ABSTRACT During oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.
The DNA damage is a major problem that leads to mutagenesis and premature aging. Intriguingly, DNA damage repair (DDR) machinery is not robust in mammalian oocytes. The underlying mechanisms of this weakened DDR are unknown. Using the mouse oocyte as a model system, we found that DDR is dysfunctional in oocytes, leading to the development of aneuploid oocytes. Our data reveal that oocyte failure to repair damaged DNA is due to the inability of DDR protein, RAD51, to access altered "closed" chromatin conformation in DNA-damaged oocytes. Our data also demonstrate that, unlike somatic cells, oocytes fail to activate autophagy in response to DNA damage, which is the cause of altered chromatin conformation and inefficient DDR. Importantly, autophagy induction rescued DDR function and decreased aneuploidy in both DNA-damaged oocytes and oocytes from maternally aged mice which are prone to severe DNA damage. Our findings provide evidence that reduced autophagy contributes to weakened DDR in oocytes, especially in those from aged females, and offer scope to improve assisted reproductive therapy in women with compromised oocyte quality.Funding Information: This research was supported by laboratory start-up funding from the University of Missouri and a research grant from the American Society for Reproductive Medicine to A.Z.B. P.S was funded by USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative Competitive Grants no. grant number 2020-67015-31017 and seed funding from the College of Agriculture, Food and Natural Resources, University of Missouri (P.S.)Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Mice were kept and experiments were performed in accordance with the Animal Care and Use guidelines of the University of Missouri (Animal Care Quality Assurance). This study was approved by the Animal Care Quality Assurance committee at the University of Missouri (Animal Care Quality Assurance Ref. Number, 17180)
During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a new class of MT organizing centers in the oocyte that does not contribute to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a new model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a new player in regulating spindle positioning, an indispensable function to avoid aneuploidy, the leading genetic cause of miscarriage and congenital abnormalities.
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