Institute of Stem Cell and Regenerative Biology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction (Huazhong Agricultural University), Ministry of Education, Wuhan, Hubei, China.
Autophagy. 2023 Jan;19(1):163-179. doi: 10.1080/15548627.2022.2063005. Epub 2022 Apr 25.
Macroautophagy/autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cells from women of advanced maternal age, implying a potential negative correlation between autophagy levels and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. Mechanistically, we found that ACLY (ATP citrate lyase), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for selective autophagic degradation. In human follicles, the autophagy level in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting ACLY during oocyte maturation. 3-MA: 3-methyladenine; ACLY: ATP citrate lyase; AMA: advanced maternal age; CG: cortical granule; CHX: cycloheximide; CQ: chloroquine; CS: citrate synthase; COCs: cumulus-oocyte-complexes; GCM: granulosa cell monolayer; GV: germinal vesicle; MII: metaphase II stage of meiosis; PB1: first polar body; ROS: reactive oxygen species; shRNA: small hairpin RNA; SQSTM1/p62: sequestosome 1; TCA: tricarboxylic acid; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild-type.
自噬是一种细胞和能量稳态机制，有助于维持原始卵泡的数量、生殖细胞的存活和抗卵巢衰老。然而，尚不清楚颗粒细胞中的自噬是否会影响卵母细胞成熟。在这里，我们显示出高龄产妇的人类颗粒细胞中自噬水平明显降低的趋势，这暗示着自噬水平和卵母细胞质量之间可能存在负相关。因此，我们建立了一个共培养系统，并表明在颗粒细胞中通过药理学抑制或基因敲除自噬会对卵母细胞质量和受精能力产生负面影响。此外，我们的代谢组学分析表明，自噬损伤对卵母细胞质量的不良影响是通过下调柠檬酸水平介导的，而外源性补充柠檬酸可以显著恢复卵母细胞成熟。在机制上，我们发现 ACLY（三磷酸柠檬酸裂解酶），一种催化柠檬酸裂解的关键酶，优先与 K63 连接的泛素链结合，并被自噬受体蛋白 SQSTM1/p62 识别，进行选择性自噬降解。在人类卵泡中，颗粒细胞中的自噬水平随着母体年龄的增长而降低，同时卵泡液中的柠檬酸减少，这表明柠檬酸代谢与卵母细胞质量之间存在潜在的相关性。我们还表明，猪卵泡液中柠檬酸水平的升高可以促进卵母细胞成熟。总之，我们的数据揭示了在卵母细胞成熟过程中，颗粒细胞中的自噬通过选择性靶向 ACLY 来维持一定程度的柠檬酸，是一种有益的机制。3-MA：3-甲基腺嘌呤；ACLY：三磷酸柠檬酸裂解酶；AMA：高龄产妇；CG：皮质颗粒；CHX：环己酰亚胺；CQ：氯喹；CS：柠檬酸合成酶；COCs：卵丘-卵母细胞复合物；GCM：颗粒细胞单层；GV：生发泡期；MII：减数分裂中期 II 期；PB1：第一极体；ROS：活性氧；shRNA：短发夹 RNA；SQSTM1/p62：自噬相关蛋白 1；TCA：三羧酸循环；TOMM20/TOM20：外膜线粒体转运蛋白 20；UBA：泛素相关结构域；Ub：泛素；WT：野生型。
