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Loss of acentriolar MTOCs disrupts spindle pole Aurora A and assembly of the liquid-like meiotic spindle domain in oocytes. Wang X et al. (2021) Oocyte-specific knockdown of pericentrin (PCNT) in transgenic (Tg) mice disrupts acentriolar microtubule-organizing center (aMTOC) formation, leading to spindle instability and error-prone meiotic division. Here, we show that PCNT-depleted oocytes lack phosphorylated Aurora A (pAURKA) at spindle poles, while overall levels are unaltered. To test aMTOC-associated AURKA function, metaphase II (MII) control (WT) and Tg oocytes were briefly exposed to a specific AURKA inhibitor (MLN8237). Similar defects were observed in Tg and MLN8237-treated WT oocytes, including altered spindle structure, increased chromosome misalignment and impaired microtubule regrowth. Yet, AURKA inhibition had a limited effect on Tg oocytes, revealing a critical role for aMTOC-associated AURKA in regulating spindle stability. Notably, spindle instability was associated with disrupted γ-tubulin and lack of the liquid-like meiotic spindle domain (LISD) in Tg oocytes. Analysis of this Tg model provides the first evidence that LISD assembly depends expressly on aMTOC-associated AURKA, and that Ran-mediated spindle formation ensues without the LISD. These data support that loss of aMTOC-associated AURKA and failure of LISD assembly contribute to error-prone meiotic division in PCNT-depleted oocytes, underscoring the essential role of aMTOCs for spindle stability.//////////////////Loss of aMTOCs disrupts spindle pole Aurora A and assembly of the liquid-like meiotic spindle domain (LISD) in oocytes. Wang X et al. (2021) Oocyte-specific Pericentrin (PCNT) knockdown in transgenic (Tg) mice disrupts acentriolar microtubule organizing center (aMTOC) formation, leading to spindle instability and error-prone meiotic division. Here, we show that PCNT-depleted oocytes lack phosphorylated Aurora A (pAURKA) at spindle poles, while overall levels are unaltered. To test aMTOC-associated AURKA function, MII control (WT) and Tg oocytes were briefly exposed to a specific inhibitor (MLN8237). Similar defects were observed in Tg and MLN8237-treated WT oocytes, including altered spindle structure, increased chromosome misalignment and impaired microtubule regrowth. Yet, AURKA inhibition had a limited effect on Tg oocytes, revealing a critical role for aMTOC-associated AURKA in regulating spindle stability. Notably, spindle instability was associated with disrupted γ-tubulin and lack of the liquid-like meiotic spindle domain (LISD) in Tg oocytes. Analysis of this Tg model provides the first evidence that LISD assembly depends expressly on aMTOC-associated AURKA, and that Ran-mediated spindle formation ensues without the LISD. These data support that loss of aMTOC-associated AURKA and failure of LISD assembly contribute to error-prone meiotic division in PCNT-depleted oocytes, underscoring the essential role of aMTOCs for spindle stability.//////////////////Error-prone meiotic division and subfertility in mice with oocyte-conditional knockdown of pericentrin. Baumann C et al. (2017) Mouse oocytes lack canonical centrosomes and instead contain unique acentriolar microtubule-organizing centers (aMTOCs). To test the function of these distinct aMTOCs in meiotic spindle formation -Pericentrin (Pcnt), an essential centrosome/MTOC protein, was knocked down exclusively in oocytes using transgenic RNAi. Here we provide evidence that disruption of aMTOC function in oocytes promotes spindle instability and severe meiotic errors that lead to pronounced female subfertility. Pcnt-depleted oocytes from transgenic (Tg) mice are ovulated at metaphase-II, but show significant chromosome misalignment, aneuploidy and premature sister chromatid separation. These defects were associated with loss of key Pcnt-interacting proteins (γ-Tubulin, Nedd1 and Cep215) from meiotic spindle poles, altered spindle structure, and chromosome-microtubule attachment errors. Live cell imaging revealed disruptions in the dynamics of spindle assembly and organization together with chromosome attachment and congression defects. Notably, spindle formation was dependent on Ran-GTPase activity in Pcnt-deficient oocytes. Our findings establish that meiotic division is highly error-prone in the absence of Pcnt and disrupted aMTOCs, similar to defects reported in human oocytes. Moreover, these data underscore crucial differences between MTOC-dependent and independent meiotic spindle assembly.//////////////////
Depletion of pericentrin in mouse oocytes disrupts microtubule organizing center function and meiotic spindle organization. Ma W 2014 et al.
Accurate chromosome segregation is dependent on the formation and stability of the microtubule spindle apparatus. Meiotic spindle assembly in oocytes differs from the process used during mitosis, and is regulated by unique microtubule organizing centers (MTOCs) that lack centrioles. To gain insight into the molecular composition and function of acentriolar MTOCs in mouse oocytes, we assessed the role of a key MTOC-associated protein, pericentrin (PCNT). In somatic cells, pericentrin functions as a scaffold that binds specific proteins at MTOCs, including ?-tubulin, which is necessary for microtubule nucleation. Pericentrin is expressed in oocytes, but the conservation of its function is not known. Pericentrin localizes specifically to MTOCs during prophase-I arrest in mouse oocytes recovered from pre-ovulatory ovarian follicles, and remains associated with MTOCs at spindle poles during metaphase-I and -II. To test function, specific siRNAs were used to knock down Pcnt transcripts in mouse oocytes. Efficient protein depletion was confirmed by Western blot as well as immunofluorescence analysis. Notably, meiotic spindle structure and chromosome alignment were disrupted in Pcnt-depleted oocytes. Disorganized spindle structures with reduced microtubule density and misaligned chromosomes were observed in the majority of these oocytes (~70%). In addition, ?-tubulin localization to MTOCs was significantly reduced and microtubule regrowth, following cold treatment, was delayed in Pcnt-depleted oocytes. Thus, pericentrin is a key functional component of the unique acentriolar MTOCs of mouse oocytes, and plays an important role in regulating meiotic spindle assembly and/or stability. Mol. Reprod. Dev. 2014. 2014 Wiley Periodicals, Inc.
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