Oocytes mount a noncanonical DNA damage response involving APC-Cdh1–mediated proteolysis

Abstract

In mitotic cells, DNA damage induces temporary G2 arrest via inhibitory Cdk1 phosphorylation. In contrast, fully grown G2-stage oocytes readily enter M phase immediately following chemical induction of DNA damage in vitro, indicating that the canonical immediate-response G2/M DNA damage response (DDR) may be deficient. Senataxin (Setx) is involved in RNA/DNA processing and maintaining genome integrity. Here we find that mouse oocytes deleted of Setx accumulate DNA damage when exposed to oxidative stress in vitro and during aging in vivo, after which, surprisingly, they undergo G2 arrest. Moreover, fully grown wild-type oocytes undergo G2 arrest after chemotherapy-induced in vitro damage if an overnight delay is imposed following damage induction. Unexpectedly, this slow-evolving DDR is not mediated by inhibitory Cdk1 phosphorylation but by APC-Cdh1–mediated proteolysis of the Cdk1 activator, cyclin B1, secondary to increased Cdc14B-dependent APC-Cdh1 activation and reduced Emi1-dependent inhibition. Thus, oocytes are unable to respond immediately to DNA damage, but instead mount a G2/M DDR that evolves slowly and involves a phosphorylation-independent proteolytic pathway.

Sirt1 sustains female fertility by slowing age‐related decline in oocyte quality required for post‐fertilization embryo development

“…OoSirt1 ΔEx4/ΔEx4 GVBD rates did recover over the ensuing hour but remained lower than OoSirt1 +/+ oocytes (Figure 5a). Since Sirt1 has been implicated in DNA repair (Wang et al, 2008) and longstanding DNA damage delays GVBD in mouse oocytes (Subramanian et al, 2020), we next investigated DNA damage levels. We immunostained oocytes for γH2AX, a known marker of DNA double‐strand breaks and found significantly higher γH2AX staining in OoSirt1 ΔEx4/ΔEx4 oocytes (Figure 5b).…”

“…Genotyping was performed as described previously (Subramanian et al, 2020). Ear clippings and oocytes were lysed using Proteinase K (ThermoFisher) and incubated overnight at 55°C followed by inactivation at 85°C for 45 min.…”

“…Ovarian immunohistochemistry was performed as described previously (Subramanian et al, 2020). Ovaries were fixed in 4% paraformaldehyde (w/v) (Sigma), embedded in paraffin and serially sectioned at 5 μm and mounted on microscope slides (ThermoFisher).…”

“…To study maturation, oocytes were allowed to resume meiosis by culturing them in micro‐drops of IBMX‐free M16 culture medium (Sigma) under embryo‐tested mineral oil (Sigma) at 37°C in an atmosphere of 5% CO 2 and 5% O 2 . Generation of histone 2B (H2B)‐RFP cRNA (250 ng/µl) and microinjection into GV‐stage oocytes was performed as described previously (Subramanian et al, 2020; Wei et al, 2018; C. Zhou et al, 2019).…”

AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes

“…The development of the zygotes is controlled by maternal mRNA and protein that deposited in the oocytes (Tadros and Lipshitz, 2009;Langley et al, 2014). However, the function of G2/M checkpoints and DNA repair mechanisms in oocytes and zygotes may be different from somatic cells (Shimura et al, 2002;Yukawa et al, 2007;Homer et al, 2020). In…”