Name: evaluation of the spindle assembly checkpoint integrity in mouse oocytes
Uploaded: 2022-09-13T14:50:00
Description: Watch this Scientific Journal Video about Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes at JoVE.com

Funding for this project was provided by the National Institutes of Health (R35GM136340 to KS).

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
<ol>
	<li>Before starting the SAC evaluations, collect mouse oocytes following the previously ...

<ol>
	<li>Gruhn, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+errors+in+human+eggs+shape+natural+fertility+over+reproductive+life+span.">Chromosome errors in human eggs shape natural fertility over reproductive life span.</a> Science. 365 (6460), 1466-1469 (2019).</li><li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nature Reviews. Genetics. 13 (7), 493-504 (2012).</li><li>Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+spindle+assembly+checkpoint+signaling+dynamics.">The molecular biology of spindle assembly checkpoint signaling dynamics.</a> Current Biology. 25 (20), 1002-1018 (2015).</li><li>Lara-Gonzalez, P., Westhorpe, F. G., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spindle+assembly+checkpoint.">The spindle assembly checkpoint.</a> Current Biology. 22 (22), 966-980 (2012).</li><li>London, N., Biggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+dynamics+in+the+spindle+checkpoint+response.">Signalling dynamics in the spindle checkpoint response.</a> Nature Reviews Molecular Cell Biology. 15 (11), 736-748 (2014).</li><li>Kuhn, J., Dumont, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+kinetochores+count+attached+microtubules+in+a+sensitive+and+switch-like+manner.">Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner.</a> Journal of Cell Biology. 218 (11), 3583-3596 (2019).</li><li>Jones, K. T., Lane, S. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+causes+of+aneuploidy+in+mammalian+eggs.">Molecular causes of aneuploidy in mammalian eggs.</a> Development. 140 (18), 3719-3730 (2013).</li><li>Gui, L., Homer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+assembly+checkpoint+signalling+is+uncoupled+from+chromosomal+position+in+mouse+oocytes.">Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.</a> Development. 139 (11), 1941-1946 (2012).</li><li>Nagaoka, S. I., Hodges, C. A., Albertini, D. F., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-specific+differences+in+cell-cycle+control+create+an+innate+susceptibility+to+meiotic+errors.">Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors.</a> Current Biology. 21 (8), 651-657 (2011).</li><li>Sebestova, J., Danylevska, A., Novakova, L., Kubelka, M., Anger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+response+to+unaligned+chromosomes+in+mammalian+female+gametes.">Lack of response to unaligned chromosomes in mammalian female gametes.</a> Cell Cycle. 11 (16), 3011-3018 (2012).</li><li>Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P., Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomolar+concentrations+of+nocodazole+alter+microtubule+dynamic+instability+in+vivo+and+in+vitro.">Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.</a> Molecular Biology of the Cell. 8 (6), 973-985 (1997).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+oocyte+microinjection,+maturation+and+ploidy+assessment.">Mouse oocyte microinjection, maturation and ploidy assessment.</a> Journal of Visualized Experiments. (53), e2851 (2011).</li><li>Thomas, R. E., Thompson, J. G., Armstrong, D. T., Gilchrist, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+specific+phosphodiesterase+isoenzyme+inhibitors+during+in+vitro+maturation+of+bovine+oocytes+on+meiotic+and+developmental+capacity.">Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity.</a> Biology of Reproduction. 71 (4), 1142-1149 (2004).</li><li>Marin, D., Nguyen, A. L., Scott, R. T., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mouse+oocytes+to+assess+human+gene+function+during+meiosis+I.">Using mouse oocytes to assess human gene function during meiosis I.</a> Journal of Visualized Experiments. (134), e57442 (2018).</li><li>Herbert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologue+disjunction+in+mouse+oocytes+requires+proteolysis+of+securin+and+cyclin+B1.">Homologue disjunction in mouse oocytes requires proteolysis of securin and cyclin B1.</a> Nature Cell Biology. 5 (11), 1023-1025 (2003).</li><li>Solc, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+requirements+of+PLK1+during+Mouse+oocyte+maturation.">Multiple requirements of PLK1 during Mouse oocyte maturation.</a> PLoS One. 10 (2), 0116783 (2015).</li><li>Blengini, C. S., Nguyen, A. L., Aboelenain, M., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+integrity+of+the+meiotic+spindle+assembly+checkpoint+in+females+requires+Aurora+kinase+B.">Age-dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B.</a> Aging Cell. 20 (11), 13489 (2021).</li><li>Balboula, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haspin+kinase+regulates+microtubule-organizing+center+clustering+and+stability+through+Aurora+kinase+C+in+mouse+oocytes.">Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes.</a> Journal of Cell Science. 129 (19), 3648-3660 (2016).</li><li>Nabti, I., Grimes, R., Sarna, H., Marangos, P., Carroll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+age-dependent+APC/C-mediated+decrease+in+securin+causes+premature+sister+chromatid+separation+in+meiosis+II.">Maternal age-dependent APC/C-mediated decrease in securin causes premature sister chromatid separation in meiosis II.</a> Nature Communications. 8 (1), 15346 (2017).</li><li>Blengini, C. S., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+technique+to+detect+subcellular+structures+critical+to+oocyte+maturation.">Immunofluorescence technique to detect subcellular structures critical to oocyte maturation.</a> Methods in Molecular Biology. 1818, 67-76 (2018).</li><li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine.">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> Journal of Cell Biology. 190 (1), 73-87 (2010).</li><li>Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+spindle+assembly+checkpoint+signaling+dynamics.">The molecular biology of spindle assembly checkpoint signaling dynamics.</a> Current Biology. 25 (20), 1002-1018 (2015).</li><li>Wassmann, K., Niault, T., Maro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+I+arrest+upon+activation+of+the+Mad2-dependent+spindle+checkpoint+in+mouse+oocytes.">Metaphase I arrest upon activation of the Mad2-dependent spindle checkpoint in mouse oocytes.</a> Current Biology. 13 (18), 1596-1608 (2003).</li><li>Hassold, T., Hall, H., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+human+aneuploidy:+where+we+have+been,+where+we+are+going.">The origin of human aneuploidy: where we have been, where we are going.</a> Human Molecular Genetics. 16, 203-208 (2007).</li><li>Gui, L., Homer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+assembly+checkpoint+signalling+is+uncoupled+from+chromosomal+position+in+mouse+oocytes.">Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.</a> Development. 139 (11), 1941-1946 (2012).</li><li>Homer, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad2+prevents+aneuploidy+and+premature+proteolysis+of+cyclin+B+and+securin+during+meiosis+I+in+mouse+oocytes.">Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes.</a> Genes and Development. 19 (2), 202-207 (2005).</li><li>Blengini, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aurora+kinase+A+is+essential+for+meiosis+in+mouse+oocytes.">Aurora kinase A is essential for meiosis in mouse oocytes.</a> PLOS Genetics. 17 (4), 1009327 (2021).</li><li>Hagting, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+securin+proteolysis+is+controlled+by+the+spindle+checkpoint+and+reveals+when+the+APC/C+switches+from+activation+by+Cdc20+to+Cdh1.">Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1.</a> Journal of Cell Biology. 157 (7), 1125-1137 (2002).</li><li>Rodriguez-Rodriguez, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+RZZ+and+Bub1-KNL1+in+mitotic+checkpoint+signaling+and+kinetochore+expansion.">Distinct roles of RZZ and Bub1-KNL1 in mitotic checkpoint signaling and kinetochore expansion.</a> Current Biology. 28 (21), 3422-3429 (2018).</li><li>Chambon, J. P., Hached, K., Wassmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+spreads+with+centromere+staining+in+mouse+oocytes.">Chromosome spreads with centromere staining in mouse oocytes.</a> Methods in Molecular Biology. 957, 203-212 (2013).</li></ol>

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 humans1,24. Several hypothes...

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 checkpoint (SAC). This mechanism senses kinetochore (KT)-microtubule (MT) attachments and the tension is generated when chromosomes are oriented in a bipolar manner3. Unattached kinetochores trigger a SAC response which starts with the recruitment of MPS1, the master regulator of the SAC, to kinetochores3,4. MPS1 initiates the recruitment of other SAC components, acting as a platform to form the mitotic checkpoint complex (MCC). The MCC, composed of MAD1, MAD2, BUB3, and BUBR1, diffuses into the cytoplasm and inhibits APC/C activation by sequestrating its activator CDC20. Once all kinetochores are stably attached to MTs and chromosomes are aligned at the metaphase plate, the SAC is silenced, and the MCC disassembles and releases CDC20, thereby allowing APC/C activation. Active APC/C degrades Securin and Cyclin B, two key steps in triggering anaphase onset5,6. In somatic cells, the SAC is stringent because it is activated by a single unattached kinetochore and is sufficient to induce cell-cycle arrest6. However, during oocyte meiosis, the SAC is more permissive, and oocytes can enter anaphase I with one or more unattached kinetochores6,7,8,9,10. Understanding why the SAC is more permissive in oocytes is an ongoing area of focus in the field. Mechanisms that cause defects in SAC activation or SAC silencing could lead to errors in chromosome segregation or prolonged cell cycle arrest and cell death. Therefore, evaluating the mechanisms that maintain SAC integrity in oocytes is important to understanding the process of forming healthy, euploid eggs.
This protocol describes techniques to comprehensively evaluate the SAC integrity in mouse oocyte meiosis by examining different critical steps of the checkpoint. First, the evaluation of the SAC response after inducing SAC activation is described. This activation is achieved by generating unattached kinetochores using nocodazole, a drug that depolymerizes MTs11. Second, a method to monitor SAC silencing is described by tracking the dynamics of Securin degradation during oocyte maturation. Finally, an immunofluorescence-based assay is employed to measure the recruitment of MAD2, one of the MCC components, to kinetochores. Together, these assays comprehensively assess SAC integrity during oocyte meiotic maturation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl-sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey-anti-rabbit-Alexa-568</td>
			<td>Life Technologies</td>
			<td>A10042</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Auto Imaging System</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>EVOS Onstage Incubator</td>
			<td>Life Technologies</td>
			<td></td>
			<td>Incubator chamber</td>
		</tr>
		<tr>
			<td>Glass Bottom 96- well plates N 1.5 uncoated</td>
			<td>MatTek Corporation</td>
			<td>P96G-1.5-5-F</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-human-Alexa-633</td>
			<td>Life Technologies</td>
			<td>A21091</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3537</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-ACA</td>
			<td>Antibodies Incorporated</td>
			<td>15-234</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 equipped with a 63&#215;, 1.40 NA oil immersion objective</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H20</td>
			<td>Sigma</td>
			<td>M7774</td>
			<td></td>
		</tr>
		<tr>
			<td>Milrinone</td>
			<td>Sigma</td>
			<td>M4659</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S2429</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldhyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti- MAD2</td>
			<td>Biolegend</td>
			<td>924601</td>
			<td>Dilution 1/1000; previously Covance #PRB-452C</td>
		</tr>
		<tr>
			<td>Reversine</td>
			<td>Cayman Chemical</td>
			<td>10004412</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the spindle assembly checkpoint integrity in mouse oocytes

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.

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-treat...

Watch this Scientific Journal Video about Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes at JoVE.com

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

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.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

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