The mechanism of acentrosomal spindle assembly in human oocytes

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes. Description Organizing for meiotic success Proper organization of the microtubules is vital for ensuring that daughter cells end up with the appropriate complement of chromosomes in both mitosis and meiosis. In somatic cells undergoing mitosis, this task is performed by centrosomes. By contrast, meiosis does not involve centrosomes in many animal species, but the specific methods of organizing the microtubules differ between animals. In particular, the mechanism of spindle organization in human oocytes has not been previously understood. Wu et al. detected a protein structure that they named the human oocyte microtubule organizing center and identified several of its constituent proteins. They then showed that mutations in one of these proteins are responsible for the clinical infertility associated with oocyte maturation arrest in human patients. —YN Meiotic spindle assembly in human oocytes is initiated by a microtubule organizing center required for oocyte maturation. INTRODUCTION Spindle assembly is essential for ensuring accurate chromosome transmission in both meiosis and mitosis. In somatic cells, mitotic spindle assembly is mediated by duplicated centrosomes, but canonical centrosomes are absent in the oocytes of many species. In rodents, acentriolar microtubule organizing centers (aMTOCs) are responsible for meiotic spindle assembly, but it has long been supposed that human oocytes lack prominent aMTOCs on the meiotic spindle, and the exact mechanism of acentrosomal spindle assembly in human oocytes has remained unclear. RATIONALE Microtubule nucleation and ensuring spindle assembly are core events regulating oocyte nuclear maturation. To identify the potential proteins driving spindle microtubule nucleation in human oocytes, we systematically localized 86 human centrosome and microtubule-related proteins by immunofluorescence or three-dimensional high-resolution live cell imaging in more than 2000 human oocytes. We then tracked the dynamic migration of identified microtubule nucleators at different time points before and after nuclear envelope breakdown (NEBD). We further down-regulated corresponding proteins to confirm their role in microtubule nucleation and spindle assembly. Given that spindle microtubule nucleation defects result in impaired spindle assembly and abnormal oocyte maturation, we screened for mutations in genes encoding components of microtubule nucleators in a cohort of 1394 infertile female patients characterized by oocyte maturation arrest. RESULTS First, we found that in human oocytes the nucleation of spindle microtubules is initiated from kinetochores from 2 to 4 hours after NEBD. We showed the process of spindle microtubules nucleating from kinetochores in human oocytes. We then found that there are 43 proteins localized in the meiotic spindle, among which four proteins—centriolar coiled-coil protein 110 (CCP110), cytoskeleton-associated protein 5 (CKAP5), disrupted in schizophrenia 1 (DISC1), and transforming acidic coiled-coil–containing protein 3 (TACC3)—exhibited both kinetochore and spindle microtubule localization. The localization of the four proteins was notably different from their localization in human mitotic cells and in mouse oocytes. Together, the four proteins formed an unusual structure that was surrounded by microtubules in human germinal vesicle (GV) oocytes just before NEBD. We refer to this potential nucleating structure as the human oocyte microtubule organizing center (huoMTOC). We found that a single huoMTOC is formed at the cortex of human GV oocytes and migrates to the nuclear envelope before NEBD. After NEBD, the huoMTOC becomes fragmented and is recruited to kinetochores to initiate spindle microtubule nucleation. Down-regulation of huoMTOC components caused considerably impaired spindle microtubule nucleation and spindle assembly in human oocytes. This structure was not detected in the oocytes of other mammalian species such as mice and pigs. We finally identified two oocyte maturation arrest patients with compound heterozygous mutations in the key huoMTOC component TACC3. All mutations disrupted the normal function of TACC3, resulting in the absence of the huoMTOC structure and completely impaired spindle assembly in the patients’ oocytes. CONCLUSION Our study shows that human oocytes possess an aMTOC-like structure, the huoMTOC, that serves as a major site of microtubule nucleation and is required for spindle assembly. The huoMTOC shows drastically different characteristics in terms of number, localization, and composition compared with aMTOCs in mouse oocytes. These findings suggest that a distinct mechanism for the initiation of microtubule nucleation and spindle assembly has evolved in human oocytes. We found that mutations in TACC3 cause defects in spindle assembly by disrupting the structure of the huoMTOC, which leads to clinical oocyte maturation arrest. This suggests that the huoMTOC might be an important biomarker for evaluating the quality of human oocytes. Our discovery of huoMTOC provides insights into the physiological mechanism of microtubule nucleation and spindle assembly in human oocytes. These findings also improve our understanding of the pathological mechanisms of oocyte maturation arrest. The huoMTOC structure in a human oocyte. The human GV oocyte shown here was matured for ~5 hours and fixed for immunofluorescence before NEBD. The huoMTOC (TACC3, magenta) was surrounded by numerous microtubules (green) on the nuclear envelope. The dashed square shows the magnification region. The arrow highlights the huoMTOC.