Meiosis is a unique division process that occurs in gametes (oocytes and sperm) and involves two successive divisions without intervening DNA synthesis to segregate homologous chromosomes and sister chromatids during meiosis-I and meiosis-II, respectively1. Errors in chromosome segregation during meiotic division in oocytes can result in aneuploidy, which is inherited by the embryo during fertilization. Notably, the incidence of aneuploidy in developing embryos increases with advancing maternal age and is a major cause of congenital birth defects as well as pregnancy loss in women1,2, thus, underscoring an important need to understand the molecular basis of aneuploidy during meiotic division.
During cell division, chromosome segregation is crucially dependent on assembly of the microtubule spindle apparatus and establishment of stable chromosome-microtubule interactions for correct attachment to opposite spindle poles. Importantly, meiotic spindle formation in mammalian oocytes differs from mitosis in somatic cells, and is regulated by unique microtubule-organizing centers (MTOCs) that lack centrioles3,4. Essential proteins necessary for microtubule nucleation and organization localize to oocyte MTOCs, including γ-tubulin that catalyzes microtubule assembly. In addition, pericentrin functions as an essential scaffolding protein, which binds and anchors γ-tubulin as well as other factors at MTOCs5. Notably, our studies demonstrate that depletion of key MTOC-associated proteins disrupts meiotic spindle organization and leads to chromosome segregation errors in oocytes, which are not fully resolved by the spindle assembly checkpoint (SAC)6,7. Therefore, defects in spindle stability, that do not trigger meiotic arrest, pose a significant risk in contributing to aneuploidy. Despite their essential role in spindle assembly and organization, oocyte MTOC protein composition and function remains poorly understood.
Testing the function of specific target proteins in mammalian oocytes is challenging, as the cells become transcriptionally quiescent shortly before the resumption of meiosis8,9. Hence, pre-ovulatory oocytes rely on maternal mRNA stores to resume meiosis and support meiotic division as well as the first cleavage divisions after fertilization10,11. The efficacy of RNA interference (RNAi) mediated degradation of mRNA transcripts in mammalian oocytes is well established and maternal RNAs recruited for translation during meiotic maturation are particularly amenable to siRNA targeting 12-14. Therefore, microinjection of short interfering RNAs (siRNAs) into oocytes provides a valuable approach to deplete target mRNAs for functional testing.
Here, we describe methods for the isolation of mouse oocytes and siRNA-mediated depletion of specific transcripts to test the function of an essential MTOC-associated protein, pericentrin. In addition, we describe immunofluorescence analysis conditions to evaluate meiotic spindle formation in oocytes.
The overall goal of this procedure is to assess myotic spindle formation and organization in Cytes following irna mediated depletion of key MTOC associated proteins such as perrin. This method can help answer key questions in a reproductive biology field, such as the mechanisms regulating chromosome segregation and potential disruptions that lead to aneuploidy in all sites. The main advantage of this technique is that it results in efficient transcript, knockdown, and specific immunofluorescence analysis of individual cytes.
This procedure can also be adapted to study almost any target protein 48 hours before the collection day treat The female mice used for oocyte collection with five international units of PMSG injected intraperitoneal after collecting the ovaries using standard practices. Transfer to a dish of prewarm equilibrated, MEM media supplemented with BSA and mil reone and prepare to isolate the cytes under a dissecting microscope. Release the cumulus cyte complexes or COCs by fixing an ovary to the bottom of the culture dish using a 27 gauge needle and use a second needle to puncture the antral follicles using oocyte aspiration or standard mouth pipetting practice.
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