CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development

Abstract

Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

H3K27me3 is an epigenetic barrier while KDM6A overexpression improves nuclear reprogramming efficiency

“…However, we observed that EZH2 deficiency diminished genomic stability and induced apoptosis in cloned embryos. It was previously shown that EZH2 acts as an epigenetic determinant by inhibiting TNF-a-mediated apoptosis in colitis (47) and maintained T-cell genomic integrity by modulating DNA damage-induced apoptosis (34). Our findings highlight the important role of EZH2 for maintaining genomic stability and inhibiting apoptosis after SCNT reprogramming.…”

“…3J). g-H2AX is a marker for DNA double-stranded breaks (34), whereas ataxia-telangiectasia and Rad3related (ATR) and ataxia-telangiectasia mutated (ATM) are 2 genes that are activated in response to DNA damage (35). The g-H2AX level was higher in EZH2 knockdown 8-cell embryos compared with the control (Fig.…”

Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development

“…Most PA embryos that were treated with the RAD51 inhibitor or knockdown were arrested at approximately the four-cell stage on day 5, and these embryos showed increased DNA damage. In response to DNA damage, cell cycle checkpoints (G1, S, G2/M) are activated, stopping cell cycle progression to allow time for repair, thereby preventing the transmission of damaged or incomplete replicated chromosomes (Wang & Kim 2016). In somatic cells, insufficient DNA repair usually activates cell cycle checkpoints via the ATM and p53-p21 pathways (Espinosa et al 2003, Arias-Lopez et al 2006, Giono & Manfredi 2006.…”

Quercetin delays postovulatory aging of mouse oocytes by regulating SIRT expression and MPF activity

“…Western blotting was performed as described previously [30, 31], using the following antibody dilutions: anti-BCL-2, 1:500; anti-CDK1-pY15, 1:500; anti-cyclin B, 1:500; anti-β-actin, 1:2000.…”