Causes and consequences of mitochondrial dysfunction in oocytes and cumulus cells

总结 女性生育力的主要决定因素是卵母细胞的“质量”：即发育能力。卵母 随着母亲年龄的增长和体重指数的增加， 环境毒素在不孕症治疗期间，卵母细胞在体外培养，因此容易受到任何影响。 不利的文化条件。目前还不清楚是什么生物学机制决定卵母细胞 质量，这阻碍了治疗，使其难以：a）识别和选择最好的卵母细胞， 受精和后续移植;和B）开发提高卵母细胞质量的方法。线粒体是 负责细胞呼吸和各种其他生物合成过程的关键细胞器，Ca 2 + 调节、凋亡和活性氧的产生。线粒体对卵子发生至关重要 和胚胎发育，两者都通过它们在卵母细胞中的直接功能和它们对卵母细胞的代谢调节来实现。 围绕着卵丘细胞。然而，线粒体功能障碍在多大程度上有助于女性 不孕症仍不清楚。在这个项目中，线粒体功能障碍的原因和后果， 将研究卵母细胞及其相关的卵丘细胞。 这项工作将：a）使用小鼠卵母细胞研究特异性干扰对发育的影响， B）研究临床相关因素，包括母亲年龄，BMI， 和培养条件影响捐赠的线粒体，否则丢弃的人类卵母细胞; 这些变化与成熟缺陷相关的程度;和3）检查临床相关因素， 包括母亲的年龄、体重指数和卵巢储备功能的下降，会影响 接受IVF的患者;以及这些变化在多大程度上与成功妊娠相关。 这项研究将通过两种新技术实现：a）大规模并行测序协议， 线粒体DNA（mtDNA）含量，以检查序列，并定量异质性水平;和B） 代谢成像与荧光寿命成像显微镜（FLIM）的线粒体功能。的 使用这些方法将允许定量表征线粒体功能和基因组 内容线粒体功能障碍对随后发育的潜在影响， 卵母细胞成熟到活产，也将被量化，包括：非整倍体，减数分裂的形态计量学， 纺锤体、染色体和动粒，以及胚胎发育和存活的动力学和保真度 出生（小鼠中）。总之，这项工作将建立如何线粒体在卵母细胞和卵丘 细胞受到临床相关因素的干扰;卵母细胞的发育能力如何受到 卵母细胞和卵丘细胞中的线粒体;以及线粒体功能障碍在多大程度上解释了 降低卵母细胞质量。除了产生基本的洞察力，这项研究可能会导致短期的 改善IVF患者的治疗。

SUMMARY The primary determinant of female fertility is oocyte “quality”: i.e. developmental competence. Oocyte quality declines with advancing maternal age and increasing body mass index, and in response to environmental toxins. During infertility treatment, oocytes are cultured in vitro, and so are vulnerable to any adverse culture conditions. It is unclear what biological mechanisms are responsible for determining oocyte quality, which impedes treatment by making it difficult to: a) identify and select the best oocytes for fertilization and follow-on transfer; and b) to develop methods to improve oocyte quality. Mitochondria are key organelles responsible for cellular respiration, and a variety of other biosynthetic processes, Ca2+ regulation, apoptosis, and the production of reactive oxygen species. Mitochondria are crucial for oogenesis and embryonic development, both by their direct function in oocytes and their metabolic modulation of the surrounding cumulus cells. However, the extent to which mitochondrial dysfunction contributes to female infertility remains unclear. In this project, the causes and consequences of mitochondria dysfunction in oocytes, and their associated cumulus cells, will be investigated. The work will: a) use mouse oocytes to study the consequences on development of specifically perturbing mitochondria during maturation; b) investigate how clinically relevant factors, including maternal age, BMI, and culture conditions influence mitochondria in donated, otherwise discarded human oocytes; and to what extent these changes are associated with maturation defects; and 3) examine how clinically relevant factors, including maternal age, BMI, and diminished ovarian reserve, influence mitochondria in cumulus cells of patients undergoing IVF; and to what extent these changes are associated with successful pregnancy. The research will be enabled by two novel techniques: a) massive parallel sequencing protocols to measure mitochondria DNA (mtDNA) content, to examine sequence, and to quantify levels of heteroplasmy; and b) metabolic imaging with Fluorescence Lifetime Imaging Microscopy (FLIM) for mitochondria function. The use of these approaches will allow a quantitative characterization of mitochondria function and genomic content. The resulting potential impact of mitochondrial dysfunction on subsequent development, from oocyte maturation to live birth, will also be quantified including: aneuploidy, morphometrics of the meiotic spindle, chromosomes, and kinetochores, and the kinetics and fidelity of embryonic development and live births (in the mouse). Taken together, this work will establish how mitochondria in oocytes and cumulus cells are perturbed by clinically relevant factors; how oocyte developmental competence is influenced by mitochondria in oocytes and cumulus cells; and the extent to which mitochondria dysfunction explains reduced oocyte quality. In addition to producing fundamental insight, this study may lead to near-term improvement in treatment of IVF patients.