(46) DUXA Modulates Embryonic Cell Survival but is not Essential for Blastocyst Formation in Pigs

Fernanda L. Facioli1, Werner G. Glanzner¹, Karina Gutierrez¹, Mariana P. de Macedo¹, Zigomar da Silva¹, Vanessa Guay¹, Luke G. Currin¹, María E. C. Herrera¹, Vilceu Bordignon<sup>1

1</sup> Department of Animal Science, McGill University, Sainte-Anne de Bellevue, Quebec, Canada

Abstract Text:

Embryo genome activation (EGA) and an effective DNA damage response (DDR) are crucial for normal embryo development and preservation of genome integrity. The double homeobox (DUX) transcription factor is a key regulator of EGA in mice and human embryos and may also be implicated in DDR. However, its role in other species remains unclear. This study investigated the function of the porcine DUXA variant during early embryo development and explored its potential link to genome integrity in pigs. In the first study, we assessed DUXA mRNA expression in porcine embryos at different developmental stages. Our results revealed that DUXA mRNA is expressed from days 2 to 7 of development, peaking around the EGA stage (day 3). In the second study, we investigated the impact of attenuating DUXA mRNA on embryo developmental outcomes, cell viability and allocation, and mRNA expression of EGA indicators. We produced DUXA-attenuated embryos by microinjecting DsiRNA into MII oocytes. While DUXA attenuation did not affect blastocyst formation, inner cell mass or trophectoderm allocation, or EGA indicators expression, it led to an increased total cell count in both blastocysts and embryos that cleaved but arrested before blastocoel formation. Additionally, DUXA-attenuated embryos exhibited higher levels of DNA damage before the blastocyst stage (days 4 and 5). To explore the underlying mechanisms, we assessed the expression of cell cycle and DDR-related genes at day 4 of development. DUXA-attenuation resulted in reduced mRNA expression of CHEK1, BRCA1 and KU-70. Lastly, we evaluated the impact of DNA damage induction in DUXA-attenuated embryos by exposing them to UV light on day 4 of development. This treatment abolished the increased blastocyst cell numbers observed in DUXA-attenuated embryos and upregulated DDR and cell cycle-related gene expression. Overall, our findings demonstrate that, while DUXA is not essential for blastocyst development in pigs, it plays a role in embryonic cell survival, potentially by regulating DDR and cell cycle processes.