光控制的小鼠卵母细胞线粒体转运及应用

Mitochondrial DNA diseases are common and severest. They are hereditable, and transmitted maternally. The mitochondrial genome is much less stable than the nuclear genome, accruing random DNA mutations much easier. As many as 1 in 5,000 to 10000 children are born with diseases caused by these mutations. Preimplantation or prenatal diagnosis are not helpful for the women who carried homoplasmic mt-DNA mutations. Options available for families carrying these mutations and desired to have unaffected children are either oocyte donation or mitochondrial replacement at the oocyte or zygote stage. However, GV transfer, spindle transfer and pronuclear transfer cannot avoid mutated mtDNA transmitted from the nuclear donor oocytes or embryos to the recipient ones. Polar bodies contain few mitochondrial, and could be used to replace the genome of the recipient oocytes to prevent inherited mtDNA disease. However, it is not fully understood whether the genomes in the polar bodies carry the same DNA mutations, DNA damages and epigenetic modifications as those of the sibling oocytes. Therefore, we use the optogenetic tools LOV-ePDZ to transport the mitochondrial in the oocyte from the perinuclear area to the peripheral area. These optogenetic modified oocytes could be used as the nuclear donor oocytes to avoid/reduce the mutated mtDNA transmit to the recipient oocyte. Eventually optimizing the mitochondrial replacement therapy methods of GV transfer and spindle transfer, and preventing the inheritance of the mitochondrial DNA disease.

线粒体DNA疾病是一类常见又极为严重的疾病，是母系遗传的可遗传性疾病。线粒体DNA随机突变率远远高于细胞核DNA突变率。每5000-10000个新生儿就会有1个发生线粒体DNA突变，产生线粒体DNA疾病。携带纯合线粒体DNA突变的患者无法通过遗传学诊断避免患病后代出生，只能通过供卵或者在卵母细胞和受精卵阶段实施线粒体替代疗法来获得不携带线粒体DNA突变的后代。GV移植、纺锤体移植、原核移植等线粒体替代疗法很难避免将核供体中的部分突变线粒体带入细胞质受体中。极体移植能够减少核供体的突变线粒体带入，但至今还不清楚极体中的基因组在基因突变、DNA损伤和表观修饰等方面是否和卵母细胞基因组完全一致。本研究拟利用光遗传学工具LOV2-ePDZ系统将小鼠卵母细胞线粒体转运到远离细胞核的区域，避免或减少GV移植和纺锤体移植时将突变线粒体带入受体卵母细胞，降低出生后代线粒体的异质性，阻止后代发病。

线粒体是体细胞和卵母细胞中的能量工厂，为细胞代谢提供大量的ATP。哺乳动物卵母细胞中含有大量的线粒体，为卵母细胞成熟和早期胚胎发育提供大量的ATP。小鼠卵母细胞中线粒体在不同发育阶段分布并不相同，其运输的控制至今还不清楚。本项研究通过微丝微管组装特异性抑制剂，结合光漂白（Photobleaching）、光遗传学（optogenetic)等技术，研究了线粒体在卵母细胞中的转运方式。研究结果表明，线粒体在卵母细胞中的运输受到微丝和微管的协同调节，微丝主要负责线粒体的短程运输，使线粒体在细胞质中分散分布，微管使线粒体运输到细胞核周围。微丝和微管的协同互作，使线粒体在卵母细胞不同发育时期呈现出不同的分布形式。本研究揭示了线粒体在卵母细胞中不同时期的分布规律以及其作用机理，为后续线粒体替代疗法等辅助生殖技术提供了一定的理论基础。