Mechanism of Spindle Pole Organization and Instability in Human Oocytes

Approximately 25% to 50% of human eggs are aneuploid, with even higher rates among older women. Aneuploidy is a leading cause of abnormal embryonic development resulting in miscarriage and genetic disorders. Recent discoveries suggest that highly unstable meiotic spindles during meiosis 1 may be a major driver of abnormal chromosome segregation resulting in aneuploidy. Interestingly, in contrast to human oocytes, the spindles of other mammalian oocytes are stable. While the mechanism of spindle pole organization in nonrodent mammalian oocytes remains unclear, proteins including.-tubulin and NUMA (nuclear mitotic apparatus protein) have been identified at the spindle poles and may play an important role. The aim of this comparative analysis was oocytes. The microtubule cross-linking protein NUMAwas localized to microtubule minus ends and functioned to recruit the molecular motor dynein for spindle pole focusing. To test if NUMAwas essential for spindle pole organization, the NUMA protein was depleted in human oocytes, which resulted in the spindle poles becoming fully defocused. Depletion of dynein similarly splayed microtubule minus ends, suggesting that together NUMA and dynein organize the spindle poles in human oocytes. Next, NUMA protein expression in other mammalian species was analyzed for comparison. Bovine and porcine oocytes were chosen as they lack centrosomes similarly to humans, and mouse oocytes that had been artificially depleted of acentriolar microtubule organizing centers were chosen. Live imaging in this analysis revealed that each of these 3 mammalian models did not exhibit unstable spindles, suggesting another mechanism besides NUMA was exerting a stabilizing effect. The molecular motor KIFC1 (kinesin superfamily protein C1) was identified using RNA interference screen of proteins with functions related to spindle organization. Data from previous proteomics studies in mice and human oocytes only had detectable KIFC1 in the mice data sets. Data from previous RNA-seq studies showed that both human and nonhuman mammals (mouse, bovine, porcine) expressed KIFC1 mRNA from the 2- to 4-cell stage onward, but only nonhuman mammals had maternal KIFC1 mRNA in the oocyte and zygote stage. Subsequent KIFC1 protein levels were measured using on-blot total protein normalization, which resulted in readily detectable KIFC1 in HeLa cell, mouse oocyte, bovine oocyte, and porcine oocyte lystates but not in human oocyte lysate, confirming human oocytes are deficient in KIFC1. Depletion of KIFC1 in nonhuman mammalian oocytes demonstrated spindles with unstable poles and an increase in aneuploidy, essentially recapitulating the spindle instability of human oocytes. Introduction of exogenous KIFC1 stabilized spindles in human oocytes and resulted in reduced chromosomal segregation errors. This study demonstrates significant differences in spindle pole organization in different mammalian species and begins to elucidate a cause of spindle instability in human oocytes due to KIFC1 deficiency.