The Mechanism of Acentrosomal Spindle Assembly in Human Oocytes
Authors: Wu Tianyu , Dong Jie , Fu Jing , Kuang Yanping , Chen Biaobang , Gu Hao , Luo Yuxi , Gu Ruihuan , Zhang Meiling , Li Wen , Dong Xi , Sun Xiaoxi , Sang Qing , Wang Lei 
Published: March 2023

ABSTRACT
During somatic cell mitosis and meiosis, spindle assembly is an essential step for ensuring accurate chromosome transmission and is mediated via duplicated centrosomes. In nonsomatic cells, canonical centrosomes are absent in the oocytes of many species, suggesting the mechanism of female meiotic spindle assembly is not conserved between species. Human oocytes lack detectable acentriolar microtubule organizing centers at the meiotic spindle poles and the exact mechanism of acentrosomal spindle assembly in human oocyte remains unclear.
This study aimed to utilize 3-dimensional (3D) high-resolution imaging of more than 2000 human oocytes to identify and characterize key structures in the spindle assembly process. First, human oocytes were stained to visualize chromosomes, kinetochores, and microtubules before maturation. Approximately 2 to 4 hours following nuclear envelope breakdown (NEBD), the nucleation of spindle microtubules was initiated from kinetochores. To identify factors driving this spindle microtubule nucleation from kinetochores in human oocytes, 86 human centrosome and microtubule-related proteins were localized by immunofluorescence in more than 1000 fixed human oocytes. Of these, 43 proteins localized in the meiotic spindle, among which 4 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]) showed kinetochore and spindle microtubule localization. It was observed that these 4 proteins belong to the same structure (human oocyte microtubule organizing center [huoMTOC]), and a dense microtubule cluster was observed around this structure, which caused asymmetrical microtubule distribution around the nuclear envelope before NEBD.
Next, it was observed that TACC3 had over 29-fold greater expression than CCP110, CKAP5, and DISC1, and after being depleted using siRNA, there was complete disruption of the huoMTOC, and the distribution of microtubules was drastically diminished. The other 3 proteins showed similar but less severe diminishment of the huoMTOC when depleted. Next, to visualize the dynamic process of huoMTOC-regulated spindle assembly, 3D time-lapse imaging was used following NEBD. This showed the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Again, each huoMTOC component was down-regulated in human oocytes and brought through this stage, showing that with each component there was significant impairment of spindle microtubule nucleation and spindle assembly compared with the control group (P < 0.001), and spindle microtubules were greatly decreased.
It was hypothesized that given the key role of the huoMTOC in human spindle assembly, a disrupted huoMTOC resulting from mutations in CCP110m CKAP5, DISC1, or TACC3 may cause abnormal oocyte maturation in patients. A cohort of 1394 infertile female patients characterized by oocyte maturation arrest was screened for mutations through whole-exome sequencing data sets. Two patients with loss-of-function mutations in TACC3 were identified, and analysis of their retrieved oocytes found a missing huoMTOC, suggesting the disruption of the huoMTOC impaired the nucleation of microtubules in the oocytes of patients.
This study identified and characterized a structure, which was named the huoMTOC, which serves as a major site of microtubule nucleation and is required for spindle assembly in human oocytes.