中国科学院昆明动物研究所机构知识库

哺乳动物卵母细胞与体细胞和雄性生殖细胞有显著的不同，它们会经历独特的减数分裂第一次分裂前期阻滞过程。出生后，卵母细胞进入减数第一次分裂前期并阻滞在终变期。阻滞过程中卵母细胞会遭受内部和外部的毒性压力，导致DNA损伤的积累。DNA损伤主要包括单链损伤（SSB）和双链损伤（DSB）。其中，DSB是对细胞损害最严重的一种形式，这会影响基因组稳定性和细胞生存能力。如果DNA损伤修复能力同时缺陷就会导致卵母细胞凋亡，降低卵泡数量。本论文中，我们较详细地检测了FLOPED蛋白的功能。FLOPED蛋白是一种卵母细胞特异的母源效应基因。它定位于卵皮质内侧，与Mater (Nlrp5)、Filia (Khdc3)和 Tle6形成皮质下母源复合物（SCMC）。FLOPED蛋白的缺失会造成小鼠胚胎发育的2-细胞时期阻滞和卵的MI期阻滞。试验中，未处理的小鼠卵母细胞经免疫荧光染色发现，Floped基因缺失卵中r-H2AX水平明显更高，而经Etoposide处理3h再恢复6h后，我们发现Floped-/-组GV卵无法像WT卵那样有效的修复DSBs，说明Floped基因缺失卵中DNA损伤修复能力受损。同时，体外培养的卵巢实验以及腹腔注射实验结果显示，经Etoposide诱导凋亡后，Floped-/- 卵巢凋亡敏感性增加。由于卵母细胞中DSBs的修复主要依靠HR途径。我们通过IF染色和western bloting实验检测HR两个关键蛋白，p-ATM和RAD51，表达差异水平发现，Floped基因缺失GV卵中两种蛋白均明显下调，并且经显微注射Floped-GFP融合mRNA“拯救”后能够恢复p-ATM和RAD51蛋白水平，并显著修复DNA双链损伤。已有证据表明，人和小鼠自然或病理情况下生殖衰老导致的卵母细胞质量与数量的下降与HR介导的DSBs修复的缺陷有关。我们通过HE染色实验检测了4周龄和16周龄左右WT和Floped-/- 卵巢切片，发现随着年龄的增长，Floped基因缺失卵巢中原始和初级卵泡数目逐渐衰减。而通过单细胞扩增技术检测Floped基因在10月龄以上小鼠GV卵中转录水平急剧下降；同样，经western bloting实验检测年老小鼠MII卵中FLOPED蛋白水平发现，随着年龄的增长FLOPED蛋白显著下调。说明FLOPED蛋白可能参与卵巢衰老过程。目前，人类卵泡中已经发现Floped同源基因Ooep的存在，借鉴小鼠卵母细胞中Floped基因功能研究能为以后人类卵泡功能的深入研究，以及改善人类生殖质量提供理论基础。

Mammalian oocytes differ from somatic cells or male germ cells in respect to their unique prophase arrest. At birth, oocytes enter into the prophase of the first meiotic division and arrest at the dictyate. During the long time period of meiotic arresting oocytes are imposed to endogenous and exogenous genotoxic insults and tend to accumulate DNA damage. DNA damage can occur as forms of single-stranded break (SSB) or double strand break (DSB). DNA DSB is most deleterious and can substantially impair genomic integrity and cell viability. Impairment of DNA damage repair can lead to oocyte apoptosis and quantity decline. In this study, we carefully examined the function of FLOPED. Floped (official symbol, Ooep) is an oocyte-specific maternal effect gene. Floped protein localizes at the subcortical region of an oocyte and interacts with four other maternal proteins including Mater (official symbol Nlrp5), Filia (official symbol Khdc3) and Tle6 to form subcortical maternal complex (SCMC). Depletion of Floped leads to embryo arrest at 2-cell stage and oocytes arrest at MI stage. IF staining results showed us that untreated Floped-/- oocytes possessed higher levels of DNA damage than untreated WT oocytes. After treatment by Etoposideg for 3h and recovery for 6h, we found that Floped-/- oocytes could not repair DSBs easily like the wildtype counterpart. So the repair ability of Floped-/- oocytes might have been impaired. After being treated with Etoposide in culture system or intraperitoneal injection, the Floped-/- ovaries were more sensitive to this insult agent. HR is the main mode of DNA DSB repair in meiotic cells including oocytes. We then detected HR key factor, p-ATM and RAD51, by IF staining and western bloting. The levels of p-ATM and RAD51 protein were downregulated in Floped-/- oocytes, and they could be rescued by Floped-GFP mRNA. Accumulating evidence supports a causal relationship between the defeat in HR-mediated DNA DSB repair and the decline of oocyte quantity as well as quality in natural or pathological reproductive aging in mouse and human. HE staining results showed us that 16 weeks old Floped-/- ovaries possessed less primrodial and primary follicles than WT ovaries. We then detected the level of Floped mRNA by Q-PCR, and found that 10 monthes old GV oocytes had less Floped mRNA than 6 weeks old GV oocytes. Also, the level of FLOPED protein was downregulated at 10 monthes old MII oocytes.At present, ooep was found being a Floped homologous gene in human. Our research will provide a theoretical foundation for the further study in improving human follicle functions and reproductive quality.