The ability to preserve spermatozoa and eggs for a long period of time is critical for retaining fertility, addressing infertility and lengthening the reproductive life span of individuals. Although spermatozoa survive freezing with relative ease, egg (oocyte) preservation is more complex because of the cell's large size, significant water content and fragile cellular skeleton. Current methods to preserve the oocyte are far from optimal, so there is a need to explore novel approaches. This proposal examines what happens when the immature oocyte nucleus, also known as the germinal vesicle, is isolated and dried alone as a potential means of genome preservation. Specifically, we will determine if a desiccated germinal vesicle (from any oocyte collected within the ovary) can be rehydrated and then placed into a fresh (previously enucleated) donor oocyte to reanimate and recover the original maternal genome. The 'reconstructed'oocyte then could be cultured and fertilized in vitro with appropriate spermatozoa to produce a viable embryo of a desired genotype. The general hypothesis is that desiccation and storage at room temperature can preserve the structural and functional integrity of the germinal vesicle. The germinal vesicle will be studied in the domestic cat because (1) this species is an important model for human health and reproduction, and (2) our preliminary data are extensive and highly promising. An important element of the proposed project is compacting the germinal vesicle to a small diameter before drying, which will ensure optimal protection during desiccation or storage while permitting easy transfer by injection into a surrogate oocyte. Our objective is to examine, for the first time, the impact of compaction, desiccation and storage at room temperature on the germinal vesicle, including its ability to reconstitute an oocyte that can undergo meiosis, fertilization and embryo development to term (i.e., production of healthy young). Early data have revealed that (1) in vitro compaction is an excellent protector of the germinal vesicle against stressors, (2) germinal vesicle transfer into a recipient oocyte is successful and (3) desiccation in the presence of sugar safeguards nuclear content. The project challenges existing paradigms about how to preserve female fertility in an area (germinal vesicle preservation and storage) that has never been explored, but shows strong promise. This innovative strategy could circumvent the complications associated with conventional inefficient, damaging and costly freeze-storage of whole oocytes in liquid nitrogen. The required studies also will substantially increase scholarly knowledge about complex interactions between the oocyte's nucleus and cytoplasm, including the mechanisms regulating developmental competence of the germinal vesicle.
(provided by the applicant): As will be described, the drying, storage and reanimation of the oocyte's germinal vesicle represent an enormous potential for preserving human fertility. This approach also could have widespread, practical application to more effective, economical preservation, propagation and management of rare animal models used to study human diseases. The ability to efficiently perpetuate these unique genotypes allows understanding mechanisms to improve human health through translational biomedical research.
|Holt, William V (2013) Who needs cytoplasm? Genomic preservation for the 21st century. Biol Reprod 88:140|
|Comizzoli, Pierre; Wildt, David E (2013) Mammalian fertility preservation through cryobiology: value of classical comparative studies and the need for new preservation options. Reprod Fertil Dev 26:91-8|
|Graves-Herring, Jennifer E; Wildt, David E; Comizzoli, Pierre (2013) Retention of structure and function of the cat germinal vesicle after air-drying and storage at suprazero temperature. Biol Reprod 88:139|