(332) Cytoskeletal Integrity Regulates Autophagy-Dependent DNA Damage Repair in Mouse Oocytes.

Mohamed A. Ezz ^1,2, Ahmed Z. Balboula¹

¹ Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA.

² Theriogenology Department, Faculty of Veterinary Medicine, Mansoura University, Egypt

Abstract Text:

Fully grown oocytes undergo a prolonged arrest at prophase-I (germinal vesicle; GV) stage before resuming meiosis. During this arrest, the oocytes experience several endogenous and exogenous stressors compromising their genome integrity and developmental competence. Unlike mild DNA damage, the oocytes are inefficient at repairing moderate/severe DNA damage. We previously demonstrated that this weakened DNA damage response to moderate/severe DNA damage in mammalian oocytes is due to their failure to activate autophagy. However, why oocytes can repair mild, but not severe, DNA damage and whether their autophagy contributes to this phenomenon, are yet understood. We first confirmed the oocyte ability to repair varying DNA damage levels induced by etoposide, a topoisomerase-II inhibitor commonly used to induce double-strand breaks (DSBs). Post-etoposide washout, DSB levels (assessed by gH2AX intensity) significantly decreased to control levels within 6h or 12h in oocytes exposed to 5 or 50 µg/ml etoposide for 1h, respectively. In contrast, oocytes exposed to 50 µg/ml etoposide for 3h failed to repair DSBs even after 12h recovery. We then examined oocyte autophagy activity in response to DNA damage. Interestingly, oocyte treatment with a low etoposide concentration (5 µg/mL) for 1h stimulated autophagy activity as evidenced by increased LC3 and Beclin1 (autophagy markers), while a high concentration (50 µg/mL) reduced autophagy activity, which was subsequently induced at 3h post-washout. Consistent with autophagy activation, RAD51 recruitment, a major DNA damage repair (DDR) protein, occurred much earlier in oocytes exposed to 5 µg/ml etoposide (1h post-washout) compared to those to 50 µg/ml (3h post-washout). In somatic cells, functional autophagy relies on the interaction between F-actin and microtubules (MTs), two major cytoskeletal components. Therefore, we hypothesized that oocytes exposed to moderate/sever DNA damage fail to activate autophagy due to cytoskeletal alterations. To test this hypothesis, GV-arrested oocytes were exposed to etoposide at 0, 5, or 50 µg/mL for 1h. Indeed, oocytes exposed to 5 µg/ml etoposide exhibited an increase in perinuclear and cytoplasmic F-actin and MTs, but oocyte exposure to 50 µg/mL severely depolymerized F-actin and MTs. To investigate the role of F-actin and MTs in DDR response, GV-arrested oocytes were exposed to 5 µg/ml etoposide for 1h, then washed and recovered in etoposide-free medium with or without cytochalasin-D (F-actin-depolymerizing drug) or nocodazole (MTs-depolymerizing drug). Both cytochalasin-D and nocodazole delayed autophagy induction and RAD51 recruitment, and exacerbated DSB levels. To confirm that the failure to activate autophagy in response to moderate/severe DNA damage was due to cytoskeletal disruption, but not due to a direct effect of DSBs on autophagy, we employed an oocyte vitrification model, notoriously known for inducing cytoskeletal alterations. We found that autophagy activity was significantly suppressed, then gradually recovered in vitrified-thawed oocytes. Post-thawing recovery was accompanied by F-actin/MT repolymerization. Although vitrification induced a mild DNA damage (comparable to 5 µg/mL etoposide), RAD51 recruitment was delayed till 3h post-thawing (comparable to 50 µg/ml etoposide). Importantly, autophagy induction by rapamycin accelerated DDR, whereas autophagy inhibition by spautin-1 accumulated DNA damage in vitrified-thawed oocytes. Altogether, our findings strongly suggest that alterations in cytoskeletal components, F-actin and MTs, due to DNA damage contribute, at least in part, to the failure of autophagy activation in oocytes exposed to moderate/severe DNA damage or vitrified-thawed oocytes. Our results suggest that preserving cytoskeletal components in oocytes may offer a promising approach to counteract stress-induced autophagy impairment and promote efficient DDR response.