Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Summary

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Abstract

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Introduction

Aneuploidy, which arises from errors in chromosome segregation, is the leading cause of early miscarriages and is highly linked to mistakes in meiosis¹. Meiosis is distinct from mitosis because it consists of two rounds of cell division without an intervening DNA replication step. In meiosis I, homologous chromosomes separate while sister chromatids remain together. In oocytes, this step is error-prone, leading to aneuploid egg production².

To prevent chromosome segregations errors, most cell types activate a surveillance mechanism that pauses the cell cycle, called the spindle assembly checkp....

Protocol

All mice used in these protocols were housed and raised according to the Rutgers University Institutional Animal Use and Care Committee (Protocol 201702497) and National Institutes of Health guidelines. These regulatory bodies approved all experimental procedures involving animal studies. All mice used in the present study were 6-8-week-old CF-1 females.

1. Experimental preparation

1. Before starting the SAC evaluations, collect mouse oocytes following the previously published report¹². Divide collected oocytes into three evenly sized groups and keep them in Chatot, Ziomek, and Bavister (CZB....

Results

Evaluation of SAC responsiveness by nocodazole treatment
The purpose of this experiment is to evaluate SAC activation and strength. By using nocodazole to depolymerize spindle microtubules, all kinetochores will be unattached, which will cause a SAC-mediated cell-cycle arrest. In the present imaging system, DMSO-treated control oocytes extruded a polar body around 14 h after release from milrinone (Figure 1, top panels). Consistent with SAC activation, nocodazole-treated.......

Discussion

The spindle assembly checkpoint is a critical control mechanism during cell division designed to prevent chromosome segregation errors. It allows the cell to have enough time to correct improper KT-MT attachments. Meiosis in oocytes is an error-prone process, where most of the chromosome mis-segregation occurs during meiosis I, leading to the generation of aneuploid eggs that are the main cause of early miscarriages and infertility in humans^1,24. Several hypothes.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine serum albumin (BSA)	Sigma	A4503
DAPI	Life Technologies	D1306
Dimethyl-sulfoxide (DMSO)	Sigma	D5879
Donkey-anti-rabbit-Alexa-568	Life Technologies	A10042
EVOS FL Auto Imaging System	Life Technologies		Fluorescence microscope
EVOS Onstage Incubator	Life Technologies		Incubator chamber
Glass Bottom 96- well plates N 1.5 uncoated	MatTek Corporation	P96G-1.5-5-F
goat-anti-human-Alexa-633	Life Technologies	A21091
HEPES	Sigma	H3537
Human anti-ACA	Antibodies Incorporated	15-234	Dilution 1/30
ImageJ	NIH
KCl	Sigma	P5405
KH2PO4	Sigma	P5655
Leica SP8 equipped with a 63×, 1.40 NA oil immersion objective	Leica
MgSO4·7H20	Sigma	M7774
Milrinone	Sigma	M4659
Na2HPO4	Sigma	S2429
NaCl	Sigma	S5886
NaN3	Sigma	S2002
Nocodazole	Sigma	M1404
Paraformaldhyde (PFA)	Sigma	P6148
PIPES	Sigma	P6757
Rabbit anti- MAD2	Biolegend	924601	Dilution 1/1000; previously Covance #PRB-452C
Reversine	Cayman Chemical	10004412
Triton-X	Sigma	274348
Tween-20	Sigma	X100
Vectashield	Vector laboratories	H-1000