Jonathan L. Tilly - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

PROJECT SUMMARY Based on the well-documented relationship that exists between ovarian function and women?s health, for decades hormone therapy was considered by many as the standard-of-care for the management, and even the prevention, of post-menopausal health complications. However, recent clinical trials have raised concerns over hormone therapy and increased risks for coronary heart disease, stroke, blood clots, and cancer. While the general thinking behind hormone therapy ? viz. provision of ovarian hormones lost at menopause would serve as a ?replacement? for failed ovarian function ? seems reasonable, it is perhaps not surprising that replacement of only one (estrogen) or two (estrogen-progestin) of the multitude of bioactive factors produced by the ovaries during reproductive life would ultimately prove insufficient. We have been working on the idea that ?ovarian therapy? would serve as a far better ?replacement? for alleviating health consequences of failed ovarian function with age. We eventually generated a mouse model that maintains an adequate reserve of oocyte-containing follicles, and an ensuing continuation of ovarian function, well into advanced chronological ages. Long-term follow-up work showed this strategy indeed yielded immense health benefits in aging females without an increase in cancer in any tissue. While we were excited to demonstrate this key proof-of-concept in mammals, there was a major downside: the ?gene knockout? approach used was not amenable for translation to women. Nonetheless, the work underscored the importance of continuing efforts to elucidate mechanisms underlying endowment and depletion of oocyte-containing follicles in the context of ovarian aging. This led us to discover the existence of female germline or oogonial stem cells (OSCs) in mice and then in humans, and the role these cells play in supporting ovarian function during adulthood. Of the many new areas this paradigm shift opened, one of the most exciting revolves around the use of regenerative medicine to extend functional ovarian lifespan into later ages of life. Achieving this, however, will require a detailed understanding of cues that drive OSC differentiation, and how aging impacts on these cues such that the ability of ovaries to sustain their oocyte reserves becomes compromised with age. To this end we found that application of mechanical ?strain? to OSCs activates their differentiation into oocytes. We also found that ovarian matrix proteins, which directly influence biomechanical properties of tissue, not only decline with age but also serve as direct activators of OSC differentiation. Here we have designed a number of in-vitro and in-vivo studies to rigorously test the hypothesis that progressive loss of mouse and human OSC function in ovaries with age is directly tied to a reduction in biomechanical stimulatory signaling occurring concomitant with aging-associated changes in matrix proteins. Completion of these studies will both support our central hypothesis as well as more broadly highlight the significance of mechanotransduction to adult stem cell function in the context of organ failure with age.

The principal function of mitochondria is the generation of cellular energy (ATP) by oxidative phosphorylation using proteins encoded for by both the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA) to assemble the machinery needed for mitochondrial respiration. Paradoxically, mtDNA is a macromolecular target of reactive oxygen species (ROS), which are mutagenic by-products generated during ATP production. Unlike nuclear DNA, which has a high-level of proofreading, error detection and correction that coordinately function to prevent passage of harmful DNA mutations to offspring, mtDNA mutations are not subject to the same degree of detection and correction. Indeed, past studies have shown that mtDNA sustains a high mutational burden that progressively increases in cells with age. This paradigm raises a fundamentally critical question when one considers that mitochondrial passage from one generation to the next is uniparental through the female germline (egg): how are detrimental mtDNA mutations that accumulate in maternal germ cells over time prevented from being passed to the next generation, the generation after that, and so on? Two distinct, but likely interrelated, processes have been offered to explain this phenomenon ? the mtDNA bottleneck and germline-purifying selection. However, large gaps in knowledge still exist regarding both. For example, several different mechanisms have been proposed for how the mtDNA bottleneck works ? none of which have been proven, and its position in germline development is debated. Likewise, it is not known where germline-purifying selection takes place relative to the bottleneck or even if it depends on the bottleneck. We recently developed an innovative technology combining PACBIO third-generation sequencing with unique molecular identifiers, which enables high-fidelity mutational analysis of entire individual mtDNA molecules. With this in hand, we are now uniquely positioned to map how transgenerational passage of high-quality mtDNA molecules is accomplished. To do this, we propose the following Specific Aims (SAs). SA1: Define the structure and dynamics of the mtDNA bottleneck. We will build detailed phylogenetic trees of mtDNA molecules within individual germline cells, and compare these trees to those simulated from proposed bottleneck models. SA2: Determine the timing and mechanisms of germline-purifying selection. We will compare ?synonymity? of mutations from phylogenetic tree branches to identify when purifying selection occurs, and how it interfaces with the bottleneck. SA3: Determine if individual mtDNA molecules carrying no/low versus high mutational burdens are preferentially allocated during early (preimplantation) embryogenesis. We will evaluate mtDNA genealogies in individual blastomeres of preimplantation embryos at the 2-, 4- and 8-cell stages, as well as in the inner cell mass (embryo proper) and trophectoderm (extraembryonic) of blastocyst-stage preimplantation embryos, to determine if there exists evidence of differential allocation of mtDNA mutations.