SUN-479 CENP-A Levels as a New Biomarker for Normal Embryo Development in Mice.

Abstract E.L. Ferrell: None. G. Manske: None. C.A. Tower: None. S. Hammoud: None. Objective: The centromere region functions as an organizing node for spindle formation for faithful chromosome segregation in mitosis and meiosis. While aneuploidies are thought to occur in somatic tissues once in every 50 division, incidences of aneuploidy occur much more frequently during meiosis and early embryonic development. An estimated 30-50% of preimplantation stage embryos are aneuploid and about 50% of first-trimester spontaneous miscarriages are due to these aneuploidies. Previous studies have found centromeres to contain a histone H3 variant named centromere protein A (CENPA). The objective of this study is to better understand the role of CENPA function in preimplantation embryos as a potential predictor of aneuploidy. Materials & Methods: A mouse model was selected and CRISPR/Cas9 was used to tag endogenous CENPA locus with red fluorescent protein, mScarlet. These transgenic lines allowed us to monitor CENPA dynamics both in gametes and through fertilization to cell division. Monitoring and quantification of dynamics, distribution, and abundance at centromere was performed using Fiji/ImageJ software. A series of molecular and genetic techniques such as knockdown, transcription, translation, and DNA replication inhibitors were used to determine the role of CENPA and impact of CENPA levels. Results: When comparing CENPA levels across gametogenesis, we observed a number of interesting observations: 1) the germline stem cell precursors in male have significantly higher levels of CENPA as compared to testis somatic cells or adult stem cells, but the levels of CENPA are significantly decreased upon germline stem cell commitment to meiosis - as a result, mature sperm cell genome retains ∼12% of the original CENPA present in the germline stem cell. 2) Unlike the decrease in CENPA observed during spermatogenesis, the level of CENPA in oocyte precursors and mature MII oocytes are maintained with only a slight reduction in CENPA - consistent with genome ploidy. This difference in CENPA levels between the maternal and paternal genome is maintained up to the Pronuclear Stage 2 embryos after fertilization. However, eight hours after fertilization, most embryos have equalized the amount of CENPA in both the maternal and paternal genomes. Failure to equalize leads to chromosome segregation defects. Conclusion: Mature sperm and egg genomes have significantly different levels of CENPA, with the paternal genome containing only ∼10% of the maternal genome levels. Upon fertilization, maternal nuclear CENPA was redistributed to the paternal genome to equalize parental CENPA levels prior to first cell division. This accumulation within the paternal genome was independent of new CENP-A formation. Failure to equalize leads to chromosome segregation defects. The results suggest that CENPA levels in oocytes and embryos could be a marker for normal chromosome segregation in embryos or oocytes. Sunday, June 2, 2024