Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing
Summary
Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.
Abstract
Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down’s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.
Introduction
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.
Protocol
Representative Results
Discussion
While there are multiple methods for exogenous nucleic acid transfer into somatic cells, such as electroporation and transfection, microinjection is the optimal method for delivery of RNA molecules into transcriptionally quiescent mouse oocytes. The current protocol provides an effective approach for in vitro depletion of specific mRNAs that enable the functional testing of different spindle and/or MTOC-associated factors in oocytes. This approach results in efficient transcript depletion and is highly adaptable…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.
Materials
| Reagents | |||
| Pregnant Mare's Serum Gonadotropin (PMSG) | EMD Biosciences | 367222 | |
| Minimal Essential Medium (MEM) | *Recipe outlined in Table 1 | ||
| Earle's Balanced Salt Solution (10x) | Sigma | E-7510 | |
| Sodium Bicarbonate | Sigma | S-5761 | |
| Pyruvic Acid, sodium salt | Sigma | P-5280 | |
| Penicillin G, potassium salt | Sigma | P-7794 | |
| Streptomycin Sulfate | Sigma | S-9137 | |
| L-Glutamine | Sigma | G-8540 | |
| EDTA, disodium salt dihydrate | Sigma | E-4884 | |
| Essential Amino Acids (50x) | Gibco | 11130-051 | |
| MEM Vitamin Mixture (100x) | Sigma | M-6895 | |
| Phenol Red solution | Sigma | P-0290 | |
| Bovine Serum Albumin (BSA) | Sigma | A1470 | |
| Milrinone | Sigma | M4659 | |
| Fetal Bovine Serum (FBS) | Hyclone | SH30070.01 | |
| EmbryoMax M2 Media with Hepes | EMD Millipore | MR-015-D | |
| siRNAs targeting Pericentrin | Qiagen | GS18541 | |
| Negative control siRNAs | Qiagen | SI03650318 | |
| Paraformaldehyde (16% solution) | Electron Microscopy Sciences | 15710 | |
| Triton-X | Sigma | T-8787 | |
| Phosphate Buffered Saline (PBS) | Hyclone | SH30028.02 | |
| Anti-Pericentrin (rabbit) | Covance | PRB-432C | |
| Anti-acetylated a-tubulin (mouse) | Sigma | T-6793 | |
| Goat anti-rabbbit Alexa Fluor 488 | Invitrogen | A-21430 | |
| Goat anti -mouse Alexa Fluor 555 | Invitrogen | A-11017 | |
| Major Equipment | |||
| Stereomicroscope (SMZ 800) | Nikon | ||
| Upright Fluorescent Microscope | Leica Microsystems | ||
| Inverted Microscope | Nikon | ||
| Femtojet Micro-injections System | Eppenforf | ||
| Micro manipulators | Eppendorf | ||
| Micro-injection needles (femtotips) | Eppendorf | 930000035 | |
| Holding pipettes (VacuTip) | Eppendorf | 930001015 | |
| Plasticware | |||
| 35mm culture dishes | Corning Life Sciences | 351008 | |
| 4-well plates | Thermo Scientific | 176740 | |
| 96 well plates | Corning Life Sciences | 3367 | |
| 0.45 mm CA Filter System | Corning Life Sciences | 430768 |
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