Double‐strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos

Abstract

DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA-dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulation of the HR pathway in mammalian embryos.-Bohrer, R. C., Dicks, N., Gutierrez, K., Duggavathi, R., Bordignon, V. Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos.

Histone Lysine Demethylases KDM5B and KDM5C Modulate Genome Activation and Stability in Porcine Embryos

“…The fluorescence intensity for H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K9me2, and KDM5B was quantified on each nucleus and corrected to the background in the cytoplasm of each embryo using the SimplePCI imaging software (Compix, Sewickley, PA, United States). The same software was used to determine the number of DSBs by counting the number of H2AX139ph foci ≥0.3 µm 3 in each nucleus (Bohrer et al, 2018).…”

“…The developmental competence of embryos, including mice (Wang et al, 2013), humans (Kort et al, 2016), and pigs (Bohrer et al, 2015(Bohrer et al, , 2018Dicks et al, 2017), is affected by DNA damage. Moreover, treatment with epigenetic modulators, such as histone deacetylates inhibitors, during embryo culture promoted DNA repair and improved embryo development (Bohrer et al, 2014).…”

Tauroursodeoxycholic acid acts via TGR5 receptor to facilitate DNA damage repair and improve early porcine embryo development

“…Disrupted TUDCA/TGR5 signaling has a significant impact on the GDR. During early embryo development, it has been shown that embryos primarily utilize the HR pathway in response to DSBs caused by UV radiation, with DsiRNA targeting ATM, but not DNAPK, significantly reducing the proportion of blastocysts that developed and their quality (Bohrer et al, ). Considering the superior fidelity and efficiency of the HR pathway (Blackford & Jackson, ; Khanna & Jackson, ), it is not surprising that embryos would preferentially employ this pathway in preventing replication of damaged DNA and the subsequent deleterious effects.…”

“…Considering there was no difference seen in cell number and, in fact, increased XIAP mRNA abundance to prevent cell death, these changes can be interpreted as a successful adaptation from the perspective of the embryo cell, or blastomere (Dicks et al, ) allowing survival despite being stressed and damaged. On the other hand, at the organismal level, the upregulation of NHEJ over HR in response to nuclear stress could represent a pathological adaptation (Dicks et al, ), raising concerns of impaired genome integrity and abnormal embryo development (Bohrer et al, ). Survival of these blastomeres carrying DNA damage will likely have deleterious consequences that will manifest during embryonic or subsequent life stages.…”

“…The HR pathway is often considered a more efficient process since it uses the sister chromatid as a template to ensure precise repair of the defect (Torgovnick & Schumacher, ). As the primary pathway involved in GDR during early embryo development (Bohrer, Dicks, Gutierrez, Duggavathi, & Bordignon, ), it also represents the repair mechanism operating under normal physiological conditions. Conversely, the NHEJ pathway is considered more error‐prone since it does not utilize a template to direct repair (Mimitou & Symington, ).…”