Abstract
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 γ-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, γ-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.
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