Progress 10/07/16 to 09/30/17
Outputs
Target Audience:Our results will be of direct interest to scientists and clinicians alike. Both human embryologists and animal scientists will benefit from the information thai is to emerge from these studies. Post-doctoral scientists, Ph.D. students and Veterinary students in the field of Animal Reproduction. Eventually, Dairy Cattle producers could benefit from incorporating novel protocols to prevent hormonal disruption of chromosome sgregation during super ovulation protocols and the finding of novel markers for oocyte quality and developmental potential. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Notably, until now, no experimental model has been described to determine the origin of complex chromosomal rearrangements. We have recently generated a novel transgenic mouse model demonstrating that the chromatin remodeling protein ATRX regulates the cellular response to chromosome instability in the early pre-implantation embryo. Importantly, our studies indicate that ATRX is conserved in human, bovine and equine oocytes. Here, we test the role of this critical epigenetic factor on genome integrity in the bovine embryo. Our long-term goal is to develop a physiologically relevant bovine embryo model to determine the mechanisms responsible for inducing chromosome instability during epigenetic reprogramming at the oocyte to embryo transition in vivo and in vitro. We test the hypothesis that ATRX is a novel epigenetic marker of bovine oocyte quality and developmental potential that regulates chromosome structure and function. With the support of our initial seed grant we have made rapid progress and successfully completed proof of principle experiments in our previous aims. Our newly developed methods and results provide strong support for our hypothesis and also lead to exciting new directions. For example, using microinjection of recombinant histone H2B protein for chromosome tracking, we have developed a system for live cell imaging and real-time analysis of chromosome segregation during bovine oocyte maturation . Importantly, we have validated the use of this system to detect both numerical and structural chromosome aberrations. Our system will prove invaluable to test next-generation in vitro oocyte maturation protocols to reduce pregnancy loss in cattle. Importantly, we have established novel methods for state of the art super resolution chromatin analysis in bovine oocytes and embryos. Using structured illumination super-resolution (SR-SIM) analysis and Stochastic Optical Reconstruction (STORM) we provide the first analysis of bovine oocyte chromatin with nano-scale resolution . SR-SIM and STORM are the two most powerful strategies that go beyond the diffraction barrier of even the most powerful conventional confocal microscopes to analyze sub-chromosomal compartments with a 100 nm and 30 nm structural resolutions, an unprecedented view of mammalian chromosomes that is already changing our understanding of genome dynamics in mammals. Our results provide the first evidence demonstrating a conserved centromeric localization of ATRX in bovine oocytes and suggest that ATRX is a critical and conserved epigenetic marker of oocyte quality and developmental potential in bovine oocytes and embryos. Moreover, using live cell imaging and super resolution chromatin analysis we provide novel evidence for a striking chromatin remodeling event in bovine oocytes that can be used as a potential marker for oocyte quality and meiotic competence. Super-resolution chromatin analysis revealed a striking nucleolar 'anchoring' of kinetochore proteins detected by the CREST antiserum and suggest the intriguing possibility that nucleolar association of kinetochore proteins may be important to coordinate chromosome-microtubule interactions and chromosome segregation during bovine oocyte maturation. These results have important implications for both human and veterinary medicine as recent studies indicate that kinetochore proteins may also be associated with the nucleolus in human cells. Our ongoing studies are focused on establishing the molecular mechanisms of chromosome instability and the biogenesis of translocations in the bovine pre-implantation embryo. Importantly, this research has provided essential preliminary data for an RO1 application NIH-USDA Dual purpose-dual benefit program for the use of agriculturally important domestic animals in biomedical research that has been selected for funding by USDA. Our studies are in direct response to one of the priority areas described by this funding opportunity: Identification of molecular mechanisms that regulate oocyte and embryo competency and validation of markers to determine oocyte-embryo quality and genome integrity. Elucidating the role of conserved chromatin remodeling proteins as epigenetic markers of oocyte quality and developmental potential is a pressing need and an issue of major significance for the rational design of clinical procedures to prevent pregnancy loss in domestic animals.
Publications
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