State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.
Department of Biotechnology and Biomedicine, Yangtze Delta Region Institutes of Tsinghua University, Jiaxing, Zhejiang, China.
Autophagy. 2022 Mar;18(3):643-660. doi: 10.1080/15548627.2021.1946739. Epub 2021 Jul 7.
There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what's the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7 or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality. ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.
越来越多的证据表明，自噬是一种专门降解和清除长寿命或受损线粒体的形式，其在衰老和与年龄相关的疾病中受损。先前的研究表明，由于不能激活自噬，暴露于肥胖的卵母细胞会积累和传递受损的线粒体。然而，目前尚不清楚卵母细胞中是否存在自噬功能，以及卵母细胞衰老的调节机制是什么。在这项研究中，当完全成熟的卵母细胞用 CCCP（一种解偶联剂，可诱导自噬）处理时，我们发现 PRKN 介导的自噬途径的激活伴随着减数分裂中期 I 阶段的阻滞。我们的结果表明，它与 RAB7 活性的降低有关，并且 CCCP 处理的卵母细胞中观察到的所有缺陷都可以通过微注射编码活性 RAB7 的 mRNA 或用 RAB7 激活剂 ML098 处理来有效挽救。进一步的研究表明，PRKN 蛋白水平是促进 RAB7 及其 GEF（鸟嘌呤核苷酸交换因子）复合物 CCZ1-MON1 通过泛素-蛋白酶体系统降解的限速因素。在卵巢衰老过程中收集的 GV 卵母细胞中，我们发现与年龄相关的 PINK1 和 PRKN 蛋白增加，RAB7 显著减少，导致噬线粒体形成缺陷和受损线粒体积累。体内用 ML098 治疗后，与年龄相关的女性生育力衰退得到改善。因此，RAB7 活性对于维持自噬和染色体稳定性之间的平衡是必需的，RAB7 激活剂是改善与年龄相关的卵母细胞质量恶化的良好候选物。ATG9：自噬相关 9A；ATP：三磷酸腺苷；CALCOCO2/NDP52：钙结合和卷曲螺旋域 2；CCCP：羰基氰化物 3-氯苯腙；CCZ1：CCZ1 液泡蛋白转运和生物发生相关；GAPDH：甘油醛-3-磷酸脱氢酶；GAPs：GTPase- 激活蛋白；GEF：鸟嘌呤核苷酸交换因子；GV：生发泡；GVBD：生发泡破裂；LAMP1：溶酶体相关膜蛋白 1；MI：减数分裂 I 期；MII：减数分裂 II 期；Mito：MitoTracker；mtDNA：线粒体 DNA；MON1：MON1 同源物，分泌转运相关；OPTN：optineurin；PINK1：PTEN 诱导的假定激酶 1；PRKN：parkin RBR E3 泛素蛋白连接酶；RAB7：RAB7，RAS 癌基因家族成员；ROS：活性氧；TEM：透射电子显微镜；TOMM20/TOM20：外线粒体膜 20 转位酶；TUBB：微管，β；UB：泛素。
