules contribute to oocyte quality by regulating mRNAs that localize to them.