# Chapter 17

## Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes

### Binyam Mogessie

#### Abstract

Chromosome segregation is conserved throughout eukaryotes. In most systems, it is solely driven by a spindle machinery that is assembled from microtubules. We have recently discovered that actin filaments that are embedded inside meiotic spindles (spindle actin) are needed for accurate chromosome segregation in mammalian oocytes. To understand the function of spindle actin in oocyte meiosis, we have developed high-resolution and super-resolution live and immunofluorescence microscopy assays that are described in this chapter.

Key words Actin, Microtubules, Chromosomes, Meiosis, Oocytes, Eggs, Spindle, Fertility, Highresolution live microscopy

#### 1 Introduction

Every mammalian life begins with the fertilization of an egg by a sperm cell. For the resulting genetically unique zygote to grow into a healthy offspring, both the egg and sperm should first contain the correct number of chromosomes. However, for reasons we are only starting to understand, eggs are often likely to have additional or missing copies of certain chromosomes before fertilization—they are aneuploid [1]. Embryos formed from fertilization of aneuploid eggs frequently die, leading to pregnancy failures, or result in offspring with genetic disorders such as Down's syndrome. Importantly, the rate of egg aneuploidy increases dramatically with advancing maternal age [2]. This phenomenon, often referred to as "the maternal age effect," is highly attributed to errors in meiosis, the specialized form of cell division that generates eggs from oocytes [3]. Indeed, a number of factors including oocyte

The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/ 10.1007/978-1-0716-0219-5\_22

Helder Maiato (ed.), Cytoskeleton Dynamics: Methods and Protocols, Methods in Molecular Biology, vol. 2101, https://doi.org/10.1007/978-1-0716-0219-5\_17, © The Author(s) 2020, corrected publication 2020

chromosome cohesion [4] and microtubule dynamics [5] are known to deteriorate with increasing maternal age.

Meiotic chromosome segregation is driven by a spindle machinery that is assembled from microtubules and separates the chromosomes in two rounds of cell division [3]. Interestingly, we recently discovered that the actin cytoskeleton plays a vital role inside the meiotic spindle—actin filaments embedded inside the spindle help to organize microtubules into functional bundles that can accurately separate the chromosomes [6]. This finding constitutes an important safety mechanism in mammalian meiosis that prevents aneuploidy in oocytes and eggs. The association of actin filaments with meiotic spindles of mouse oocytes has long been known [7, 8], but its functional analyses had been thwarted due to lack of high spatial and temporal resolution microscopy assays. This chapter discusses in detail the live and immunofluorescence microscopy assays that have for the first time enabled highly resolved visualization and functional analysis of spindle actin in mammalian oocyte meiosis.

#### 2 Materials

2.1 Ovary Dissection and Oocyte Isolation 1. N6,2<sup>0</sup> -O-dibutyryladenosine 3<sup>0</sup> ,5<sup>0</sup> -cyclic monophosphate sodium salt (dbcAMP). 2. 35 mm Tissue culture dishes. 3. Pastettes (e.g., Alpha Laboratories, LW4206). 4. Paraffin (mineral oil). 5. 37 -C incubator. 6. 1.5 mL microcentrifuge tubes. 7. Mouth aspirator tube assemblies for calibrated microcapillary pipettes (e.g., Sigma, A5177-5EA). 8. 100 μL micropipettes (e.g. PIP3022, Scientific Laboratory Supplies).


#### 2.2 Stock Solutions for Oocyte Culturing and In Vitro Maturation M2 Medium


2.3 mRNA Synthesis and Preparation for Microinjection

	- 2. Hydrochloric acid solution.
	- 3. Microwave.
	- 4. Absolute ethanol.
	- 5. Tungsten carbide scriber (glass cutter) (e.g., Micro-Mark, 50299).
	- 6. Microinjection chamber constructed from plastic according to dimensions specified in Subheading 3.2, step 2.
	- 7. Scotch double-sided tape, 12.7 7.9 mm.
	- 8. P-1000 micropipette puller fitted with a box filament (World Precision Instruments).
	- 9. Zeiss AxioVert A1 microscope equipped with Narishige MN-4, MO-202 U and NZ-19-2 micromanipulators, and a CellTram 4r Oil hydraulic manual microinjector (Eppendorf).
	- 10. Mercury.

2.5 High-Resolution and Super-Resolution Live Imaging, and Drug Addition Experiments


2.6 Immunofluorescence Microscopy of Fixed Oocytes


#### 3 Methods

3.1.1 Preparation of Oocyte Transfer and Washing Dishes

#### 3.1 Oocyte Isolation, Culturing, and In Vitro Maturation

Mouse ovaries are typically collected from 6- to 12-week-old females (e.g., 129 s, CD1, C57BL/6). Depending on the strain used, typically 30–50 oocytes can be obtained from each animal. This is normally sufficient for one live imaging or immunofluorescence experiment. Ovaries should be dissected and processed with as little delay as possible to maximize the number of meiosiscompetent oocytes that can be isolated. All culturing medium and dishes should be prepared and stored at 37 -C for prompt transfer of dissected ovaries. Oocytes should be cultured in dbcAMP (a non-hydrolyzable analog of cAMP) to keep them arrested in prophase during micromanipulation.


Fig. 1 Preparation of oocyte culturing and in vitro maturation dishes. Dishes should be prepared well ahead of oocyte isolation and microinjection experiments and stored at 37 -C

Fig. 2 Ovary dissection and puncturing. (a) A mouse ovary surrounded by fat tissue. (b) Mouse ovaries after extraction from surrounding fat tissue. (c) Intact (left) and punctured (right) mouse ovaries


3.1.2 Isolation and Preparation of Oocytes for Microinjection

Fig. 3 Mouth pipette production and assembly for aspiration of mouse oocytes. (a) A fully assembled mouth pipette containing mouth piece-fitted tubing and a pulled glass micropipette separated with a 0.22 μm filter. (b) The process of cleaning isolated oocytes from debris by washing through nine droplets of M2 + dbcAMP. (c) Production of glass micropipettes for mouth pipetting using fire-assisted manual pulling techniques. Micropipettes with sharp or broken edges should be discarded to avoid oocyte death during mouth pipetting


Microinjection of oocytes is a principal method for studying mammalian meiosis by high-resolution live imaging. We routinely perform quantitative microinjection of oocytes using a modification of a method that was described in great detail by Jaffe and Terasaki [9]. In this section, technical details in the context of mouse oocyte microinjection are provided along with visual aids.

Visualization of spindle actin, microtubules, and chromosomes in live oocytes is achieved by microinjection of in vitro-transcribed mRNAs that encode their fluorescently labeled markers. We routinely use the calponin-homology domain of utrophin (UtrCH) to mark actin filaments, the microtubule-binding domain of MAP4 (MAP4-MTBD) to mark meiotic spindles, and histone H2B to mark chromosomes. To avoid degradation of mRNAs, they should be prepared for microinjection as follows only after isolation of oocytes from ovaries. In addition, all mRNA samples should be handled on RNase-free bench with gloved hands and using pipettes thoroughly cleaned with 70% ethanol.


3.2 Studying Spindle Actin Function Using High-Resolution and Super-Resolution Live Microscopy

3.2.1 Preparation of mRNAs for Microinjection

Fig. 4 Preparation of mRNA for microinjection. (a, b) Procedures to produce a small contraption to hold capillaries during mRNA loading. Fold a small piece of cardboard at the indicated perforations. (c) Stick folded capillary holder on a 25 75 mm glass slide using tape and place a dollop of grease to hold the capillary in place. (d, e) Load into the capillary the indicated volumes of oil, mRNA, and oil and place on capillary holder grease. (f) Place loaded capillary on 25 75 mm glass slide containing a dollop of grease. Transfer the capillary-containing slide into a 15 cm petri dish overlaid with tissue and place the petri dish on ice


11. Load 0.5 μL of capillary oil into the capillary and place it on ice inside the capillary storage dish by gently laying it over the grease dollop (Fig. 4d).

3.2.2 Preparation of Microinjection Chamber A U-shaped microinjection chamber can be constructed from clear plastic with little effort by most workshops according to the dimensions in Fig. 5a. In this chamber, oocytes are lined up for microinjection inside a small glass shelf created by spacing two specially cleaned coverslips with a double-sided tape. The cleanliness of coverslips used for microinjection strongly affects oocyte health. We use the following steps to thoroughly clean and store coverslips:


Fig. 5 Preparation of chamber and oocytes for microinjection. (a) Dimensions and sides of a microinjection chamber that can be constructed from plastic. (b) Cutting of coverslips into smaller pieces for construction of microinjection shelves. The non-cut (non-sharp) edge of shelving coverslips should be stuck on double-sided tape as an overhang to create space between the two coverslips. The side of the coverslip to which the shelf is affixed with double-sided tape is marked with asterisk symbol to aid with orientation. (c) Apply a thin layer of grease and mount a 22 22 mm coverslip on the back of the microinjection chamber. (d) Apply a thin layer of grease and mount a microinjection shelf on the front of the chamber. The shelving coverslip should face the medium dam. (e) Fill the dam space created on the chamber with M2 + dbcAMP. (f) Load oocytes into microinjection shelf by mouth pipetting. (g) Apply a thin layer of grease into the holding groove and mount mRNA capillary

We then prepare a microinjection shelf and chamber using the following steps:

(a) Using fine tweezers, place a double-sided tape roughly 5 mm away from either side of a 22 22 mm coverslip (Fig. 5b) and firmly stick it down by pressing on it using the non-sharp end of the tweezers.




Fig. 6 Positioning of oocytes, mRNA capillary, and chamber for microinjection. (a) Oocytes loaded into microinjection shelf by mouth pipetting. (b) Microinjection needle holder connected to hydraulic manual injector. (c) Needle, chamber, and mRNA-loaded capillary positioned on microinjection microscope


Fig. 7 Loading of microinjection needle with mRNA. (a–c) Steps of breaking the tip of the microinjection needle and moving mercury to the front of the needle. (d–k) Steps of needle loading with oil and mRNA

that the needle is not sufficiently broken. Repeat the tip breaking step and try to move the mercury to the front of the needle again.


Fig. 8 Microinjection of mouse oocytes. (a) Positioning of microinjection needle in focus with oocytes. (b) Microinjection needle in contact with oocyte at a focal plane that is not ideal for injection (above the hemispheric region of the oocyte). (c) Microinjection needle in contact with oocyte at the ideal focal plane. (d) Piercing into oocyte with microinjection needle followed by expulsion of oil and mRNA into the oocyte cytoplasm. When the front oil portion and mRNA volumes are expelled, mercury can be seen returning to the needle's front


the adjusted position, it should not be necessary to move the needle in the Z-axis throughout the rest of the microinjection experiment.

24. After microinjection, transfer oocytes to a prewarmed dish of M2 + dbcAMP droplets prepared earlier for mRNA expression.

For live imaging of meiosis in mammalian oocytes, we have routinely used confocal microscopes from Zeiss (LSM 710, LSM 780, LSM 800, LSM 880) and Leica (SP8). To achieve maximum resolution during imaging, we use 40 and 63 water immersion objectives (1.1–1.2 NA). In addition, we are able to achieve high signal-to-noise ratio using the Airyscan super-resolution module on LSM 800 and LSM 880 microscopes. Combined, these setups allow us to perform four-dimensional (x,y,z,t) live-cell imaging of meiosis without compromising oocyte viability.

To perform live imaging of microtubules, chromosomes, and spindle actin, oocytes must first be released from prophase arrest by washing out dbcAMP. Depending on the mouse strain used, the release from prophase arrest can take 30 min to 1 h. We find that oocytes from 129-s mouse strains release from prophase arrest faster than oocytes from CD1, C57BL/6, and FVBN strains. Achieving a temperature of 37 -C inside the imaging dish, close to the objective lens where oocytes eventually settle, is absolutely critical for oocyte development (see Note 18). To visualize the entire process of meiosis starting from nuclear envelope breakdown, oocytes should be transferred to the microscope immediately after washing out dbcAMP.


3.2.4 High-Resolution Live Imaging of Spindle Actin Assembly and Chromosome Segregation During Meiosis I and II

Fig. 9 Live imaging of spindle actin, microtubules, and chromosomes during mouse oocyte meiosis. (a) Single confocal section images of surrounded and non-surrounded nucleolar configurations of chromosomes (magenta) in prophase-arrested mouse oocytes. (b) Optimal signal-to-noise ratio of microtubules (gray) and chromosomes (magenta) in an oocyte-resuming meiosis after dbcAMP washout. (c) Spindle actin (white) and chromosomes (magenta) in a mouse oocyte progressing through meiosis I. The signal-to-noise ratio of spindle actin, cytoplasmic actin, and cortical actin indicates optimal mRNA expression level of EGFP-UtrCH that does not cause stabilization of actin filaments and perturb meiotic maturation. (d) Single-plane super-resolution (Airyscan) live imaging of actin (green), microtubules (gray), and chromosomes (magenta) in a mouse oocyte during spindle bipolarization. Faint labeling of actin filaments during spindle bipolarization indicates optimal expression of EGFP-UtrCH that will not interfere with spindle relocation and chromosome segregation due to actin stabilization


Fig. 10 High-resolution live imaging of meiotic spindle assembly and chromosome segregation in a mouse oocyte. Upon release from prophase arrest, a meiotic spindle is assembled from microtubules (gray). Homologous chromosomes (magenta) are captured by the spindle and transported to the oocyte surface where they are segregated. After a second meiotic spindle assembles and aligns the chromosomes, the egg is arrested in metaphase of meiosis II until fertilization

> conditions should allow continuous live imaging of oocytes from nuclear envelope breakdown through meiosis I to anaphase I and metaphase II spindle assembly without compromising oocyte health (Fig. 10) (see Note 23).

3.2.5 Pharmacological Drug Addition Experiments Cytoskeletal loss-of-function assays can be readily performed in oocytes by addition of widely used cytoskeletal inhibitors to the culture medium. Cytoskeletal disruption is reversible when oocytes are treated with drug concentrations we routinely use. We typically treat oocytes with 5 μg/mL cytochalasin D to disrupt the actin cytoskeleton, with 5 μM nocodazole to disrupt microtubules and 5 μM SiR-actin to stabilize actin. In addition, cytochalasin D and nocodazole can be combined to simultaneously disrupt actin filaments and microtubules in loss-of-function assays. Drugs can be added before releasing oocytes from prophase arrest. Alternatively,

Fig. 11 Cytoskeletal disruption assays for studying spindle actin function in mouse oocytes. Oocytes should be washed through 9 prewarmed 20 μL droplets of DMSO (control), cytochalasin D (to disrupt spindle actin), or nocodazole (to disrupt microtubules) before being placed in a glass-bottom imaging dish containing a droplet of DMSO or the respective cytoskeletal drug. For simultaneous drug treatment experiments, as many as four droplets can be placed in the imaging dish without risking cross-contamination

acute cytoskeletal disruption or stabilization can be achieved by addition of drugs at any desired stage of meiosis (e.g., spindle relocation, immediately before anaphase I or II, or before and after metaphase II spindle assembly). For analysis of spindle actin function in meiosis II chromosome alignment and segregation, cytochalasin D or SiR-actin should be acutely added at least 4 h after metaphase II spindle assembly to ensure that all chromosomes are fully aligned first.


3.2.6 Parthenogenetic Activation of Mouse Eggs to Visualize Spindle Actin and Chromosome Segregation During Anaphase II

6. Transfer oocytes to the imaging dish containing DMSO and drug droplets accordingly and proceed with microscopy.

7. If the experiment involves acute perturbation of spindle actin in meiosis II, perform the steps above with oocytes that have matured into eggs and have been arrested at metaphase II for at least 4 h (see Note 25).

After release from prophase arrest, mouse oocytes progress through meiosis I, segregate the homologous chromosomes in anaphase I, and become arrested in metaphase II until fertilized, when the sister chromatids are segregated in anaphase II. Importantly, fertilization can be chemically mimicked in vitro to induce anaphase II and observe sister chromatid separation. We follow these steps to efficiently achieve release from metaphase II arrest in mouse eggs:


3.2.7 Quantification of Chromosome Alignment and Segregation Errors from Live Imaging Datasets

Drug-mediated disruption of spindle actin or its genetic disruption in formin-2 knockout oocytes leads to chromosome alignment and segregation errors that lead to oocyte aneuploidy [6]. High temporal resolution is critical for quantification of chromosome misalignment and segregation. 3–6-min time-lapse live imaging datasets allow us to capture oocyte chromosome alignment and segregation in detail and to reproducibly quantify errors. These chromosomal defects can be quantified in two ways—firstly through manual quantification using well-defined criteria and secondly through automatic detection of chromosomes in live imaging datasets using Imaris software (Bitplane).

The first method takes into account the meiotic spindle length in maximum intensity projection confocal images to define outliers of chromosome alignment. As such, it should be used for quantification only in oocytes where the meiotic spindle is positioned parallel to the imaging plane (Fig. 12a). Z-projection of meiotic spindles that are oriented at various angles relative to the imaging plane will lead to inaccurate measurement of spindle length and the perception of chromosome misalignment where there is none (Fig. 12a). Alignment should be quantified in the metaphase frame immediately before anaphase onset. Anaphase onset is defined as the first frame where homologous chromosomes (anaphase I) or sister chromatids (anaphase II) noticeably start to move apart (Fig. 12b).


The second method uses the three-dimensional surface reconstruction module of Imaris software (Bitplane) to automate the detection of misaligned and lagging chromosomes from highresolution live imaging datasets. This approach can be used to independently confirm results from manual analyses. We have confirmed that the steps described here can be performed using Imaris versions 7.0–9.2.


#### 288 Binyam Mogessie

Fig. 12 Quantification of meiotic chromosome misalignment and segregation errors from high-resolution live imaging datasets. (a) Maximum intensity projections of microtubules (gray) and chromosomes (magenta) in a spindle that is parallel (left) and nonparallel (right) to the imaging plane. Because the spindle length appears shorter and the metaphase plate much wider, projection of nonparallel spindles gives the impression of chromosome misalignment where there is none, and thus should be avoided. (b) The frame of anaphase onset is determined as the time at which chromosomes (magenta) clearly begin to move toward opposite spindle poles. (c) Criteria for quantification of misaligned chromosomes and lagging chromosomes during meiosis are shown. Misaligned chromosomes are classified based on spindle length measurement as described in Subheading 3.2.7. Lagging chromosomes are defined as those chromosomes that fail to clear the central spindle region within 12 min of anaphase onset. Chromosomes that remain in the central spindle region 18 min after anaphase onset are classified as severely lagging. (d, e) Automated identification of misaligned (d) and lagging or severely lagging (e) chromosomes by three-dimensional iso-surface reconstruction of chromosomes (magenta) and using criteria described in (c)


For best results, mouse oocyte fixative solution should be prepared and prewarmed at 37 -C prior to fixation. In addition, the extraction and washing buffers should be prepared beforehand and stored at 4 -C when not being used.


#### 3.3 Studying Spindle Actin Function in Fixed Mouse Oocytes

3.3.1 Fixation and Preparation of Mouse Oocytes for Immunofluorescence

3.3.2 Labeling of Spindle Actin, Chromosomes, and Microtubules in Fixed Oocytes


#### 4 Notes


between imaging each color. Before nuclear envelope breakdown, only some acentriolar microtubule organizing centers (aMTOC) can be seen with MAP4-MTBD. UtrCH will produce a clear labeling of cortical actin and fainter labeling of cytoplasmic actin filaments, which will allow for later detection of spindle actin without stabilizing actin filaments (Fig. 9c). In meiosis-competent oocytes, chromosomes labeled with H2B-mRFP generally adopt a surrounded nucleolar (SN) configuration (Fig. 9a). In some oocytes, a non-surrounded nucleolar (NSN) configuration will be seen (Fig. 9a). Oocytes that achieve the surrounded nucleolar configuration before nuclear envelope breakdown are most likely to progress through meiosis. Since computing storage space is often a limitation with long-term live imaging of meiosis, it might be beneficial to only select those oocytes that have SN configuration for the overnight live imaging experiment. Although the microinjection techniques described here are highly quantitative and reproducible, some oocytes will often express more fluorescently labeled proteins than others. It is important to avoid imaging oocytes with much brighter fluorescence of actin, microtubules, and chromosomes as overexpression of each marker by itself will significantly perturb meiosis. In particular, overexpression of EGFP-UtrCH will stabilize actin filaments and prevent spindle relocation while overexpressed EGFP-MAP4- MTBD and SNAP-MAP4-MTBD will stabilize microtubules and lead to chromosome segregation errors. Similarly, overexpression of H2B-mRFP will interfere with chromosome individualization and segregation.


#### Acknowledgments

I would like to thank Kathleen Scheffler and Sam Dunkley for their input and critical reading of this manuscript. This work was supported by a Wellcome Trust and Royal Society Sir Henry Dale Fellowship.

#### References


Floros V, Adelfalk C, Watanabe Y, Jessberger R, Kirkwood TB, Hoog C, Herbert M (2010) Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2. Curr Biol 20 (17):1511–1521. https://doi.org/10.1016/j. cub.2010.08.023


meshwork of actin filaments. Curr Biol 18 (19):1514–1519. https://doi.org/10.1016/j. cub.2008.08.044

9. Jaffe LA, Terasaki M (2004) Quantitative microinjection of oocytes, eggs, and embryos. Methods Cell Biol 74:219–242

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