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.
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.
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.
This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Georgia.
1. Preparations
2. Mouse Oocyte Collection
3. Oocyte Microinjection
4. Oocyte Culture for Meiotic Maturation
5. Immunofluorescence Analysis
Microinjection of siRNAs provides an effective approach for mRNA degradation and subsequent protein depletion in oocytes, which enables efficient and highly specific functional testing of different target factors in vitro. Subsequently, immunofluorescence is used for specific phenotype analysis as well as to validate protein depletion in siRNA-injected oocytes. In the current example, fluorescent labeling of individual oocytes with DAPI together with anti-tubulin and anti-pericentrin antibodies enabled: (i) confirmation of pericentrin depletion as well as (ii) a comprehensive assessment of both chromatin and meiotic spindle configurations in control and PCNT–depleted oocytes.
Representative immunofluorescence images of oocytes microinjected with non-specific control or Pcnt siRNAs are shown in Figure 27. Following a 17 hr culture, most control oocytes reached the metaphase-II stage and contain organized meiotic spindles with aligned chromosomes and bright pericentrin labeling at the spindle poles (Figure 2A). Notably, pericentrin was not detected in oocytes injected with Pcnt siRNA, confirming efficient protein knockdown in these cells. Moreover, the majority of PCNT-depleted oocytes remained at the metaphase-I stage and exhibit disorganized spindle structures with misaligned chromosomes (Figure 2B). The few PCNT-depleted oocytes that progressed to metaphase-II also exhibited disrupted spindles (Figure 2C).
Figure 2. Immunofluorescence analysis of meiotic spindle organization in mouse oocytes. Representative images of oocytes injected with either (A) control non-specific siRNAs or with (B, C) specific Pcnt-siRNAs. Oocytes were double-labeled with anti-pericentrin (red) together with an anti-acetylated α-tubulin antibody for the detection of microtubules (green). DNA was labeled with DAPI and is shown in blue. The inset shows a 2X magnification of the spindle pole area. Arrows denote misaligned chromosomes. *Spindle pole. Pb: First polar body. Scale bar of 10 µm. This figure has been modified from Ma and Viveiros, 20147. Please click here to view a larger version of this figure.
Table 1. Recipe for Preparation of MEM. Listing of all reagents and specific amounts needed to prepare 250 ml of MEM. The media is filter sterilized using a 0.45 μm cellulose acetate (CA) membrane filter unit and stored at 4 °C for a maximum of 7 days.
Chemical | Amount |
Earle's Balanced Salt Solution (10x) | 25 ml |
Sodium Bicarbonate | 0.550 g |
Pyruvic Acid | 0.0063 g |
Penicillin G | 0.0188 g |
Streptomycin Sulfate | 0.0125 g |
L-Glutamine | 0.073 g |
EDTA, Disodium Salt Dihydrate | 0.1 mg |
Essential Amino Acids (50x) | 5.0 ml |
MEM Vitamin Mixture (100x) | 2.5 ml |
Phenol Red Solution | 0.2 ml |
Bovine Serum Albumin (BSA) | 0.75 g |
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. Though siRNAs were used to specifically target Pcnt transcripts, similar conditions can be employed to target almost any gene of interest.
Several aspects of this protocol, including oocyte collection, culture and manipulation require practice and experience to preserve oocyte quality and to avoid technical artifacts. Fluctuations in the pH and/or temperature of the culture media must be avoided by working expediently and carefully. Microinjection of small groups of oocytes, and the availability of a heated stage in the microinjection system will limit detrimental temperature changes. The use of pre-validated siRNAs is recommended and efficient target protein depletion requires confirmation by immunofluorescence or Western blotting. Some steps will require modification to target different genes of interest. For example, the recommended 24 hr ‘hold period’ in milrinone-supplemented medium is effective for various siRNAs. However, this time period may require adjustments for highly abundant mRNAs and/or target proteins with particularly long half-lives, or could be shortened for less abundant targets. Yet, it is important to consider that oocyte quality and meiotic potential are best preserved using hold periods of 24 hr or less. Caution with oocyte handling is also needed during fixation and immunofluorescence analysis. Oocyte fixation at 37 °C limits microtubule depolymerization, which occurs with lower temperatures, and better preserves meiotic spindle structure. However, these conditions may need to be adjusted depending on the target protein of interest. In addition, optimal conditions for immunofluorescence (i.e., antibody concentrations as well as incubation time and temperature) also need to be established for all antibodies of interest, and tested rigorously prior to conducting the siRNA microinjection.
This technique may not effectively deplete highly abundant proteins within a reasonable timeframe that maintains oocyte quality and competence to undergo meiotic division, as oocyte viability can only be sustained for a limited time in culture. Hypomorph phenotypes may be observed with partial knockdown of target transcripts. However, co-injection of specific morpholinos may be used as a parallel strategy for additional inhibition of translation. There are also some limitations as to potential downstream analysis that are feasible with microinjected oocytes. For example, the required handling and extended culture can limit the oocyte capacity to undergo successful in vitro fertilization (IVF) and preclude the study of functional effects on pre-implantation embryo development. Moreover, large sample sizes are difficult to achieve with this approach that are needed for some downstream applications, such as immunoblotting or biochemical assays.
Despite some limitations, this approach promotes effective in vitro depletion of specific mRNAs that enables the functional testing of different factors in oocytes and has been used successfully by different research groups. Loss-of-function analysis in oocytes usually involves the generation of (conditional) knockout or transgenic RNAi mouse models. Both approaches are labor and time-intensive. In contrast, in vitro siRNA microinjection can be readily used for functional testing with significantly less time and resources. It is a particularly valuable approach to gain insight of a target protein’s functional implications before embarking on the task of generating a knockout/transgenic mouse model.
The combination of siRNA microinjection and immunofluorescence analysis provides a powerful analytical tool for the study of protein expression and subcellular distribution patterns during different stages of meiotic progression in response to siRNA-mediated target protein depletion. Potential differences in siRNA efficacy from oocyte to oocyte are readily identifiable and allow for the accurate correlation between successful target protein depletion and phenotypical/functional analysis. Further development of protocols for the co-injection of specific siRNA molecules with capped messenger RNAs encoding histone H2B-GFP fusion-proteins (or other fluorescently labeled markers) will enable the assessment of target protein function in real-time by live cell imaging.
In summary, with optimized conditions, siRNA microinjection for transcript silencing provides a valuable mechanistic approach to test the function of specific target proteins in mouse oocytes on a cell per cell basis, especially when combined with other powerful analytical tools such as immunofluorescence analysis. This combined approach was used to successfully deplete MTOC-associated proteins and evaluate meiotic spindle formation.
The authors have nothing to disclose.
This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.
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 |