Department of Obstetrics & Gynaecology, University of Melbourne, Parkville, Australia.
Ovarian Biology Laboratory, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, 3800, Australia.
J Assist Reprod Genet. 2021 Jun;38(6):1405-1417. doi: 10.1007/s10815-021-02184-3. Epub 2021 Apr 16.
Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma mice in response to DSBs caused by the chemotherapy agent cisplatin.
Adult C57BL/6 WT and Puma mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised.
Puma mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role.
PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.
基因毒性化疗和放疗会导致原始卵泡（PMF）卵母细胞中的 DNA 双链断裂（DSB），随后发生细胞凋亡。部分原因是对 PMF 卵母细胞中 DNA 修复的了解有限，这阻碍了有效新的生育力保存剂的发展。本研究调查了在化疗药物顺铂引起的 DSB 作用下，WT 和凋亡缺陷型 Puma 小鼠卵泡中经典 DSB 修复途径的诱导。
成年 C57BL/6 WT 和 Puma 小鼠经腹腔注射生理盐水或顺铂（5mg/kg）；8 或 24 小时后收获卵巢。计数卵泡，并使用 H2A 组蛋白家族成员（γH2AX）免疫荧光显示 DSB。使用 RAD51 同源物 1（RAD51）和 DNA 依赖性蛋白激酶，催化亚基（DNA-PKcs）免疫荧光鉴定所利用的 DNA 修复途径。
顺铂处理后 24 小时，Puma 小鼠保留了 100%的卵泡。处理后 8 小时，γH2AX 免疫荧光显示 Puma 小鼠的卵泡各阶段均有 DSB；5 天内 PMF 中的染色恢复到对照水平，表明在此窗口内修复了 PMF 卵母细胞。顺铂处理 8 小时后，WT 和 Puma 小鼠的受损细胞类型中 RAD51 免疫荧光均为阳性，表明诱导了同源重组途径。相比之下，PMF 中很少观察到 DNA-PKcs 染色，表明非同源末端连接的作用不大。
PMF 卵母细胞能够对化疗过程中积累的 DNA 损伤进行高保真修复。因此，抑制细胞凋亡为接受治疗的女性提供了一种可行的生育力保存策略。
